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Contents

INTRODUCTION. . . . . . . . . . . . . . . . . 568THE MECHANISM OF

SITE-SPECIFICRECOMBINATION: ANOVERVIEW . . . . . . . . . . . . . . . . . . . . 569

TWO FAMILIES OFRECOMBINASES WITHDISTINCT MECHANISMS OFSTRAND BREAKAGE,EXCHANGE, ANDREUNION . . . . . . . . . . . . . . . . . . . . . . 570

TYROSINE RECOMBINASES . . . . . 572The Process of Strand Exchange

via a Holliday JunctionIntermediate . . . . . . . . . . . . . . . . . . 572

Structural Insights into Synapsisand Strand Exchange . . . . . . . . . . 574

Controlling Catalytic Activity:Half-of-the-Sites Reactivity . . . . 577

Controlling the Outcome ofRecombination . . . . . . . . . . . . . . . 578

SERINE RECOMBINASES . . . . . . . . 582Recombination by a Process of

Double-Strand Break, Switch,and Rejoin . . . . . . . . . . . . . . . . . . . . 584

Structural Insights into Synapsis,Cleavage, and Strand Exchange. 586

Complex Systems of SerineRecombinases: Regulating theOutcome of Recombination . . . . 592

SUMMARY AND PERSPECTIVES. 600

INTRODUCTION

Site-specific recombination describes a va-riety of specialized recombination processesthat involve reciprocal exchange between de-fined DNA sites. In its strictest definition(as used in this review), site-specific recom-bination involves (a) two DNA partners,(b) a specialized recombinase protein that isresponsible for recognizing the sites and forbreaking and rejoining the DNA, and (c) amechanism that involves DNA breakage and

reunion with conservation of the phosphodi-ester bond energy (i.e., lacking a requirementfor either DNA synthesis or a high-energynucleotide cofactor). The prototypes of site-specific recombination (thus defined) are theintegration of bacteriophage λ into the Es-cherichia coli chromosome (1), the resolutionof cointegrates derived from transposition ofTn3-related transposons (2), and the DNA in-versions responsible for flagellar phase varia-tion in Salmonella (3). [The strict definitionexcludes several other specialized recombina-tion processes that have, on occasion, been de-scribed as site-specific; these include (a) VDJrecombination catalyzed by the RAG1/2 pro-teins during the development of the immunesystem; (b) most DNA transposition events(even when a specific target site is used), in-cluding integration of retroviral cDNAs; and(c) the “homing” of mobile introns.]

Depending on the initial arrangement ofthe parental recombination sites, site-specificrecombination has one of three possible out-comes: integration, excision, or inversion(Figure 1). Integration results from recom-bination between sites on separate DNAmolecules (provided that at least one of theparental chromosomes is circular) and oc-curs with a uniquely defined orientation.For sites located on the same chromosome,the outcome is determined by their rela-tive orientation. Thus, excision results fromrecombination between sites in a head-to-tailorientation, whereas inversion results fromexchange between inverted (head-to-head)sites. The three structural outcomes are usedfor a wide variety of purposes in biologi-cal systems. Most commonly, the use of site-specific recombination by an organism or agenetic element is driven by a primary needto physically join or separate DNA segments.In addition to phage integration and excision,and cointegrate resolution, examples includereduction of replicon dimers to monomersand DNA transposition (see Table 1). How-ever, site-specific recombination is also usedas a means of activating or switching geneexpression as well as a means of generating

568 Grindley ·Whiteson · Rice

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genetic diversity through the acquisition ofadvantageous genes or gene segments.

THE MECHANISM OFSITE-SPECIFICRECOMBINATION: ANOVERVIEW

The process of site-specific recombinationcan be divided into a series of conceptu-ally simple steps. The recombinase bindsto the two recombination sites. The tworecombinase-bound sites pair, forming asynaptic complex with crossover sites juxta-posed. The recombinase then catalyzes cleav-age, strand exchange, and the rejoining of theDNA within the synaptic complex. Finally,the synaptic complex breaks down, releasingthe recombinant products.

From this description, it follows that theminimal components of a site-specific recom-

Excision (resolution)

Integration

Inversion

+

Figure 1The three possibleoutcomes ofsite-specificrecombination.

bination system are a recombinase and a pairof recombination sites. The simplest sites areshort duplex DNA segments, 20 to 30 bpin length, which contain an inverted pair ofrecognition sequences and bind one dimer(or two monomers) of the recombinase. Suchsites contain at their center the point of DNAbreakage and joining, and these are often re-ferred to as the crossover sites. In nature, how-ever, many recombination sites are more com-plicated, containing not only a crossover site,

Table 1 Site-specific recombination: a sampling of enzymes and functions

Recombinase Biological functionTyrosine recombinase family

λ Int and many other phage integrases Integration and excision of phage genomesInt of Tn916/Tn1545 Integration and excision: transposition of circular transposonsIntI Integration and excision of gene cassettes in integronsCre Excision: dimer reduction in phage P1 plasmidsXerC/D Excision: dimer reduction in the E. coli chromosome as well as in many other

bacterial chromosomes and some plasmidsTnpI of Tn4430 Excision: resolution of cointegrates resulting from transposition of Tn4430FimB, FimE Inversion: alternation of gene expression (fimbrial phase variation in E. coli)Rci of R64 Inversion of shufflon segments in plasmid R64, producing various forms of piliXisA, XisC Excision: for developmentally regulated gene activation in AnabaenaFlp Inversion: for amplification of yeast 2-μm plasmid

Serine recombinase familyTnpR of Tn3/γδ and related transposons Excision: resolution of cointegrates resulting from transpositionSin of Staphylococcus aureus Excision: dimer reduction in staphylococcal plasmidsParA of RP4 Excision: dimer reduction in plasmid RP4Hin Inversion: alternation of gene expression (flagellar phase variation) in SalmonellaGin, Cin Inversion: alternation of gene expression (tail fiber proteins) in phages Mu and P1OrfA of IS607/IS1535 Integration and excision: transposition of the Helicobacter pylori element IS607 (and

others?)Int of φC31/Bbv1/φRv1a Integration and excision of Streptomyces and mycobacterial phagesTnpX of Tn4451a Integration and excision: transposition of Tn4451 in ClostridiumSpoIVCA (CisA)a Excision: for developmentally regulated gene activation in Bacillus subtilisXisFa Excision: for developmentally regulated gene activation in Anabaena

aMembers of the large serine recombinase subfamily.

www.annualreviews.org • Site-Specific Recombination 569

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but also additional sequences spanning 100 ormore base pairs. Such a complex site may op-erate in combination with a simple crossoversite [as with λ integrase (λ Int) during integra-tion] or with another complex partner (as withγδ resolvase). The extra DNA contains ad-ditional sites of protein recognition and maybind more copies of the recombinase or otherprotein factors encoded by the host cell or bythe genetic element (e.g., phage or transpo-son) associated with the recombination sys-tem. The purpose of these additional DNA-bound proteins may be regulatory, structural,or both. They may initiate or stabilize thepairing of recombination sites or inhibit in-appropriate pairings; they may deliver recom-binase catalytic domains to the crossover site;they may activate the recombinase; and theymay determine the directionality of recombi-nation (for example, promoting deletion butpreventing inversion, or vice versa).

As indicated above, breakage and rejoin-ing of DNA in site-specific recombinationoccur with no loss or gain of nucleotidesand with strict conservation of phosphodi-ester bond energy. To achieve this, a mech-anism analogous to that of a topoisomeraseis used; DNA strands are broken not byhydrolysis but rather by direct phosphoryltransfer to a side chain of the recombinase.This side chain, a tyrosine or a serine in allcharacterized cases, directly attacks the DNAsugar-phosphate backbone at the crossoversite in a transesterification reaction, form-ing a covalent recombinase-DNA interme-diate on one side of the break and a freehydroxyl group on the other. Rejoining theDNA strands is accomplished by reversingthe process; the free hydroxyls from one re-combination partner directly attack the phos-phodiester linkage between recombinase andDNA of the other partner, releasing the re-combinase and sealing the breaks to pro-duce recombinant products. Intriguingly, thedetails of the process differ depending onwhether the recombinase uses a tyrosine ora serine as the attacking nucleophile (seebelow).

TWO FAMILIES OFRECOMBINASES WITHDISTINCT MECHANISMS OFSTRAND BREAKAGE,EXCHANGE, AND REUNION

Despite the many and distinct roles that site-specific recombination plays in biology andthe large number of systems that have beenidentified, comparisons of the recombinaseamino acid sequences indicate that nearly allfall into two families—the tyrosine recom-binases (also known as the λ integrase fam-ily) (4) and the serine recombinases (alsoknown as the resolvase family, named af-ter the cointegrate-resolving recombinase en-coded by transposons such as Tn3 and γδ)(5).Intriguingly, the tyrosine recombinases arealso related to the eukaryotic type IB topoi-somerases. The two recombinase families areunrelated in protein sequence or structureand employ different recombinational mech-anisms; each family appears to have arisen andevolved separately.

There are interesting similarities and dif-ferences in the catalytic mechanims usedby these recombinases. In both cases, DNAis cleaved by nucleophilic displacement ofa DNA hydroxyl by a protein side chain(Figure 2). From studies of model com-pounds and other enzymes, the phosphotrans-fer reactions themselves are assumed to occurthrough the in-line nucleophilic displacementof one hydroxyl group by another with a pen-tacoordinate transition state. The degree ofexcess negative charge on the nonbridgingoxygens in the transition state (relative to theground state) depends on the degree of simul-taneous bond formation to the nucleophileand the leaving group. For phosphodiesters,such as DNA, both bonds are probably onlypartially formed in the transition state (6).In the tyrosine recombinases, the DNA’s 5′

bridging O is displaced to create a phospho-tyrosyl bond to the 3′ end of the broken DNAstrand, whereas in the serine case, the 3′ bridg-ing O is displaced to form a 5′ phosphoserinelinkage. Unlike many phosphotransferases,


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Figure 2Phosphoryl transfer reactions catalyzed by (a) tyrosine and (b) serine recombinases. Cleavage is assumedto proceed through the in-line nucleophilic displacement of a DNA hydroxyl by the relevant protein sidechain, and ligation by the reverse reaction. The cleavage direction may be assisted by a general base toaccept a proton from the attacking side chain and by a general acid to protonate the leaving DNA oxygen(and vice versa for the ligation reaction). Their probable identities are discussed in the text for thetyrosine recombinases and are as yet unkown for the serine family. Both enzymes also position conservedarginine residues near the scissile phosphate, which may localize it and may stabilize the transition stategeometrically and/or electrostatically. The nonbridging oxygens in the transition state are each given aformal negative charge for artistic simplicity; the degree and distribution of charge is not known in detail.(c, d) The constellation of conserved residues surrounding the scissile phosphate in (c) Flp and (d) γδresolvase. Figures 2c-d as well as Figures 4, 10, 12, and 13 were made with Ribbons (156). Protein DataBank (PDB) identification numbers (IDs) are 1M6X for Flp and 1ZR4 for γδ.


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neither of these recombinase families exploitdivalent metal ions for catalysis. Instead, theysurround the scissile phosphate with severalhighly conserved positively charged aminoacid side chains. Both recombinase types mayalso employ general/acid base catalysis, al-though experimental evidence for this is lim-ited so far to the tyrosine family (discussedbelow).

The serine recombinases introducedouble-strand breaks at both crossover sites;all strands are broken before any exchangeis initiated (7). In contrast, the tyrosinerecombinases only cleave one strand of eachduplex at a time: After each crossover site isnicked, it must be joined to its partner beforethe second strand can be cut. This producesa cross-strand intermediate called a Hollidayjunction (8, 9).

TYROSINE RECOMBINASES

Tyrosine recombinases are most widespreadamong prokaryotes but are also found in ar-chaea and even eukaryotes, where exampleshave been described in fungi, ciliates, and,most recently, certain families of retrotrans-posons (4, 10). The size of the family is il-lustrated by a recent iterative PSI-BLASTsearch (10a) that yielded ∼1000 clearly relatedsequences.

The tyrosine recombinases share a cat-alytic domain with recognizable sequence mo-tifs (4, 11). Structural studies have shown thatthe fold of the entire domain is well con-served even when the sequence identity out-side of the active site region is insignificant.Although some family members, such as FimBand FimE, contain only this domain, in mostthe catalytic domain is preceded by a variableN-terminal domain that helps bind DNA.Some, such as λ Int, have a second N-terminaldomain that binds different DNA sites, andsequencing projects are sure to reveal evenmore variety: For instance, database searchesfound two tyrosine recombinases of unknownfunction whose catalytic domains are followedby putative molybdate-binding domains (12).

