3 recombinase of plasmid psm 19035 binds to two adjacent sites

© 7995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 16 3181-3188

The (3 recombinase of plasmid pSM 19035 binds totwo adjacent sites, making different contacts at eachof themFernando Rojo1 and Juan C. Alonso1-2*

1 Centra Nacional de Biotecnologi'a, CSIC, Campus de la Universidad Autbnoma de Madrid, Cantoblanco, 28049Madrid, Spain and 2Max-Planck-lnstitut fur molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany

Received May 22, 1995; Revised and Accepted July 10, 1995

ABSTRACT

The (3 recombinase from plasmid pSM19035 catalyzesIntramolecular site-specific recombination betweentwo directly or Inversely oriented six sites In thepresence of a chromatin-associated protein (Hbsu, HUor HMG-1). The six site is a DNA segment containingtwo binding sites (I and II) for p protein dimers. Weshow that p recombinase binds sequentially to bothsites, having a different affinity for each one. Hydroxylradical footprints show a different protection pattern ateach site. Positions critical for (3 protein binding havebeen identified by methylation interference and mis-sing nucleoside assays. The results indicate that theprotein recognizes each site in a different way. Com-parison of the (3 protein recombination site with that ofDNA resolvases and DNA invertases of the Tn3family,to which it belongs, shows that these sequences canbe divided into two regions. One corresponds to thecrossover point and is similar for all recombinases ofthe family. The other region differs in the differentsubfamilies and seems to have an architectural role Inaligning the crossover sites at the synaptlc complex.The different ways to assemble this complex couldexplain why each system leads to a particular recom-bination event: DNA resolution (resolvases), Inversion(invertases) or both (p recombinase).

INTRODUCTION

(3 recombinase, encoded by the Gram-positive, broad host rangeplasmid pSM 19035, participates in replication and orderedpartition of the plasmid. It catalyzes conversion of dimers orhigher oligomeric forms into monomers and mediates a DNAinversion process between the inverted repeated arms of theplasmid molecule (1 -4, see 5 for a discussion). In the presence ofa chromatin-associated protein, such as Bacillus subtilis HBsu,Escherichia coli HU or mammalian HMG-1, purified P recombi-nase is able to catalyze both deletions (resolution) and inversions

between two specific recombination sites {six sites), dependingon their relative orientation (5,6).

In terms of sequence homology the (3 protein belongs to the Tnifamily of DNA recombinases (6). These proteins can be dividedinto three major subfamilies: DNA resolvases, DNA invertasesand resolvase-invertases. The P recombinase and the ResPprotein of plasmid pAMP 1 are the only members of the last groupthat have been analyzed in vitro (5,7). DNA resolvases are highlyspecialized in catalyzing deletions between two directly orientedrecombination sites, named res sites (8,9; Fig. 1). DNA invertaseshave the opposite characteristic: they promote inversions betweentwo inversely oriented target sites, but deletions between twodirectly oriented sites occur at a very low frequency (10,11).Conversely, P recombinase can catalyze both deletions (resolu-tion) and inversions between two specific recombination siteswith comparable efficiency (5,6).

DNA resolvases do not require accessory factors to promoterecombination (9,12), but DNA invertases and resolvase-invertases demand an accessory protein to mediate recombination(Fig. 1). Efficient inversion by DNA invertases requires thepresence of a stimulating sequence in cis (an enhancer), to whichFis protein binds (reviewed in 10,11). The chromatin-associatedprotein required by P recombinase to mediate both DNAresolution and DNA inversion is needed in stoichiometricamounts: -1-2 Hbsu dimers/DNA molecule are sufficient tostimulate the reaction (5). Hbsu seems to bind to the recombina-tion region (the six site) in a specific way, since at protein-DNAmolar ratios of -1:6 it cannot be competed for by a supercoiledDNA lacking the six site (13). The P protein does not require Hbsuto bind to DNA (6). We have recently shown that Hbsu protein isrequired for synaptic complex formation (13). Altogether, thesedata suggest that the role of the chromatin-associated protein is torecognize and stabilize a DNA structure at the six site that isrequired for assembly of the synaptic complex, rather than tointeract with P recombinase.

The different requirements for accessory factors by each classof enzymes is likely to be related to the organization of theirrecombination sites. DNA resolvases bind to a DNA segmenttermed res, containing three adjacent binding sites with dyad axissymmetry (I, II and III, see Fig. 1; 12). DNA resolution occurs

* To whom correspondence should be addressed at: Centre Nacional de Biotecnologfa, CSIC, Campus de la Universidad Autonoma de Madrid, Cantoblanco,28049 Madrid, Spain

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3182 Nucleic Acids Research, 1995, Vol. 23, No. 16

Rejotvuei (y6 res site)

Eeiotvut-InvtrUiti (p" six site)

} • - / / - - : I : - •//• •

invtrUiei (Hin hixl site)

12 2 12

Figure 1. Structure of the binding sites for DNA recombinases of the Tnifamily. DNA resolvases, exemplified by y5 protein, bind to targets (named ressites) composed of three adjacent sites (I, II and III; reviewed in 8). Each siteconsists of an imperfect inverted repeat (open arrows). Resolvases do notrequire any accessory factors. Resolvase-invertases (the p recombinase) bindto a target (the six site) composed of two sites (I and ID and require an accessorychromatin-associated protein, such as Hbsu (shaded discontinuous segment onthe right), to mediate DNA recombination (5,6). The P protein recognizes aninverted repeat at site I (open arrows) and an apparendy non-symmetrical targetat the low affinity site II (open box). DNA invertases, exemplified by Hinprotein, bind to targets (named Hix in this case) containing an inverted repeat(open arrows) and to mediate inversion require the presence of an enhancersequence in cis (shaded box), to which Rs protein binds (reviewed in 10,11).Numbers indicate the length of the inverted sequences (open arrows), thespacers (heavy line), the non-symmetrical sites (open boxes) or between thecenter of each site.