The catalytic domain is shared with atleast two other classes of enzyme. Membersof the first, type IB topoisomerases, functionas monomers to release supercoiling tensionin DNA by cleaving and religating just onestrand of DNA, but they do so through asimilar 3′ phosphotyrosine intermediate withan almost identical active site. Enzymes ofthe second class, termed either telomere re-solvases or protelomerases, maintain the co-valently closed hairpin ends of the linear repli-cons found in certain prokaryotes and viruses(13, 14)

The Process of Strand Exchange via aHolliday Junction Intermediate

Each tyrosine recombinase has a specificDNA site, which is minimally comprised ofa pair of inverted enzyme-binding sites sep-arated by a 6–8-bp spacer, although manysystems also include accessory sites whereregulatory proteins can exert their influence.Cleavage and religation take place at the 5′

boundaries of the spacer. Although the se-quences of the spacers can vary, there is gen-erally a requirement for identity between re-combination partners.

The requirement for sequence identityin the crossover region was originally inter-preted to mean that each site would align withits partner, and the Holliday junction inter-mediate would branch migrate through thisregion, exchanging one base pair at a time(15). However, careful biochemical experi-ments led away from this toward a “strand-swapping isomerization” model, in which 2–3 bp adjacent to the cleavage site are melted,and the free end anneals to the complemen-tary sequence in its recombination partner(16–18).

Current understanding of the reactionmechanism comes from many years of suchbiochemistry studies and just under a decadeof structural knowledge. A generalized mech-anism is cartooned in Figure 3. Recombi-nation is initiated when one strand of eachduplex is cleaved by a nucleophilic tyrosine,


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Cleavage / ligation IsomerizationExchange Ligation / cleavage

Figure 3Cartoon of tyrosine recombinase–catalyzed strand exchange. The synaptic complex comprises two DNAduplexes bound by four recombinase protomers assembled in a head-to-tail fashion. Blue stars representactive catalytic centers in the active protomers (pale yellow). One strand from each duplex is cleaved,exchanged, and ligated to form a Holliday junction (rightmost two panels). Isomerization of this junctionalternates the catalytic activity between the two pairs of protomers.

creating covalent DNA-protein phosphotyro-sine linkages at the 3′ ends of the DNA andfree hydroxyls at the 5′ ends. The energy fromthe phosphodiester bond in the DNA back-bone is transferred to the phosphotyrosine.Although recombination requires synapsis oftwo sites, cleavage of a single strand, at leastin some cases, requires only a dimer (19, 20).The next step involves an exchange where thefree 5′ ends attack the 3′ phosphotyrosines ofthe opposing DNA substrates to form a Holl-iday junction. The complex can then isomer-ize so that the inactive monomers become ac-tive and vice versa. This enables a repeat ofthe whole process, i.e., the second, untouchedstrand is attacked, and the new 5′ ends migrateover and attack their partners’ 3′ phosphoty-rosine linkages, freeing the protein, resolvingthe Holliday junction, and completing the re-action. The approximately square planar con-formation of the recombinase-bound DNAallows exchange of the DNA ends with re-

markably little rearrangement of the proteincomponent. The utility of this conformationwas pointed out in 1989 (21) and firmly es-tablished by the Cre-DNA complex structure(22).

The phosphotransfer reaction catalyzed bytyrosine recombinases and type IB topoiso-merases is diagramed in Figure 2. Sequencecomparisons and mutagenesis studies haveidentified five highly conserved active siteresidues that cluster (in three dimensions)near the critical tyrosine: RKHRH [see previ-ous reviews (23–25)]. Studies of the topoiso-merase from vaccinia virus implied that theinvariant lysine acts as a general acid, pro-tonating the leaving 5′ hydroxyl during thecleavage reaction (26). The first arginine isalso important in this process, although itsrole is debated: It may form part of a protonshuttle, it may lower the pKa of the nearby ly-sine, or it may act as the general acid itself withassistance from the lysine (27, 28). The first


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histidine may act as a general base, acceptinga proton from the attacking tyrosine (29; Y.Chen, K.L. Whiteson, P.A. Rice, unpublishedresults ). However, this residue is not as highlyconserved as the others, and its mutagenesisis often not as deleterious to the reaction rate(1, 24). The need for a general base may notbe as strong as the need for a general acid be-cause the pKa of tyrosine is lower than that ofa 5′ hydroxyl. The positively charged catalyticresidues may play multiple roles, such as lo-calizing the scissile phosphate and stabilizingthe pentacoordinate transition state both ge-ometrically and electrostatically (28, 30). Thesecond histidine also makes an important hy-drogen bond to the scissile phosphate in thetransition state of the vaccinia topoisomerasereaction (31). However, in Flp, the analo-gous tryptophan, W330, was found to havea largely architectural role, stabilizing the po-sition of the helix containing the nucleophilictyrosine (32). As more distantly related en-zymes are studied in detail, new variations onthese themes may emerge. For example, ex-periments with the hairpin telomere resolvaseResT did not find evidence that the lysine isacting as the general acid, although its mu-tation did nearly obliterate activity (33), anddespite the presence of a serine in place ofthe normally crucial first arginine, wild-typeCTnDOT integrase is fully functional (34).

Hydrolysis of the phosphotyrosine inter-mediate is normally much slower than religa-tion. The Shuman group recently suggestedthat electrostatic repulsion of water by thephosphate itself plays an important role inpreventing hydrolysis (30). When one of thenonbridging oxygens of the scissile phosphatewas replaced with a methyl group, which hasa similar size but lacks a charge, they foundthat vaccinia topoisomerase became a nucle-ase. Why is the incoming DNA’s 5′ hydroxylnot similarly deterred by electrostatic repul-sion? It may be that it is localized and ori-ented by other contacts within the complex(e.g., by base pairing with the opposite strand),whereas an incoming water molecule wouldhave no such help. Many phosphotransferases

that efficiently mediate the attack of water ona phosphodiester bond include divalent metalions at the active site that can coordinate theattacking water.

Structural Insights into Synapsisand Strand Exchange

X-ray crystallography has provided a wealthof molecular detail on tyrosine recombinases.Structures are now available for the completesynaptic complexes of Cre, Flp, and λ Int, formonomeric DNA complexes of human topoi-somerase I and λ Int, and for XerD, HP1 Int,λ Int, and vaccinia virus topoiosomerase inthe absence of DNA (16, 22, 35–41). Interest-ingly, in the absence of DNA, the active sitetyrosine-containing helices are disordered ormisoriented (with the exception of HP1 inte-grase, where a sulfate ion bound in the activesite may mimic the scissile phosphate). In a re-cent crystallographic triumph, two structureswere determined of full-length λ Int com-plexed with not only crossover but also ac-cessory site DNAs (36).

Cre, Flp, and λ Int all form C-shapedclamps around the DNA substrate (Figure 4).The larger and mostly helical C-terminal do-mains are highly conserved and contain thecatalytic residues, whereas the preceding do-mains are structurally varied. The latter inter-act with the major groove of the DNA nearthe substrate crossover region and can formsignificant protein-protein interfaces with theother protomers in an assembled tetramericcomplex, as we have seen in the structuresof Cre, Flp, and λ Int. The C-terminal do-mains interact with consecutive minor andmajor grooves on the opposite face of theDNA. The monomeric human topoisomeraseI exhibits a similar architecture. It may bethat the catalytic domain is the common an-cestor of the tyrosine recombinases and thatthe N-terminal domains have been added in-dependently to aid complex formation andregulation.

The overall architectures of the synap-tic complexes of Cre, Flp, and λ Int are


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Figure 4Comparison of Cre, Flp, and λ Int tetramers. (a) Tetramers viewed with their N-terminal domains in theforeground and catalytic domains in the background (Flp, left; Cre, center; λ Int, right). The catalyticallyactive protomers are yellow, the inactive protomers are blue, and the nucleophilic tyrosines, wherevisible, are drawn in red. PDB IDs are Flp, 1M6X; Cre, 3Crx; lambda, 1Z19. (b) View of each rotated 90◦such that their N-terminal domains are above the DNA and catalytic domains are beneath.

strikingly similar: The tetramers have twofoldand pseudo fourfold symmetry, and hold theHolliday junction intermediate in a nearlysquare planar conformation (Figure 4). Thestructures also show that the catalytic do-mains interact by swapping part of the C ter-minus with a neighboring protomer. In allknown cases, the segment immediately fol-lowing the tyrosine-bearing helix (Figure 5)crosses from one protomer to the next. InCre, the final helix nestles into a pocket onthe neighboring subunit within the tetramer.In the HP1 apoprotein structure, a similar C-terminal helix swap occurs across a dimer. TheC-terminal segment exchanged by λ Int formsa short beta strand when packed into the adja-cent monomer in the synapsed complex. Flpdisplays a mechanistically important variationon this theme: The tyrosine-containing he-

lix itself is swapped, so that each active site isassembled in trans (after this helix the chainreturns to its original protomer) (42, 43). Inaddition to other Flp-like proteins from fun-gal plasmids, the thermophilic SSV1 integrasehas also been reported to domain swap its ty-rosine (44, 45). As discussed below, these transsegments are usually critical in enforcing half-of-the-sites activity.

The contributions of the N-terminal do-mains to the synaptic complexes vary. Cre’scomplex is the most rigid with close contactsbetween its globular N-terminal domains. Flpis more flexible: Its N-terminal domains in-teract through a second trans helix that packsinto a hydrophobic groove in the neighbor’sN-terminal domain but is connected to itsown protomer by flexible turns. λ Int com-plexes, although still roughly square planar,


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Figure 5Comparison of interfaces within the Flp, Cre, and λ Int tetramers. (a) Two interacting protomers fromeach tetramer (viewed roughly as in Figure 4a). Only the catalytic domains and DNA are included. Thecatalytic tyrosines are shown in red, as is the lysine in Cre, and the scissile phosphates are marked byorange spheres. Even though the Cre and λ Int Holliday junctions shown here were trapped with inactivemutants, all the catalytic residues cluster near the scissile phosphate in the catalytically active protomers(yellow), whereas in the inactive protomers (blue) either the tyrosine (in Flp and λ Int) or the lysine (inCre) is displaced. After Holliday junction resolution from this conformation, both protomers shownwould be bound to the same product duplex. PDB IDs are Flp: 1M6X; Cre, 3Crx; and λ Int, 1Z1G.(b) The alternative interfaces from each tetramer, colored as in Figure 4. After Holliday junctionresolution from this conformation, the protomers shown would be bound to different product duplexes.Made with PyMOL (157).

have much more skewed pseudo fourfold sym-metry and little contact between the domainsequivalent to Flp and Cre’s N-terminal ones,which is consistent with λ’s dependence on theaccessory sites for synapsis. However, its addi-tional N-terminal accessory site-binding do-mains (not shown in Figure 4) do make con-siderable contact with one another and withtheir neighbors’ central domains.

The sequence specificity of these recombi-nases is of interest to those who exploit themas genetic tools as well as to basic scientists.In the Flp, Cre, and λ Int-DNA structures,each domain flanking the crossover site DNAinserts a helix into a major groove, but di-

rect side-chain-base contacts are rather sparseand can rationalize only some of these pro-teins’ sequence specificity (22, 35, 46, 48). λ

Int binds this DNA rather weakly anyway,but in the Flp and Cre cases, water-mediatedcontacts and indirect readout of the DNA’ssequence-dependent conformational parame-ters undoubtedly play important roles. Severalclever approaches have recently been used toselect Flp and Cre variants with relaxed and/oraltered specificity (49–51). Structural stud-ies from the Baldwin group (52) have high-lighted the complexities of sequence recogni-tion: The protein-DNA interface is a largehydrogen-bonded network involving many


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water molecules, and the connectivities of thisnetwork can shift in unexpected ways in re-sponse to mutation. Furthermore, specificitycan be enforced at the catalytic step as well asat the binding step of the reaction (53).

Controlling Catalytic Activity:Half-of-the-Sites Reactivity

A recurring feature of the tyrosine recombi-nases is half-of-the-sites reactivity: Only alter-nating protomers within the synaptic tetramerare active at any given time. First noted byenzymologists in the 1960s (54), the prac-tical consequence of this phenomenon fortyrosine recombinases is that double-strandbreaks are avoided; one strand must be reli-gated before its partner can be cleaved. Thisalso avoids the formation of side products suchas hairpins and three-way junctions that canresult when adjacent protomers within a com-plex are simultaneously active (55–57).

In those cases that have been studied indetail, the key to mediating half-of-the-sitesactivity lies in the geometry of the synap-tic tetramer. It has true twofold symmetry,such that protomers on opposite arms havesimilar conformations, and approximate four-fold symmetry, such that the isomerization be-tween states is relatively straightforward. Thealternating interfaces between protomers di-rectly affect the active site conformations, butexactly how they do so varies in each case.

Flp’s activity is primarily controlled by lo-calization of the tyrosine nucleophile, whichis donated in trans from an adjacent protomer.Within the synaptic tetramer, two interfaces(termed type I) allow proper docking of thetyrosine-bearing helix, while the alternate in-terfaces (type II) place the catalytic domainstoo far apart (Figure 5). Several lines of ev-idence imply that the remainder of the Flpactive site is preassembled around the scissilephosphate.

1. Hydroxyl radical footprinting showedhypercleavage resulting from nucle-ophilic peroxide attack at the phosphate

that lies in the active site, even in thecontext of Flp-binding sites that shouldnot assemble into tetramers (58).

2. Structures of Flp revealed no significantdifferences among active site conforma-tions other than the presence or absenceof the tyrosine.

3. Kinetic studies imply that when the ty-rosine is supplied on a synthetic pep-tide, only a monomer of Flp Y343F isneeded for catalysis (K.L. Whiteson &P.A. Rice, unpublished results).

4. Flp can readily disassemble a syntheticthree-armed DNA junction into a du-plex and a hairpin product, which re-quires the simultaneous activity of adja-cent Flp protomers (56, 59, 60).

Model building confirmed that, although ad-jacent type I interfaces could be formed withina trimer of Flp, addition of a fourth could onlybe accommodated by alternating type I inter-faces with looser type II interactions (61). Thisimplies that a Flp protomer is active wheneveran exogenous tyrosine can reach into its activesite.

λ Int is also regulated largely by posi-tioning of the tyrosine. New structures ofInt synaptic complexes show an even moreskewed fourfold symmetry than Flp’s (36).Here the tyrosine-bearing helix itself is in cis,but the polypeptide chain immediately fol-lowing it crosses to the neighboring protomer,where it forms a short stretch of β strand.In two interfaces, the linker between thesetwo secondary structure elements forms in-teractions that stabilize the tyrosine’s posi-tion, whereas in the other two, the stabilizinginteractions are disrupted, and the tyrosine-bearing helix is disordered (Figure 5).

Int’s C-terminal tail also represses cat-alytic activity when the protein has notformed an appropriate oligomer. Mutationsand deletions in this region greatly en-hance topoisomerase-like DNA relaxationand cleavage by monomeric Int (62–64). Inthe crystal structure of the catalytic domainin the absence of DNA, this tail makes cis in-teractions similar to the trans ones seen in the


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tetramer, but in this context, they trigger arepacking of the previous segment, which re-moves the tyrosine from the active site (38,48). NMR data found that the tail is flexibleand is not altered by the addition of a single Intmonomer-binding site (65). However, activityof the wild-type monomer can be restored byadding an excess of a peptide that mimics theC-terminal tail (66). Such negative regulationmay be particularly important for Int, whichbinds crossover site DNA with relatively weaksequence specificity.

Structures of Cre-DNA complexes showmore subtle differences between the two typesof interfaces. Here, in the context of one typeof interface, the catalytic lysine is displacedfrom the active site, whereas in the other typeof interface, it lies near the scissile phosphate(16) (Figure 5). Cre’s tyrosine-bearing helixis also in cis and is followed by a helix thatpacks in trans against the neighbor’s catalyticdomain. The linker between these two helicesadopts different conformations as it crossesthe two types of interface, which may affectthe dynamics of the tyrosine-bearing helix butnot its general placement. Cre will reluctantlyrecombine three-way junctions and has evenbeen crystallized as a trimeric complex (57). Inthis structure, all three protomers are crystal-lographically identical, and although in someways the active site most closely resemblesthat of the active protomers of tetramers, thecatalytic lysine and the loop on which it liesare disordered.

In the XerCD system, the fourfold symme-try is broken not only by conformation butalso by sequence: Here alternate monomersare actually different proteins (albeit closelyrelated—36% identical) that bind differentDNA sequences, one on each side of thecentral spacer. Sequence similarities suggestthat both of these proteins contain a trans-packing C-terminal segment similar to Cre’s,and biochemical studies have shown that theinteractions made by these segments playa key role in regulating catalytic activity(67, 68).

Controlling the Outcome ofRecombination

How do these systems specifically produce in-versions, deletions, or insertions, and how dosome of them drive the reaction in one direc-tion despite the lack of obvious energy sourcessuch as ATP?

Key factors include the conformation ofthe DNA in the initial synaptic complex andthe stabilization of one conformation overanother in the product complex. First, acrossover site’s central spacer must adopt oneof two possible bends to accommodate con-tacts between the two protomers that bindit (Figure 6a). The conformation that isadopted in the substrate complex determineswhich strand will be cleaved first. Second,the synaptic complex can form, and strandcleavages can be initiated with the asymmet-ric spacers in either relative orientation, butonly the antiparallel orientation is productive(Figure 6b). After the second set of strandexchanges, the spacers in the resulting prod-uct complex will display the alternate bends tothose in the substrate. In the simplest cases,the substrate and product are isoenergetic,and the reaction reaches equilibrium whenthere is 50% of each. However, as describedbelow, many systems have evolved tricks (usu-ally involving accessory factors) to tip the bal-ance toward the desired product.

Simple systems: random synapsis and re-liance on spacer complementarity. Synap-tic complexes formed with parallel spacers areunproductive: Ligation of the product strandswould produce mismatches. These inhibit lig-ation, particularly near the cleavage sites, pre-sumably by misorienting the attacking 5′ hy-droxyl (69). The back reaction is thus favored,and the complex can dissociate. In simple sys-tems, the sequence of the spacers is in fact theonly feature determining the overall polarityof the sites (shown as arrows in Figure 1),which in turn determines the overall choice ofinversion vs deletion/insertion reactions. This


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is not utterly foolproof; however, if the prod-ucts of incorrect (parallel) Cre synapses areselected for in vivo, they can readily be found(70).

Flp- and Cre-mediated recombination israther simple and does not require accessoryfactors, nor is it strongly biased in one direc-tion. Despite an asymmetric spacer sequence,Flp does not even display a significant pref-erence for initial cleavage at one end of thespacer vs the other. Cre, however, does. In as-says using 5′ bridging phosphorothioate sui-cide substrates, Cre preferentially cleaves atthe GpC end of the spacer rather than theApT end (19). Interestingly, cleavage of thepreferred strand was more strongly stimulatedby synapsis than cleavage of the other. Whenthe reaction is started with Holliday junctionsubstrates, Cre preferentially resolves them bycleavage at the ApT end. Combined, thesepreferences may improve the overall effi-ciency of recombination by minimizing theaccumulation of junctions and biasing theirresolution toward products (71). Cre’s cat-alytic preferences probably reflect a balanceof the sequence-dependent flexibility of theDNA itself and the interactions of the pro-tein with its substrate DNA (72, 72a). Crystalstructures of Cre-DNA complexes show strik-ingly asymmetric bends in the spacer regionsthat vary with sequence (16, 73, 74).

Using accessory proteins and other tricksto control outcome. Phage λ integration isa paradigm for the use of accessory proteins tohelp assemble the correct initial complex andto drive the overall reaction in the desired di-rection. Many other integrases appear to usesimilar overall schemes for regulating the di-rectionality of recombination, although thedetails vary (75–79). The λ Int protein has anadditional domain at its N terminus that bindstightly to “arm” DNA sites found on bothsides of the crossover (“core”) site within thephage attachment site, attP (Figure 7). DNA-bending proteins help bring the two types ofsite into close proximity. The bacterial attach-ment site, attB, lacks accessory sites, but the

Antiparallel spacers;both with bend a

Antiparallel spacers;both with bend b

Bend a Bend b

Attempted strand exchangecreates mismatches

a

b

Figure 6Controlling the outcome of recombination. (a) Determining which strandwill be cleaved first. The initial recombinase-bound duplex bends in one oftwo different ways, and this determines which strand will be cleaved first(blue stars). In all cases studied, the activated protomer is the one whoseC-terminal tail is bound by the other. (In Flp, this corresponds to thepolypeptide chain returning after the trans tyrosine-bearing helix.) (b)These two differently bent duplexes can be combined into three differentsynaptic complexes: two productive ones with antiparallel spacers (top) andone unproductive one with parallel spacers (bottom). Note that if aproductive synaptic complex initially has bend a in the spacers, its productwill display bend b, and vice versa.


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Figure 7Cartoon of λ att sites. The crossover site within the phage attachment site, attP (top), is flanked bydifferent arrays of binding sites for accessory proteins (IHF, Xis, and Fis) and for the N-terminal domainof Int (arm sites). Integrative recombination of attP with the simple bacterial attB requires IHF andcreates attR and attL as products (bottom). Excisive recombination is not the direct reversal of thisreaction and requires Xis and Fis as well as IHF.

Int protomers whose catalytic domains bindhere also interact with the arm sites of attPvia their N-terminal domains. Although theaccessory proteins can only dictate the ini-tial bend direction of attP, suicide substratesusing bridging 5′ phosphorothioate linkagesshowed that synapsis with attB is not ran-dom (18). This implies that the initial bendin attB is determined by its DNA sequence.The strand that is preferentially cleaved firstis also exchanged first (80, 81).

After integration, the recombinant sitesflanking the phage are referred to as attL andattR. Excisive recombination, however, is notthe simple reversal of integration: It is stim-ulated by different relative concentrations ofDNA-bending proteins, leading to formationof an initial complex whose geometry differsfrom that of the integrative product complex.In both reactions, the same strand is cleavedand exchanged first, presumably because ofsimilar bends in the core DNA (82).

The arm-binding domains of Int do morethan just help deliver it to the crossover sites;they also regulate catalysis. If complexes areassembled with full-length Int on crossover-

site Holliday junctions, addition of short du-plexes bearing arm DNA sites not only stim-ulates resolution but also biases it towardappropriate duplex products rather than aber-rant hairpins and three-armed junctions (55).The crystal structure of an entire Int tetramerbound to both core and arm DNA has re-cently been reported (36). The N-terminalarm-binding domains form an additional, in-tertwined layer of protein displaying twofoldbut not even pseudo fourfold symmetry. EachN-terminal domain interacts more closelywith the core-binding domain of the neigh-boring protomer than its own. These inter-actions presumably bias the overall complextoward the product conformation, in a senseusing product-binding energy to drive for-ward an otherwise isoenergetic reaction.

Accessory proteins also play crucial rolesin XerCD-mediated recombination, but herethe details vary greatly with context (67).However, one common feature is thattwo recombinases’ preference for hetero-rather than homomeric interactions tidilyavoids the formation of unproductive paral-lel synpatic complexes. Recombination at the


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chromosomal recombination site, dif, requiresFtsK, an ATP-dependent DNA translocasethat also plays other roles in chromosomalsegregation. Without FtsK, XerC catalyzescleavage and strand exchange, but the result-ing Holliday junction can only be resolvedbackward to the substrate; XerD remains inac-tive. However, in the presence of FtsK, XerDis activated and actually catalyzes strand cleav-age and exchange before XerC, implying adifferent initial synaptic complex is formed(83). FtsK’s effects in vitro require only a shortstretch of DNA extending past the XerD-binding site and involve direct contacts withXerD as well as ATP hydrolysis by FtsK (84).

XerCD-catalyzed resolution of dimers ofthe plasmids ColE1 (through its action atcer sites) and pSC101 (at psi sites) has alsobeen well characterized (67). These systemsdo not require FtsK. However, their recom-bination sites are much larger than dif and in-clude binding sites for accessory factors: theDNA-binding peptidase/transcription factorPepA in both cases, plus the arginine repressorArgR for ColE1 and the phosphorylated formof the anaerobic growth regulator ArcA forpSC101 (Figure 8). These accessory proteinsform a topologically defined synaptic complex

that, much like the synaptic complexes formedby the canonical serine recombinases, dictatesintra- rather than intermolecular recombina-tion by a mechanism termed a topological fil-ter. Depending on the spacer, the reaction iseither completed by the sequential action ofboth XerC and XerD, or Holliday junctionsformed by XerC action are resolved by othercellular enzymes. The conversion of negativesupercoils, trapped within the synaptic com-plex, into intermolecular nodes, linking thecatenated products, may also help drive theoverall reaction forward. The X-ray structureof the hexameric PepA revealed a positivelycharged groove that is important for DNAbinding and has greatly aided modeling of thesynaptic complex (85, 86).

The synaptic complex formed by PepA notonly channels but also stimulates recombi-nation. It probably does so simply by aid-ing synapsis of the crossover sites by bringingthem into close proximity; there is no evidencefor direct recombinase-accessory protein con-tacts, and the XerC- and XerD-binding sitescan be swapped without the deleterious ef-fects that might be expected if such con-tacts were important (87). Furthermore, if theXerCD crossover site is replaced with the Cre

PepA XerDXerC

ArgRor ArcA

PepA

=

Figure 8Cartoon of XerCD–mediated resolution of plasmid dimers. (top) Arrangement of protein-binding sites ina dimerized ColE1 or pSC101 plasmid (not to scale). (bottom) Synapsis as proposed by Reijns et al. (86) ismediated by a single PepA hexamer (yellow), which interacts with binding sites in both direct repeats.ArgR (at ColE1 cer sites) or ArcA (at pSC101 psi sites) assists synapsis. XerCD-mediated recombinationof these substrates produces four-noded catenanes as products.


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crossover site (loxP), Cre-mediated recombi-nation also becomes dependent on PepA whenCre’s protein-protein contacts are mutation-ally compromised (88). Surprisingly, it was re-cently reported that ArgR and PepA are re-quired for stable maintenance of the plasmidstate of phage P1 in vivo, although their mech-anism of action on P1 has not yet been directlydetermined (89).

Vibrio cholerae phage CTXφ exploits itshost’s XerCD system for integration but re-quires none of the accessory proteins de-scribed above (90). The secondary structure inthe single-stranded phage DNA creates a du-plex recombination site with a bulged spacer(91). As shown previously for Flp-mediatedrecombination, such mismatches adjacent tothe scissile phosphate will favor cleavage overreligation of the substrate (69). However, afterXerC-mediated cleavage, the new 5′ end doesmatch the chromosomal dif site spacer, so thata covalent Holliday junction can be formed.XerD’s presence, but not its catalytic activity,is required; the junction is resolved by replica-tion and/or repair. The resulting XerCD sitesflanking the integrated prophage no longerhave matching spacers, thus blocking excision(unlike λ, integration of this phage is a one-way street). Directionality in this case is thusdictated by the ability of the single-strandedphage to form a bulged recombination site.

Although the mechanistic details are lessclear, the integrases that mobilize the genecassettes of integrons may also exploit un-usual DNA structures (92). The attC attach-ment site (also known as the 59-bp element)contains a set of repeats capable of forming acruciform structure, and in vitro studies sug-gest that the enzyme preferentially binds suchDNA hairpins (93, 94).

Formation of hairpins is the raison d’etrefor hairpin telomere resolvases (also knownas protelomerases), a novel type of tyrosinerecombinase. These enzymes convert a singleinverted repeat-containing site into two du-plexes with hairpin ends. They contain mostof the canonical conserved catalytic hexad,including the tyrosine that forms a cova-

lent protein-DNA intermediate (33, 95). Re-cent work suggests that ResT from Borre-lia burgdorferi combines this active site witha hairpin-binding module that may resem-ble one found in otherwise unrelated DNAtransposases that form hairpin intermediates(96).

SERINE RECOMBINASES

The serine recombinases are a rather het-erogeneous family of proteins, ranging insize from 180 to nearly 800 amino acid (aa)residues, and with unexpected variations indomain organization (Figure 9a) (5).

Most of our information regarding serinerecombinase domain structure and functionhas come from the prototypical recombi-nase, γδ resolvase, which has been charac-terized extensively, both biochemically andstructurally, by X-ray crystallography. This183-residue protein has an N-terminal cat-alytic domain of ∼100 residues, linked by along (36-aa) α-helix (the E-helix) and an un-structured segment (10 aa) to a typical helix-turn-helix DNA-binding domain at the Cterminus (Figure 10) (97). The serine nu-cleophile is close to the N terminus at po-sition 10. γδ resolvase is a dimer in solution,with the N-terminal portion of the E-helixforming the bulk of the dimer interface. DNAbinding (at least to the crossover site) in-volves not only the H-T-H domain but alsothe C-terminal portion of the E-helix andthe intervening segment. The dimer’s H-T-Hdomains bind symmetrically to the DNA,making sequence-specific major groove con-tacts ∼10 bp from the central cleavage point;E-helix residues (particularly the conservedArg-125) hold the DNA (via phosphate andminor groove contacts) close to the cleavagesite and the 3′ end of the DNA after cleav-age (98); and the unstructured segment snakesalong the minor groove between the two (seeFigure 10) (97). The H-T-H domain appearsto play no important roles outside of DNAbinding because it could be replaced by azinc finger DNA recognition domain in Tn3


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Figure 9(a) Domain structure of serine recombinases. This figure shows the catalytic domain and E-helix (blue),with S∗ showing the position of the serine nucleophile; the DNA-binding domain (green) containing arecognizable helix-turn-helix (H-T-H) motif; and conserved domains of unknown function found insubsets of recombinases (magenta, yellow, and red) (5). (b) Conserved motifs within the catalytic domainand dimerization helix (αE) of serine recombinases. Motifs A and C contain the critical active siteresidues of the recombinase. Motif D, contained within the C-terminal portion of the E-helix plus a fewresidues beyond, is mostly involved with binding the DNA in the region abutting the cleavage site. MotifB forms a rather mobile loop whose function remains mysterious despite the remarkable conservation ofthe Ser-39, Gly-40, and Arg-45 residues.

resolvase without loss of recombinationactivity (99).

Although the well-studied DNA inver-tases, Hin and Gin, are similar in size andorganization to γδ resolvase (and many oth-

ers), some members of the serine recom-binase family are considerably larger, andyet others have switched the DNA-bindingdomain to the N terminus of the protein(5). Despite these variations, all members


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Figure 10Structure of the γδ resolvase bound to its crossover site (97). Residues shown as ball-and-stick shapes areSer-10 (yellow), Gly-101 and Glu-102 (green, labeled hinge), Arg-2, Arg-32, Glu-56, and lys54 (purple,labeled 2-3′), and Glu-124 and Arg-125 (blue). Cα atoms of positions of some informative cysteinesubstitutions (see text) are shown as colored balls: Met-106 (pink), Thr-73 and Ser-112 (red), Asp-95 andAla-113 (gold), Ala-74 (dark gray), and Val-114 (blue). P atoms at the cleavage sites are shown as magentaballs. E marks the long E-helix at the dimer interface. N marks the N termini of resolvase. Figure madefrom PDB ID 1GDT.

contain a readily recognizable catalytic do-main with two clusters of conserved residues,including the serine nucleophile, that formthe recombinase active site (Figure 9b). Thelarge recombinases also contain an obviousanalogue of the E-helix immediately follow-ing the catalytic domain and in most caseshave E-helix residues equivalent to the γδ re-solvase residues Glu-118, Arg-119, Glu-124,Arg-125, and Gly-137 (those that are mostconserved in the E-helices of the conven-tional resolvases and DNA invertases). Eventhe “domain-switched” recombinases appearto have an E-helix analog (with equivalents toArg-119 and Arg-125), presumably allowingthem to form dimeric assemblies and bind the3′ end of the cleaved recombination site in amanner similar to resolvase.

Recombination by a Process ofDouble-Strand Break, Switch,and Rejoin

The salient features of the processes of DNAcleavage and strand exchange have been re-

vealed by detailed biochemical and DNAtopological analyses, primarily of the re-solvases from Tn3 and γδ, and the DNA in-vertases, Hin and Gin (2, 3, 21, 100–103).

All catalytic processes usually occur withina synaptic complex with two crossover sitesand four recombinase subunits (although theSin recombinase appears to be at least oneexception to this). It is now clear that, insynaptic complexes formed by the serine re-combinases, the crossover sites are locatedon the outside, separated by the catalytic do-mains; this is in stark contrast to the synap-tic complexes formed by the tyrosine recom-binases. The idea that the recombinase wasat the center of the synapse with DNA onthe outside was an explicit feature of the ear-liest “precrystal structure” model of the re-solvase synaptic complex (104, 105) and grewout of pioneering topological studies (21, 101,102, 106), but lacked direct evidence. Sub-sequently, evidence for this arrangement wasprovided by three distinct and complementaryexperiments. First, recombination by an acti-vated γδ resolvase between a pair of crossover


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sites separated by an IHF-induced DNA U-turn was shown to be sensitive to the helicalphasing of the sites; the positions of maxi-mum recombination efficiency indicated thatthe catalytic domains formed the core of thesynapse with the DNA outside (107). A similarconclusion was obtained from low-angle X-ray and neutron scattering experiments per-formed with an activated Tn3 resolvase thatformed a stable synaptic complex with two un-cleaved crossover sites; moreover, these dataindicated a substantial separation of the DNAs(108). Finally, an entirely different approach,chemical cross-linking at the sites of intro-duced cysteine residues in an activated Hinmutant, showed that the surface of the cat-alytic domain around the beginning of theE-helix (around residue 100) was at or veryclose to the synaptic interface (109). The re-cently solved crystal structure of a minimalsynaptic complex formed by γδ resolvase haselegantly confirmed the “DNA-out” config-uration of the crossover site synapse and hasthrown new light on the processes of synapsisand strand exchange (see below) (98).

Once a synaptic complex is formed, thefour recombinase subunits are activated toattack the two crossover sites, forming twodouble-strand breaks (Figures 2 and 11).This reaction covalently joins the four recom-binase subunits by a phosphoserine linkage tothe four 5′ ends of the broken strands, leav-ing free hydroxyls at the 3′ ends (7, 110). Thespacing of the scissile phosphates is such thatcleavage leaves a two-base single-strand ex-tension at each 3′ end (7).

Cleavage is a coupled reaction and is per-formed by the recombinase “in cis” (111).Coordination is most pronounced betweenthe two subunits bound to the same site;nicked sites are rarely seen with the wild-type recombinase and remain a minor specieseven when active and inactive subunits aresimultaneously targeted to specific halves ofa crossover site. Nevertheless, in such tar-geted experiments, nicking is seen to be highlystrand specific, occurring at the scissile phos-

phate closest to the binding site of the activesubunit.

Once both crossover sites are fully cleaved,the broken ends are rearranged to bring theminto a recombinant configuration. Studies ofthe changes in DNA topology and linkingnumber that accompany recombination in-dicate that strand exchange involves a mo-tion equivalent to a single 180◦ rotation ofone half of the complex relative to the otherhalf (21, 101, 106, 112–115). The directionof rotation is generally right handed, serv-ing to relax the natural negative superhelicityof the substrate. However, in reactions withrelaxed DNA, it appears that rotations canoccur in either direction (21, 116). Preciselyhow rotations of the broken ends occur is stilla mystery, although recent structural studiesof an active synaptic complex with cleavedcrossover sites strongly favor a particular pro-cess of subunit exchange (see below) (98). Fol-lowing the exchange, the free 3′ OH ends at-tack the 5′-phosphoseryl linkages to rejoin thecrossover sites in recombinant configurationand release the resolvase subunits. For the ini-tial cleavage step, the sequence of the central2 bp is not very important (many 2-bp se-quences can be cleaved, but not all). However,for the rejoining step, it is essential that thetwo-base single-strand extensions of the part-ner sites are able to form Watson:Crick basepairs. Thus, as in the tyrosine recombinases,asymmetry of the central 2-bp sequence canbe used to dictate recombinational direction-ality, allowing recombination of two sites inone orientation but not in the other. Becausecrossover site heterologies are only detectedwhen rejoining is attempted (that is, aftercleavage and strand exchange), the presenceof a mismatched site forces the recombinaseto proceed through a second round of strandexchange. This restores the parental configu-ration and so is without genetic consequence;however, the double rotation leaves a topolog-ical footprint on the substrate DNA, remov-ing supercoils and, in some cases, generatingknotted products (113–115).


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SSSSSS

HOSSS

OOSS

OHHSS

SOH

SSS HOS

SSS SS

SS

SS SSOHH

SS HOSS

OO

SSSSSHO

SOO

SOHH

SS

5'

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3'

5'

5'

3'

3'

5'

5'

3'

3'

Switchstrands

Cleaveboth

duplexes

Rejoin

5'

5'

3'

3'

Figure 11Mechanism of recombination by a serine recombinase. The cartoon shows a synaptic complex, formedfrom a pair of recombinase-bound crossover sites. Only the catalytic domains of the recombinase dimersare shown; these are responsible for the pairing and separate the two DNAs. The serine nucleophiles arerepresented by SOH when free (top left and bottom right) or S- when attached to the DNA 5′ ends (top rightand bottom left). The free 3′ OH groups at the cleavage sites are shown as o. During strand exchange,catalytic domains and DNAs move together because they are covalently joined.

Structural Insights into Synapsis,Cleavage, and Strand Exchange

For the serine recombinases, a molecular un-derstanding of the processes of synapsis andstrand exchange has long remained an elusivegoal. A complicating factor for analysis of theserine recombinases is that the systems of re-combination best characterized biochemicallyrequire large DNA sites in supercoiled plas-mid vectors and either multiple copies of therecombinase bound at sites in addition to thecrossover sites (as in the case of the resolvases)or an additional protein factor, FIS (as with theDNA invertases). To overcome these difficul-ties, it was first necessary to obtain mutantsof the recombinase that were able to performrecombination on short, linear crossover sitesin the absence of additional factors. Such ac-tivated mutants, first obtained with DNA in-vertases (117, 118) and subsequently with re-solvases (119, 120), have played a crucial role

in all experiments that have recently advancedour understanding of synapsis and strand ex-change.

Synapsis. Using such an activated mutant,crystal structures of γδ resolvase with cleavedcrossover sites have recently been obtainedand have provided clear confirmation of theDNA-out configuration of the synaptic com-plex (98). The activated mutant forms atetramer in solution (with or without DNA)and binds, cleaves, and recombines pairs ofisolated crossover sites (121).

Crystallization of the activated resolvasewith a symmetrized crossover site yielded twocrystal forms (98). In each, a tetramer of re-solvase was bound to two cleaved crossoversites, with phosphoseryl covalent linkagesjoining the four 5′ phosphates at the cleavagesites to the Ser-10 residues of the recombinasesubunits (Figure 12). The tetramers in the


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two crystal forms have pseudo 222 symmetry(that is, three orthogonal twofold symmetryaxes) and essentially identical conformations.The core of the tetrameric complex is formedby the resolvase catalytic domains and the E-helices while the DNA and DNA-binding do-mains are on the outside.

The synaptic tetramer has a unique andunanticipated quaternary structure (98). Itconsists of two dimeric units with a quaternarystructure related to, but distinct from, that ofthe dimer of resolvase bound to uncleaved siteI (97). It is assumed that these represent theparental dimers that have just cleaved theircrossover sites (or the recombinant dimersthat are about to rejoin their crossover sites).These two site I-bound dimers are sepa-rated by an interface (the synaptic interface)that is extensive (1780 A2 per dimer) andunexpected—unexpected because the dimersare substantially interdigitated and the inter-face is not formed by docking contiguous pre-existing surfaces of the presynaptic dimer. In-deed, many side chains at the interface werenot exposed on the surface of the presynapticresolvase dimer but were buried at the inter-face between its subunits. A prominent fea-ture of the tetramer (and one that is key toholding the complex together) is its central in-ner core, consisting of two pairs of antiparal-lel E-helices (associated over their N-terminalhalves) that cross at an angle of 100◦ to forman extended X. Within this core, each subunitinteracts with all three of its partners withinthe tetramer. In contrast, no contacts areformed between diagonally positioned pro-tomers within the tyrosine recombinase com-plexes (22, 35). Remarkably, the other inter-face that separates the tetramer into left andright halves is almost totally flat; this may havefunctional significance for strand exchange.

The synaptic tetramer could not have beenmodeled readily, since its formation is ac-companied by dramatic conformational tran-sitions from the structure of the presynap-tic resolvase dimer. These changes are seenboth in the tertiary structure of individual sub-units and in the interactions between them.

Figure 12Structure of the γδ resolvase synaptic tetramer complexed with cleavedcrossover sites (98). Magenta balls indicate the P atoms at the cleavage sites,and the yellow ball and stick side chains show the active site Ser-10residues. (a) View through the complex showing the flat interface thatseparates the complex into two halves. Presumed parental sites are labeledL and R, and L′ and R′, respectively; it is not known, however, whetherrecombination will join L to R′ or to L′. Colored balls indicate the Cαatoms of Ala-74 (dark gray) and Val-114 (blue) (see also Figure 10 and text).(b) Structure in panel a rotated 90◦ about the horizontal axis. (c) Similarview of the presynaptic dimer, to emphasize the tertiary and quaternarychanges that accompany the dimer-tetramer transition. Note theinterlocked dimeric interface that exists prior to synapsis and DNA cleavageas well as the large distance between Ser-10 residues and the cleavage sites.Figures from PDB IDs 1ZR4 and 1GDT.


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For individual subunits, if the DNA andDNA-binding portions (residues 120 to 183)of the presynaptic and synaptic states aresuperimposed, the catalytic Ser-10 residueof the activated resolvase is seen to havemoved 11 A directly toward the scissile phos-phate (Figure 13) (98). This movement con-sists chiefly of two components: substantialrotation (40◦–70◦, depending on the sub-unit of the presynaptic dimer selected forcomparison) of the catalytic domain relativeto the E-helix using residues 101–102 as ahinge (see also Figure 10) and bending ofthe E-helix itself (which moves the helix Nterminus by about 6 A). This new tertiarystructure of the resolvase subunit, with theSer-10 hydroxyl well within bonding distanceof the scissile phosphate, presumably repre-sents the activated conformation necessaryto catalyze cleavage of an initially unbrokencrossover site. In the activated conformation,the D-helix of the catalytic domain swings to-ward the E-helix (the Thr-73-Ser-112 Cα-Cα distance changes from 9.8 A to 5.9 A). Wefind it intriguing that an engineered intrasub-unit disulfide bond between T73C and S112Cin an otherwise wild-type Tn3 resolvase issufficient to activate its ability to cleave anisolated crossover site (122).

Substantial changes in the dimer inter-face accompany the subunit conformationalchanges (98). In the presynaptic state, the in-terface is formed largely by the E-helices,which cross at a 45◦ angle. In the tetramer,however, the E-helices open up like the bladesof a pair of scissors, increasing the cross-ing angle to 100◦. This scissoring of the E-helices, together with the subunit conforma-tional changes, creates an altered and muchreduced dimer interaction. Many interactionsseen across the interface of the presynapticdimer are lost (or exchanged for new, synap-tic interactions), and only a few new ones aregained in the altered dimer.

The process by which a synaptic complexmay be formed is illuminated by the struc-ture of the γδ resolvase synaptic tetramer,even though the activated mutant has cir-

Figure 13Motion of the resolvase catalytic domainassociated with formation of the synaptic tetramer.A postsynaptic subunit (yellow) is superimposed onone half of the presynaptic γδ resolvase-DNAcomplex (blue) to illustrate the change inconformation of the catalytic domain and part ofthe E-helix relative to the DNA andDNA-binding domain (superimposed residueswere 120–183). This motion brings Ser-10 (redoxygen atom) close to the scissile phosphate of theuncleaved DNA (pink ball).

cumvented the normal process of synapsisbecause it is already tetrameric. Presumably,in more natural circumstances, two crossoversite-bound presynaptic dimers of a recombi-nase initially interact via the surface centeredaround the beginning of the E-helices, per-haps as suggested in the various models ofSarkis et al. (121), Nollmann et al. (108), orDhar et al. (109). Li et al. (98) have modeleda plausible transition from this initial state to


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the interdigitated tetramer using the programMORPH (123). The transition shows that,as the N-terminal portions of the E-helicesslide past each other to establish the antipar-allel pairings at the synaptic interface, the E-helices across the dimer interface graduallyscissor open.

Crossover site cleavage. The timing andmechanism of DNA cleavage during the tran-sition are quite uncertain and are not ad-dressed by the tetramer structure becausecleavage has already occurred. Clearly the N-terminal catalytic domains must swing intothe active configuration, allowing the Ser-10residues to attack the scissile phosphates, andpresumably, this conformational change is de-pendent on synapsis. However, although theactivated tetramer can readily make double-strand breaks at the crossover site even whenlocked into the tetrameric conformation bya disulfide link between cysteines positionedin the antiparallel D helices (at position 74)(A. Sinha and N.D.F. Grindley, unpublishedresults), the precise conformation seen in thecrystal structures appears inappropriate eitherfor DNA cleavage or for ligation. Each Ser-10residue is 30 A from that of its dimer partner(compared with a distance of 16A between thescissile phosphates in the presynaptic dimersite I complex) and is 14 A from the 3′ OHthat would be the attacking group in the join-ing step.

There are several possible solutions to thisdilemma. (a) The overall structure of thesynaptic complex protein core is retained, butmotions of the DNA-binding region (residuesfrom the middle of the E-helix to C terminusof resolvase) deliver the uncleaved DNA se-quentially to the two Ser-10 nucleophiles (orthe 3′ OHs sequentially to the phosphoseryllinkages). A potential problem with this sce-nario is that the covalent linkage of the DNAto the first Ser-10 is likely to impede its move-ment to the second Ser-10. (b) The quaternarystructure of the crossed antiparallel pairs ofE-helices is retained, but additional motionsof the catalytic domains allow the two Ser-10

nucleophiles of each synapsed dimer to comecloser together, facilitating attack on the scis-sile phosphates either simultaneously or se-quentially but with minimal distortions (theDNA-binding domains would also need tomove to accommodate binding to uncleavedDNA or deliver the 3′ ends to the phospho-seryl bonds). In the tetramer structure, the di-rect path between each phosphoserine and theappropriate 3′ OH is unimpeded, adding tothe plausibility of this scenario. (c) Since onecannot simply model the uncleaved crossoversite and its associated DNA-binding domainsfrom the presynaptic structure onto the coreof the synaptic tetramer, the possibility re-mains that a structure formed on the pathwayof transition between the presynaptic dimerand the synaptic tetramer but with unknownconformation is responsible for cleavage.

Although the structures of γδ resolvasewith DNA provide two revealing views of therecombinase active site, some important de-tails are missing, and the process of chemistryremains obscure. In particular, it is not at allclear how either the serine hydroxyl or the 3′

OH are activated for the cleavage and rejoin-ing steps, respectively.

The most critical components of the activesite, in addition to the Ser-10 nucleophile, arethree arginine residues (residues 8, 68, and71) and an aspartate (Asp-67) (Figure 2d ).All these residues are very highly conserved,and mutation of any of them substantially re-duces or abolishes crossover site cleavage andrecombination. In the presynaptic structure,Ser-10, Arg-8, Arg-68, and Asp-67 of one sub-unit (along with the nonessential Glu-124′

residue provided in trans from the partner sub-unit) form a hydrogen-bonded network, butno residues appear to be appropriately posi-tioned to act as a proton acceptor for Ser-10.The phosphate of the scissile bond is distantfrom the Ser-10 (∼ 11 A), and in one subunit,Arg-71 appears to coordinate it and the phos-phate 5′ to it (97).

The synaptic tetramer is a product com-plex. The 3′ end (the leaving group of thecleavage reaction) has moved about 14 A away


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from the 5′ phosphoserine and is held by an in-teraction between its phosphate and the well-conserved Arg-125′ (of the partner subunit)(Figure 2d ) (98). Although the path betweenthe 3′ end and the phosphoserine is open,it is not clear what conformational changewould be necessary to bring them together.Arg-8 and Arg-68 interact with the nonbridg-ing oxygens of the 5′ phosphoserine, suggest-ing that this was also their role immediatelyprecleavage, and Asp-67 interacts with Arg-68. Arg-71 no longer appears to contact theDNA but instead packs with residues in themobile loop (residues 40–45); however, if itadopted the configuration seen in the presy-naptic structure, it would be well positioned tointeract with the phosphate 3′ to the cleavagesite.

Strand exchange. As the crystal structureof the resolvase synaptic tetramer indicates,strand exchange poses several substantial chal-lenges (98). The free 3′ OH ends of thecleaved DNAs are far (about 50 A) from thephosphoserine groups they must attack toproduce recombinants (and they appear to bewell bound by the C termini of each E-helix);the 5′ ends are covalently linked to the re-combinase and are not free to move with-out accompanying protein motions; the spacebetween the recombinant ends is filled withthe resolvase catalytic domains, preventingdiffusion of DNA ends across the gap; andboth strands of each crossover site are bro-ken, so that the complex is held together onlyby protein-protein interactions. In addition,topological analysis has shown that strand ex-change involves a motion equivalent to a sin-gle 180◦ rotation of one half of the complexrelative to the other half. How, then, is thismovement of the ends achieved?

Potential mechanisms of strand exchangedepend on which pairs of ends in the tetramerstructure need to be joined to form recombi-nants. The crossover sites could be approxi-mately parallel or approximately antiparallel.If the sites are parallel, then the half sites la-beled L and L′ would be joined to R′ and R, re-

spectively (Figure 12a). For antiparallel sites,however, L would be joined to L′ and R to R′.Each of these scenarios poses a very differentstrand exchange problem within the tetramerstructure.

The structure of the tetramer providesa conceptually simple and elegant way torecombine parallel crossover sites (98). Forthis scenario, the left and right halves of thetetramer are separated by a remarkably flat in-terface with essentially no interlocking com-ponents (Figure 12ab). Indeed the only spe-cific interactions that appear to hold the twohalves together in the orientation observedare those between the positive patches formedin each subunit by Arg-121 and Arg-125 andthe complementary negative patches formedin the dimeric partner subunit by Asp-95 andAsp-96. The rest of the interface is highly hy-drophobic. Li et al. (98) propose that strandexchange is accomplished simply by allowingthe two halves of the tetramer to rotate rel-ative to each other—a process called subunitrotation (21)—using the flat, hydrophobic in-terface both as a bearing and to maintain stablecontact regardless of the relative orientationof the two halves. Calculations and modelingsuggested that each flat surface can readily ad-just its precise conformation so as to avoidany steric clashes during the proposed rota-tion (98). The complementary positive andnegative patches mentioned above may act toprovide a gating mechanism that favors ro-tations in steps of 180◦ and stabilizes a statethat favors rejoining the half sites. Not onlydoes this proposed mechanism account for thetopological changes observed in recombina-tion by resolvases and DNA invertases, but italso readily accounts for the processive cyclesof 360 rotations that occur when strand ex-change of cleaved but nonidentical crossoversites creates mismatches between potentiallyrecombinant ends (114).

If the appropriate alignment of crossoversites was antiparallel, not parallel, recombi-nation by a simple process of subunit rota-tion would be sterically impossible. An alter-native mechanism—domain swapping—has


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been proposed for facilitating strand exchangeby serine recombinases (2, 120; M. Boocock,personal communication). In this process, thefour E-helices forming the core of the synapticcomplex are proposed to remain in place whilea pair of catalytic domains, for example thoseattached to the two functional left half sites,break their interdomainal contacts and, rotat-ing about the “hinge” that connects each toits E-helix, switch places. This would positionthe DNAs in a recombinant configuration. Aconceptual advantage of the domain swap pro-cess is that the continued association of the E-helices holds the synaptic complex together;a disadvantage is that the DNA-binding do-mains of the pair of subunits that switch wouldneed to release their hold on the half sites thatmove. If one looks at the tetramer as a synap-tic complex with antiparallel crossover sites,the subunit arrangement does appear to becompatible with a domain swap. The putativehinge regions of the diagonally opposed sub-units are close—a prerequisite of the domainswap process (the Cα positions of Ile-103 areonly 11 A apart). However, the antiparallel na-ture of the DNAs would mean that each mov-ing half site would have to reverse its directionduring the switch. Also, the two catalytic do-mains that would switch positions are not incontact and could not move as a single rigidbody as originally proposed. Overall, the do-main swap mechanism in the context of thetetramer structure appears rather implausible.

Since the mode of crossover site bind-ing has a profound affect on possible strandexchange mechanisms, it remains crucial todetermine whether recombination emanatesfrom a parallel or antiparallel synapse. We areunaware of any definitive evidence for paral-lel rather than antiparallel crossover sites, al-though the parallel arrangement was favoredby the phasing data of Leschziner & Grindley(107) and was also suggested to be the morelikely alternative by Li et al. (98) on the basisof structural considerations of the completeresolvase synaptosome.

A variety of cross-linking experiments sup-port the subunit rotation model. With the ac-

tivated mutant of γδ resolvase, substitution ofVal-114 with a cysteine enables efficient disul-fide bond formation across the flat interface.The cross-linked species retains DNA cleav-age activity but cannot perform recombina-tion (unless the disulfide bond is reduced) (98).This cross-link would be expected to preventsubunit rotation using the flat interface as abearing, but because it links the E-helices, itwould not prevent domain swapping. By con-trast, a disulfide between cysteines at posi-tion A74C across the synaptic interface allowsboth cleavage and recombination (A. Sinhaand N.D.F. Grindley, unpublished data); thiscross-link is not predicted to prevent recom-bination of parallel crossover sites by subunitrotation but should prevent recombination ofantiparallel sites by domain swapping.

Further strong support for the approx-imately parallel alignment of the crossoversites and for rotation at the flat interfaceas the mode of strand transfer is providedby cross-linking data with an activated, Fis-independent mutant of Hin when these dataare examined in the context of the struc-ture of the γδ resolvase synaptic tetramer.Cysteines at Hin residues S94C (or S99C)(equivalent to γδ resolvase residues Gly-96and Ser-101 in the tetramer structure) canbe readily cross-linked in synaptic complexeswith cleaved crossover sites, using a thiolreagent with an 8A linker (109). The tetramerstructure predicts that cross-links involvingeither of these residues would initially be be-tween diagonally positioned resolvase sub-units (e.g., L and R′). Following subunit ro-tation at the flat interface, L- and L′-linkedsubunits exchange places so predicted cross-links (still between diagonally positioned sub-units) would join subunits at L and R. The Hindata indeed show a time-dependent switch-ing of cross-linked pairs of subunits (identi-fied by the DNA species to which each sub-unit is covalently linked) consistent with thepredictions from the structure. Note that inthe absence of the crystal structure, these Hindata are equally consistent with a domain swapmodel of strand exchange.


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Overall, the available evidence supportsthe proposal that the flat interface separatesthe two recombining halves of the synaptictetramer, adding credence to the model ofsubunit rotation using this interface as a rota-tional bearing. That the flat interface devel-ops from interlocking dimeric interfaces dur-ing the transition from the presynaptic to thesynaptic state is fully consistent with it playinga functionally important postsynaptic role.

Complex Systems of SerineRecombinases: Regulating theOutcome of Recombination

The simplest recombination system, consist-ing of just four subunits of a single recom-binase operating on two identical minimalcrossover sites, cannot distinguish between in-termolecular and intramolecular reactions or(unless crossover sites with asymmetric cen-tral sequences are used) between inversionsand excisions. Biological reactions that re-quire specificity must impose regulatory pro-cesses on the simplest system to promote thedesired reaction and suppress the undesir-able ones. Examples of such reactions are the

Transposition(cointegrate formation)Transposase

Resolvase

Target

Donor

Cointegrate resolution

Tn3

Figure 14Two stages of Tn3 transposition: the formation of the donor-targetcointegrate, and its subsequent resolution. Transposase, responsible(along with the host cell replication machinery) for the first step, acts atthe transposon ends. The site-specific recombinase, resolvase, acts at asite, res (red rectangle), within the duplicated transposon (light blue bars).Adapted from Reference 2.

excision-specific recombination systems me-diated by the resolvases and the inversion-specific recombination systems promoted bythe DNA invertases. Regulatory processesmay operate at the level of synapsis, for ex-ample, by promoting synapsis of appropriaterecombination sites or recombination site ori-entations, or they may operate at the level ofcatalytic activation by promoting the cleavageand strand exchange steps only from appropri-ate synaptic complexes.

Resolvases: excision specificity and the“topological filter.” Serine recombinasesthat specifically promote an excision preventcatalysis of inversions and intermolecular re-combination by requiring the formation of atopologically elaborate synaptic complex foractivation of the recombinase. This process,initially elucidated for Tn3 resolvase, is con-ceptually similar to that used to constrainXerCD activity on dif and cer (see above) butuses different protein components.

The Tn3 and γδ paradigm. The first re-solvase site-specific recombination systemsdiscovered were those encoded by the re-lated bacterial transposons Tn3 and γδ, andthese remain the most thoroughly studied andbest understood. When transposons of theTn3 family move from one replicon to an-other, they form an intermediate, called acointegrate, in which the entire donor repli-con is inserted into the target with a copy ofthe transposon at each of the donor-targetjunctions (see Figure 14). The transposon-encoded resolvase protein acting at the two re-combination sites, termed res sites, within theduplicated transposons, excises the donorreplicon (along with one of the transposoncopies), leaving the other copy in the targetDNA. Two features of the resolvase systemsensure their excision specificity: the complex-ity of their recombination sites and the re-quirement that the substrates be negativelysupercoiled.

The res sites of γδ and Tn3 are nearly 120bp in length and contain three binding sites


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for dimers of resolvase (Figure 15a) (124).The point of DNA breakage and reunion, alsocalled the crossover point, lies at the center ofsite I, but the other two sites (II and III, alsocalled the accessory sites) are required for therecombination process. Spacings between thesites are critical for activity (125). A feature ofthe three binding sites, particularly unusualfor DNA sites recognized by dimeric DNA-binding proteins, is that each has a differentgeometry; all consist of a head-to-head pairof 12-bp recognition sequences, but these se-quences are separated by spacers of differentlengths (4 bp, 10 bp, and 1 bp at sites I, II,and III, respectively). This variation indicatesthat the γδ resolvase dimer has an unusualflexibility (also seen in other serine recom-binases) that enables the DNA-binding do-mains to reach over different distances alongand around the DNA helix.

The accessory binding sites play a cru-cial regulatory role in resolvase-mediated re-combination, preventing inversions or inter-molecular recombination (i.e., integration)and, thus, ensuring that resolvase promotesonly excisions. Synapsis is a prerequisite forsite-specific recombination; nevertheless, sin-gle site I-bound dimers of γδ or Tn3 resolvasecannot form synaptic complexes. Rather, theminimal synaptically competent unit is a pairof dimers bound adjacently to sites II and III,with the subunits at II-L and III-L interact-ing specifically with each other using the crys-tallographically defined 2-3′ interaction (126–129). Synapsis of two res sites is initiated whentwo such accessory site complexes interact (seeFigure 16). Formation of this initial complexpromotes the subsequent pairing of the tworesolvase-bound site Is; this step also dependson the 2-3′ interaction (this time, betweensubunits at site I-R and site III) as well as ad-ditional interactions between the site I-bounddimers (127). The role of the 2-3′ interactioncould be either to abut the subunits thus creat-ing a larger (and thus a more stable and effec-tive) interaction surface or to distort the dimerconformation into a quaternary structure thatfavors synapsis (such as the scissoring of the

Figure 15Two styles of res sites. (a) The γδ res site (a typical site) with threeresolvase-binding sites; below it is shown the sequence of the crossover site.(b) The Sin resH site (an atypical site) with two resolvase-binding sites; notethe unusual head-to-tail arrangement of the two halves of site II. Thehorizontal arrow heads represent the 12-bp binding sequences recognizedby each recombinase subunit. The numbers indicate the lengths of DNAbetween these recognition sequences. The cleavage sites (red) are indicated.

E-helices to allow interdigitation of dimers asseen in the tetramer crystal structure).

The accessory site requirement inhibits in-version and intermolecular recombination byimposing what has been called a topologi-cal filter on the formation of a productivesynapse (21, 102, 104, 105). When two res sitessynapse, they wrap around each other, trap-ping three (−) superhelical turns. Remark-ably, synapsis of just the accessory sites alsotraps three (−) nodes (126, 130). The res inter-wrapping is favored by negatively supercoiledsubstrate DNA but only under the specific cir-cumstance of pairing two res sites in head-to-tail orientation on the same supercoiled DNAmolecule (as shown in Figure 16a). In allother circumstances, for example when two ressites are inverted or are on separate molecules,negatively supercoiled DNA operates to in-hibit productive synapsis because formationof the res site interwraps imposes a compen-satory DNA tangle elsewhere in the substrate


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I

I

III

II III

II

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I I

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II II II

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IIIII

II

I

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IIIII

II

I

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I

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I II III––

–

a

+––

–

–

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+

b –

c – –

– –––

–

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+

+

Figure 16Two-step model for res site synapsis and the topological filter. (a) Two-step synapsis. Synapsis is initiatedby antiparallel pairing of subsites II and III, trapping three (−) nodes. This facilitates the productivepairing of both site Is. (b) Consequence of pairing inverted res sites. To obtain antiparallel pairing ofsubsites II and III, at least one interdomain node must be formed. The subsequent interwrapping of thetwo res sites causes a further four interdomain nodes to form. (Note that nodes formed by the resinterwrapping are (−) relative to the res site orientation but are (+) relative to the path of the DNA.)(c) Consequence of trapping two (−) interdomain nodes at the initiation stage. To compensate for thewrapping of subsites II and III around resolvase, three (+) intradomain nodes must be introduced intoone of the two substrate domains. Adapted from Reference 2.


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that would be energetically unfavorable (e.g.,Figure 16b). An interesting consequence ofthe topological filter is that it operates evenwhen the res sites are in the correct orientationon the same molecule, but the initial synap-sis passively traps two or more extra interdo-main supercoils (as would very likely occurwhen the two sites synapse by random colli-sion but are separated by many kilobases ofDNA) (see Figure 16c). This explains whyresolution in vitro yields catenated productcircles that are invariably singly linked (131).This property of resolvases contrasts dramati-cally with recombinases of the tyrosine family,such as λ Int, Flp, and Cre, which impose notopological filter and produce multiply cate-nated product circles when an excision reac-tion is performed on supercoiled substrateswith well-separated recombination sites (126,132, 133).

Molecular models for the synaptic complexformed by pairing accessory sites or completeres sites have been proposed on the basis ofcrystal structures of γδ resolvase in its presy-naptic conformation (121, 134). Despite theirstructural plausibility and their compatibilitywith experimental data for the 2-3′ interac-tion between subunits at specific sites (127),none of the models provides a satisfactory fitwith the latest crystal structure of the resolvasesynaptic tetramer. Curiously, although dataindicating a 2-3′ interaction between the siteI-bound resolvase subunits and specific sub-units within the accessory site synaptic com-plex appear to be very convincing, modelingadditional subunits onto the synaptic tetramervia 2-3′ interactions places them in positionsthat seem to make any synaptic interactionsimpossible (98). Our conclusion is that the2-3′ interactions between resolvase subunitsat site I and at the accessory sites that areneeded for assembly of the synaptic complexare likely to be broken during the formationof the synaptic tetramer.

The new structure of the synaptic tetramerraises the question of whether resolvase atsynapsed accessory sites would also adopt thepostsynaptic conformation. Existing data sug-

gest that a transition to the conformation ofthe synaptic tetramer does not occur at the ac-cessory sites. Resolvase dimers that are cross-linked by disulfide bonds, either at position106 or between residues at 95 and 113, ef-ficiently form accessory site synaptic com-plexes that readily support synapsis and re-combination by wild-type resolvase at the twocrossover sites (122, 135). Yet these cross-linksshould fix the dimers in a presynaptic state andprevent transition to the interdigitated con-formation of the synaptic tetramer. This sug-gests that there is a stable synapsed state ofresolvase that is distinct from that of the cat-alytically active tetramer. For a more completemolecular understanding of the role of the ac-cessory site-bound resolvase subunits in theassembly of the crossover site synapse and theactivation of catalysis, we await further struc-tural information.

The Sin paradigm. The Sin recombinase isthe prototype of a group of serine recombi-nases encoded by several large staphylococ-cal plasmids. The Sin recombination systemdiffers from that of Tn3 and γδ resolvasein two significant ways (136, 137). First, itsres site, although complex, is only 86 bplong and binds just two dimers of Sin (seeFigure 15), and site II consists of direct (head-to-tail) repeats of the 12-bp binding sequence.Second, recombination requires an architec-tural, DNA-bending protein such as E. coliHU or Bacillus subtilis Hbsu. Nevertheless,like the transposon-encoded cointegrate re-solvases, Sin is specific for an excision reac-tion (its biological role is likely to be reduc-ing plasmid dimers to monomers to ensuretheir stability), and the product of recombi-nation in vitro is a pair of singly linked, cate-nated circles. Furthermore, additional topo-logical analysis indicated that the synapticcomplex trapped three interdomain super-coils. Rowland et al. (136) have proposed thatSin together with Hbsu forms a synaptic com-plex topologically indistinguishable from thatof γδ resolvase and that this complex createsa topological filter to inhibit inversions and


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Figure 17The hin and gin genes and inversion systems. The crossover sites (openarrows), bracketing the invertible DNA segments, and the enhancers(black rectangles within the hin and gin genes), bound by two dimers of Fis,are shown. P indicates the promoters of the inversion-regulated genes.Note that the left crossover site for Gin lies within the S gene so that theS gene product contains a constant and a variable portion: Sc-Sv in theorientation shown and Sc-Sv’ after inversion.

integrations. In their model, they propose thatHbsu is used to facilitate a tight bend be-tween sites I and II, allowing direct interac-tions between the Sin subunits at these sites.Consistent with this, protection assays haveshown that when Sin is bound to sites I andII, Hbsu occupies the intervening DNA seg-ment. In this model, Hbsu essentially replacesthe DNA-bending role of γδ resolvase at siteII of res, and Sin at site II of resH plays a similarrole to γδ resolvase at site III.

One difference between Sin and Tn3/γδ

resolvase is that Sin is catalytically activein the absence of synapsis (presumably as adimer) and is able to cleave and rejoin isolatedcrossover sites (without site II or Hbsu) (136).Thus, for Sin, synapsis, which is essential forthe resolution reaction, may simply be a wayof bringing together a pair of recombinationsites in a controlled (that is excision-specific)manner. By contrast, for Tn3/γδ resolvase,synapsis not only brings the crossover sitestogether but also activates the recombinase.

DNA invertases: inversion specificity andthe Gin/Hin paradigm. A number of serinerecombinases specifically promote inversionof DNA segments to provide a switch betweentwo alternative and mutually exclusive geneticstates (3). The best characterized of these arethe very closely related proteins, Hin, whichis responsible for the phenomenon of flagel-lar “phase variation” in Salmonella, and Gin,

which enables phage Mu to infect alternativebacterial host strains. As shown in Figure 17,inversion promoted by Hin switches the ori-entation of a promoter and, thus, turns on oroff the expression of the adjacent genes; thehin gene lies within the 1-kb invertible seg-ment. The action of Gin inverts an adjacent3.0-kb DNA segment that contains alterna-tive phage tail fiber genes. Remarkably, theGin and Hin recombinases are interchange-able and are able to operate on each othersrecombination sites.

Complete in vitro recombination reactionswith Hin or Gin generate inversions rapidlyand efficiently, but intermolecular recombina-tion is undetectable, and excisions (with sub-strates containing directly repeated recombi-nation sites) are very rare (3, 117, 138). Whatspecifies this pronounced directional bias? Aswith the resolvases, requirements for a super-helical substrate and for complex recombina-tion sites are key determinants. However, therecombination site complexity contrasts withthat of the resolvase systems. There are norequirements for additional recombinase sub-units and binding sites; instead an additionalprotein, Fis (factor for inversion stimulation,a homodimer of 98 aa subunits), and a specificDNA sequence to which Fis binds, called theenhancer, are needed (3, 139–141). The en-hancer, which contains two binding sites forthe Fis dimer, separated by 48 bp (center-to-center), operates independent of orientationand can be placed virtually anywhere in a plas-mid substrate but must be provided in cis (3,142). The synaptic complex within which re-combination takes place (also called the inver-tasome) is a three-looped structure that trapstwo interdomain negative supercoils and mayform at the junction point of a branch in thesupercoiled substrate (Figure 18) (112, 113,143). The three DNA segments at the branchpoint consist of the two crossover sites andthe enhancer, and the complex contains twodimers of the recombinase and two dimers ofFis. Direct interaction between Fis and the re-combinase is needed to activate double-strandcleavage of the crossover sites. However,


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Figure 18The invertasome, showing the −2 topology of the productive synapse. Crossover sites (red arrows) arebound by dimers of the recombinase (green subunits); the enhancer (blue bar) is bound by two dimers ofFis (pale blue ellipses). Two negative interdomain nodes are trapped by the threading of the enhancer. Therecombinase tetramer has been drawn to show a flat interface (proposed to be the bearing for subunitexchange), separating the halves of the crossover sites to be exchanged. (inset) The invertasome, rotated90◦, showing how threading of the enhancer and the Fis-Hin interactions stabilize a proposed + node,which is necessary for appropriately orienting the Hin tetramer. Note that all other DNA crossings arenegative.

following strand exchange, the rejoining stepappears to be Fis independent (113, 142).

In contrast to the resolvase systems, theDNA invertases (along with Fis) do form ac-tive synaptic complexes with inappropriatelyoriented recombination sites. The DNA in-vertases synapse and cleave crossover sitesthat are directly repeated (with respect totheir asymmetric central dinucleotides), butinstead of producing the anticipated excisionproducts, they efficiently convert the super-coiled DNA substrate into a knotted state(113, 138). Topological analysis indicated thatthe knotted circles result from two (or an evennumber of) 180◦ cycles of strand exchange.This showed that the crossover sites, despitetheir direct orientation in the substrate, weresynapsed as if they had been inverted, form-

ing a synaptic complex identical in structureand topology to the normal invertasome. Asa consequence, a single 180◦ cycle of strandexchange resulted in pairing of mismatchedand unligatable crossover sequences (the 2-base 3′ single-strand extensions resulting fromthe double-strand cleavages), necessitating asecond 180◦ rotation to occur before the sitescould be rejoined. This second cycle restoresthe sites in their original (parental) config-uration but because of the two negative su-percoils, trapped by the synapse, converts thesubstrate into a trefoil knot (as this prod-uct is also a substrate, more complex knotsare also produced by repetitions of the pro-cess). Not surprisingly, if crossover sites aremodified to make the central dinucleotidesymmetrical, then recombination by a DNA


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invertase yields inversion products regardlessof whether the full recombination sites are in-verted or directly repeated (138).

How Fis activates catalysis by the DNA in-vertases remains a mystery, although a num-ber of important facts have been determinedby experiments with Hin (3, 144, 145). Activa-tion depends on a direct interaction betweenFis and the recombinase. The Fis componentappears to involve a surface at the tip of a flex-ibly connected β-hairpin arm formed by theN-terminal portion of Fis. Somewhat surpris-ingly, only one functional β-arm in each Fisdimer is needed for efficient activation (sug-gesting contact with only one subunit of eachrecombinase dimer). The region of Hin thatcontacts Fis remains to be determined, al-though the large separation of the two Fis-binding sites in the enhancer is most consis-tent with contacts to the outside of the Hinsynaptic complex. It has been suggested thatFis induces a conformational change in thepaired Hin dimers, which promotes cleavageand strand exchange.

Wild-type Hin, in contrast to Tn3/γδ re-solvase, can efficiently pair isolated crossoversites, but these complexes are inactive inrecombination (143). This raises the pos-sibility that Fis activates Hin by contact-ing the preformed Hin synaptic complex.However, there are problems with this sce-nario. First, activation is absolutely depen-dent on topological linkage of the enhancerand crossover sites; even high concentrationsof Fis and enhancer when added in transfail to activate Hin synaptic complexes (3,142). Second, the topology of the inverta-some, with the requirement that two neg-ative supercoils be trapped, is not easy toreconcile with action of Fis on a preformedHin complex, since capturing these super-coils either depends on Hin synapsis ran-domly trapping a single enhancer-containingDNA segment within the supercoiled invert-ible domain or requires the Hin synapse todissociate, allowing the enhancer-containingsegment to pass between the crossoversites.

The structure of the resolvase synaptictetramer and the strong hypothesis that thecrossover sites form a local negative node andexchange by subunit rotation, using the flatinterface as a swivel (98), offer a new way ofthinking about the role of the enhancer. Wesuggest that the most important role of Fisat the enhancer may be to ensure that theinverted crossover sites cross with a local neg-ative node and, thus, position the flat inter-face of the synaptic tetramer such that rota-tions will cause an inversion. This crossingcreates a positive global node (Figure 18, in-set), which would not be favored in a neg-atively supercoiled substrate in the absenceof Fis [note that although negative with re-spect to the local (roughly parallel) orienta-tions of the crossover sites, this node is glob-ally positive because one site (and the arrowthat represents it) is inverted with respect tothe overall path of the DNA]. One way to fa-cilitate assembly of this final structure wouldbe for a Hin dimer bound to one crossoversite to interact with a Fis dimer at the en-hancer, forming the first ear of the inverta-some. This half invertasome together with thesecond enhancer-bound Fis dimer would cap-ture the second Hin-bound crossover site withthe components of the initial half invertasomeensuring the correct final topology. Fis mayactivate the Hin recombination functions sim-ply by stabilizing the Hin synaptic complexand providing a platform for the strand ex-change steps, although a Fis-induced confor-mational change in the Hin tetramer remainsa possibility (perhaps even mediated by me-chanical/torsional effects imposed by twistingof the three DNA segments and Fis-invertaseinteractions).

Phage integrases: induced fit determinessynapsis specificity. Phage integrases needto distinguish between intermolecular re-combination, resulting in phage integration,and intramolecular recombination, result-ing in prophage excision. The prototypicalphage integrase (and tyrosine recombinase),λ Int, achieves this regulation by means of


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accessory sites and accessory proteins as de-scribed above.

A number of phage integrases that are ser-ine recombinases and members of the largeserine recombinase subgroup (5) appear todistinguish between integration and excisionby a remarkably different (and still poorlyunderstood) mechanism. The best studied ofthese integrases are those of the Streptomycesphage, φC31, and the mycobacteriophages,Bxb1 and φRv1. In each of these cases, theattP and attB sites are simple sites with cen-tral crossover points (attPs range from 40–52bp, attBs from 34–40 bp) (146–149). Each in-tegrase alone efficiently catalyzes integrativeattP × attB recombination in vitro; however,despite binding with similar affinity to attLand attR, it cannot recombine them or anyother pairs of sites (for example, attP × attL orattP × attR, even though these pairs have threeof the four half sites arranged in the same wayas the productive attP × attB pair). The blockto recombining all other pairs of sites appearsto be at the level of synapsis—the recombi-nase alone can only stably synapse attP withattB (149–151).

How is the attP-attB pairing specificityachieved in the absence of other factors whenall four att sites are equally well bound? A cluecomes from the sequences of the sites. attP andattB appear to be surprisingly dissimilar in se-quence; moreover, although each binds just adimer of the integrase, the difference in theirminimal lengths suggests that they bind therecombinase in a different manner. It is pro-posed that each binding site induces an att-specific conformation on the bound integrasedimer and that only the attP- and attB-specificconformations have the necessary comple-mentary interfaces to form a stable synap-tic complex (149, 150). Following recombina-tion, the conformations switch to the attL andattR specificities, the interface complemen-tarity breaks down, and the complex disso-ciates into the separate integrase-bound attLand attR sites. Because these phages can alsoexcise from their integrated state, the recom-binases must be able to catalyze attL × attR

recombination. φRv1 encodes an Xis pro-tein (and the other phages are expected to doso too), and Xis not only enables the φRv1integrase to promote attL × attR recombi-nation, but it inhibits attP × attB recombi-nation (147, 152). These actions of Xis donot require any extra DNA sequences; min-imal attL and attR sites are simply the re-combinants of minimal attP and attB sites.Thus, it seems likely that Xis interacts directlywith the att-bound integrase dimers to switchthe conformation to a synapsis-competentstate if they are bound to attL and attR butto a synapsis-incompetent state at attP andattB.

Serine recombinases as transposases:avoiding target-target recombination. Arather different regulatory issue occurs withthe transposons such as Tn4451, Tn5397,or IS607. These elements move by sequen-tial excision and insertion steps, with excisionforming a circular transposon with abuttedleft and right ends. As with the phage inte-grases, the recombination sites are relativelysmall; by contrast, however, the recombinaseis sufficient for both excision and integration(153, 154). Because the transposition processrequires that the transposase recognizes thetarget sites in addition to the ends of the trans-poson, a conceptual problem is that simulta-neous recognition and synapsis of two targetsites could result in a substantial chromosomaldeletion.

How is this avoided? TnpX, the recombi-nase from Tn4451 and a member of the largeserine recombinase subfamily, has been shownto bind to target sites with much lower affin-ity (about 100- to 1000-fold) than to eithertransposon end (153). This helps reduce un-desirable target-target interactions but (con-sidering the large excess of potential targetsites) is, alone, unlikely to eliminate them. Anadditional proposal is that binding of the re-combinase dimer to an end-end junction in-duces a conformational change that enablesit to capture and activate a second recombi-nase dimer, forming a tetrameric presynaptic


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complex poised for catalysis (153, 155). Thesecond dimer could be loosely bound to tar-get DNA (leading directly to cleavage of tar-get and transposon crossover sites as well as tostrand exchange), or it could be a free dimerthat then captures (perhaps more tightly) atarget sequence.

SUMMARY AND PERSPECTIVES

Despite performing identical biological pro-cesses, two distinct classes of recombinasehave evolved, with each family using entirelydifferent mechanisms of DNA synapsis, cleav-age, and strand exchange. One of the mostsignificant differences lies in the general na-ture of the synaptic complexes formed. Thesynapse of the tyrosine recombinases, with theDNA held within a protein scaffold, allowsstrand exchange to occur with only very mi-nor adjustments of the quaternary structure.The serine recombinase synapse, however,with a solid protein core on which the DNAsites bind, necessitates dramatic movementsof DNA-linked protein subunits to achievestrand exchange.

The relative rigidity of the tyrosine re-combinase synaptic complexes has made itpossible for structural studies to achieve analmost complete series of snapshots, greatlyincreasing our understanding of the entire re-combination process (16, 24, 36). Thus far,perhaps hampered by the dynamics of theprocess, structural studies of serine recom-binases have yielded two pictures (97, 98),and much needs to be revealed before a com-plete understanding of DNA cleavage andstrand exchange is achieved. Although thelatest crystal structure is highly suggestiveof a plausible, if unprecedented, mechanismfor strand exchange, namely subunit rotation,those with interests in either site-specific re-combination or protein motion await a directdemonstration. The process of synapsis is alsonot well understood for either family of re-combinases. What might a dimer of Cre orFlp bound to a single site look like and does

synapsis involve conformational acrobatics?How does the resolvase dimer transition intothe interdigitated structure of the synaptictetramer?

As we have indicated, the natural examplesof site-specific recombination are not only nu-merous but also highly varied in their reg-ulation, yet only a relatively small numberhave been investigated biochemically or struc-turally. Mechanisms of recombinase activa-tion are all mysterious and await a detailedexamination. Do interactions of the recom-binases with accessory proteins directly pro-mote conformational changes? Might twist-ing or bending forces on the DNA sites set upby the topological interlinking of crossoversites and accessory protein-binding sites aswell as protein-protein interactions play a sig-nificant role? Many serine recombinases dis-play a remarkable and unusual capacity to bindto DNA sites with differing sequences and ge-ometries; these differences affect the behaviorof the recombinases in intriguing but poorlyunderstood ways that deserve a thorough in-vestigation.

Many questions remain regarding the cat-alytic mechanisms of these enzymes, particu-larly the serine family. What are the roles ofthe individual residues that surround the scis-sile phosphate? How plastic are these roleswithin a family?

Finally, both families have expanded toinclude noncanonical members with unusualdomain arrangements and interesting varia-tions on the standard recombination scheme.For example, what is the huge C-terminalextension of the large serine recombinasesfor? What enables some recombinases toshow relaxed specificity for one of the part-ner sites? And does that play into the ques-tion of site-induced conformations? Howdo the telomere resolvases orchestrate hair-pin formation rather than strand exchange?How do the integron integrases utilize theirnoncanonical recombination sites? And howmany other uses are there for these versatileenzymes?


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ACKNOWLEDGMENTS

We thank many in the community of site-specific recombination researchers, including ourlab groups and especially Martin Boocock, Marshall Stark, Reid Johnson, Graham Hatfull,Satwik Kamtekar, Tom Steitz, Sean Colloms, Enoch Baldwin, and Kent Mouw for valuablediscussions. Our research is supported by grants from the National Institutes of Health.

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NOTE ADDED IN PROOF

New data strongly support a mechanism for integration of integron cassettes involving a foldedsingle-stranded substrate (159).


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Contents ARI 22 April 2006 9:16

Annual Reviewof Biochemistry

Volume 75, 2006Contents

Wanderings of a DNA Enzymologist: From DNA Polymerase to ViralLatencyI. Robert Lehman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Signaling Pathways in Skeletal Muscle RemodelingRhonda Bassel-Duby and Eric N. Olson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �19

Biosynthesis and Assembly of Capsular Polysaccharides inEscherichia coliChris Whitfield � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �39

Energy Converting NADH:Quinone Oxidoreductase (Complex I)Ulrich Brandt � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �69

Tyrphostins and Other Tyrosine Kinase InhibitorsAlexander Levitzki and Eyal Mishani � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �93

Break-Induced Replication and Recombinational Telomere Elongationin YeastMichael J. McEachern and James E. Haber � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 111

LKB1-Dependent Signaling PathwaysDario R. Alessi, Kei Sakamoto, and Jose R. Bayascas � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 137

Energy Transduction: Proton Transfer Through the RespiratoryComplexesJonathan P. Hosler, Shelagh Ferguson-Miller, and Denise A. Mills � � � � � � � � � � � � � � � � � � � � � � 165

The Death-Associated Protein Kinases: Structure, Function, andBeyondShani Bialik and Adi Kimchi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

Mechanisms for Chromosome and Plasmid SegregationSantanu Kumar Ghosh, Sujata Hajra, Andrew Paek,

and Makkuni Jayaram � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 211

Chromatin Modifications by Methylation and Ubiquitination:Implications in the Regulation of Gene ExpressionAli Shilatifard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 243

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Structure and Mechanism of the Hsp90 Molecular ChaperoneMachineryLaurence H. Pearl and Chrisostomos Prodromou � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 271

Biochemistry of Mammalian Peroxisomes RevisitedRonald J.A. Wanders and Hans R. Waterham � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 295

Protein Misfolding, Functional Amyloid, and Human DiseaseFabrizio Chiti and Christopher M. Dobson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 333

Obesity-Related Derangements in Metabolic RegulationDeborah M. Muoio and Christopher B. Newgard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 367

Cold-Adapted EnzymesKhawar Sohail Siddiqui and Ricardo Cavicchioli � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

The Biochemistry of SirtuinsAnthony A. Sauve, Cynthia Wolberger, Vern L. Schramm, and Jef D. Boeke � � � � � � � � � � � 435

Dynamic Filaments of the Bacterial CytoskeletonKatharine A. Michie and Jan Lowe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 467

The Structure and Function of Telomerase Reverse TranscriptaseChantal Autexier and Neal F. Lue � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 493

Relating Protein Motion to CatalysisSharon Hammes-Schiffer and Stephen J. Benkovic � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 519

Animal Cytokinesis: From Parts List to MechanismsUlrike S. Eggert, Timothy J. Mitchison, and Christine M. Field � � � � � � � � � � � � � � � � � � � � � � � � 543

Mechanisms of Site-Specific RecombinationNigel D.F. Grindley, Katrine L. Whiteson, and Phoebe A. Rice � � � � � � � � � � � � � � � � � � � � � � � � � � 567

Axonal Transport and Alzheimer’s DiseaseGorazd B. Stokin and Lawrence S.B. Goldstein � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 607

Asparagine Synthetase ChemotherapyNigel G.J. Richards and Michael S. Kilberg � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 629

Domains, Motifs, and Scaffolds: The Role of Modular Interactions inthe Evolution and Wiring of Cell Signaling CircuitsRoby P. Bhattacharyya, Attila Remenyi, Brian J. Yeh, and Wendell A. Lim � � � � � � � � � � � � � 655

Ribonucleotide ReductasesPar Nordlund and Peter Reichard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 681

Introduction to the Membrane Protein Reviews: The Interplay ofStructure, Dynamics, and Environment in Membrane ProteinFunctionJonathan N. Sachs and Donald M. Engelman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 707

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Relations Between Structure and Function of the MitochondrialADP/ATP CarrierH. Nury, C. Dahout-Gonzalez, V. Trezeguet, G.J.M. Lauquin,G. Brandolin, and E. Pebay-Peyroula � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 713

G Protein–Coupled Receptor RhodopsinKrzysztof Palczewski � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 743

Transmembrane Traffic in the Cytochrome b6 f ComplexWilliam A. Cramer, Huamin Zhang, Jiusheng Yan, Genji Kurisu,

and Janet L. Smith � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 769

INDEXES

Subject Index � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 791

Author Index � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 825

ERRATA

An online log of corrections to Annual Review of Biochemistry chapters (if any, 1977 tothe present) may be found at http://biochem.annualreviews.org/errata.shtml

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mechanisms of site-specific recombination*

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