between two directly oriented res sites, at the center of site I. Allthree sites are required for efficient recombination; it is believedthat the role of sites II and III is to facilitate the formation of asynaptic complex in which the two sites I (one from eachrecombination site) are in the proper relative orientation for thereaction to occur (14, reviewed in 8,9). DNA invertases bind toa single site with dyad axis symmetry, within which recombina-tion occurs (reviewed in 10,11; Fig. 1). The p recombinase bindsto a region (the six site) containing two adjacent binding sites,named I and II (6, see Fig. 1). The (3 protein, which is a dimer insolution (15), binds cooperatively to these two sites (6).Recombination takes place at site I (our unpublished data).

Therefore, the recombination sequences of the three membersof the Tni family (see Fig. 1) have in common the presence of onebinding site for the recombinase in which strand exchange occurs(the crossover site) and one or two additional sites that have anaccessory role. In the case of DNA resolvases these accessorysites are targets for further recombinase dimers. In the case ofDNA invertases there is a single accessory site, to which the Fisprotein binds. The P protein (a resolvase-invertase) accessorysites are targets for a recombinase dimer and for a chromatin-as-sociated protein.

In this report we have analyzed in detail the interaction of pprotein with its target site. Conversely to DNA resolvases, ourresults indicate that p protein makes very different contacts atsites I and II. The sequences recognized, as defined by severalfootprinting techniques, show a dyad axis symmetry at site I, butnot at site II (see Figs 1 and 2). The reported data, together withthe available information, suggest that P recombinase is a distantmember of the TnJ family of enzymes, being perhaps a linkbetween the different classes of site-specific recombinases.

MATERIALS AND METHODS

Bacteria] strains and plasmids

Plasmids pBT331 and pBT338 contain a 1040 bp Hindlll-BbrPl(coordinates 1506-2546) and a 447 bp Asel-BbrPl (coordinates2099-2546) DNA segment from plasmid pSM19O35 respectively(6; see Fig. 2). A 172 bp Taq\ DNA fragment (coordinates2186-2358) from pBT331 containing the P protein binding site(see Fig. 2) was cloned into Accl-cleaved pUC19Q (see 16),generating plasmid pREST. The orientation of the insert was suchthat site I faces the EcoRl site of the polylinker of the vector, whilethe promoter for orfa and gene P (see 17) faces the HindlU siteof the polylinker.

Proteins

Protein p was purified and its concentration determined aspreviously described (6,15). Purified Hbsu protein was a giftfrom Professor U. Heinemann (Max-Delbriick-Centrum furMolekulare Medizin, Berlin, Germany). Since both proteins aredimers in solution, their concentration is expressed as mol proteindimers.

DNAs

Band shift and footprinting assays were performed with DNAfragments containing the p protein binding site derived fromplasmid pREST (see above and Fig. 2). To label the top strand thefragment was excised with Kpn\ and HindlU and end-labeled atthe Hind\U site by filling the end with KJenow enzyme. The finallength of the fragment was 211 bp. To label the bottom strand thefragment was excised with BamHl and Pstl and the BamHl endfilled-in as described above. The final fragment length was 194 bp.

Band shift assays

The end-labeled 194 bp DNA fragment from plasmid pRESTcontaining the P protein binding site (see above and Fig. 2) wasused. The DNA (2-8 nM) was incubated at 30°C for 20 min withincreasing concentrations of P protein (26-840 nM) in 25 mMTris-HCI, pH 7.5, 10 mM MgCl2, 30 mM NaCl, 5% (v/v)glycerol, in a total volume of 20 u.1. Poly(dldC) (2 u.g) was addedas non-specific competitor DNA. Samples were transferred to iceand 3 u.1 of a solution containing 30% glycerol, 0.25% bromophe-nol blue, 0.25% xylene cyanol were added. Binding reactionswere analyzed on a 4% non-denaturing polyacrylamide gel (80:1acrylamide:bis).

DNase I footprinting of specific complexes

The DNA fragments used, containing the P protein binding site,were obtained from plasmid pREST either as a 194 bpBamW\-Pst\ fragment to label the bottom strand or as a 211 bpKpn\-Hin&\\\ fragment to label the top strand (see above). DNaseI footprinting of the individual complexes formed by the Precombinase at sites I and II was performed essentially asdescribed (18). Binding reactions were performed as for bandshift assays and the different complexes were resolved in a bandshift gel, excised from the gel and subjected to DNase Ifootprinting in the gel slice exactly as described (18). The DNAwas recovered from the gel, precipitated with ethanol andanalyzed in 6% ursa-polyacrylamide gels (19).


Nucleic Acids Research, 1995, Vol. 23, No. 16 3183

repS a(1506)

H(1656)

AcI

(2099) (2186)

A T

I

(2254)

Ac

Y////X

(2358) (2*11)

T F

LJon six

(2546)

B

Bsite I site II

10 bp 32 bp14 bp

57 bp

Figure 2. The B recombinase binding site of plasmid pSM 19035. (A) Physical map of the 1040 bp HinAWl-BbrPl DNA fragment of pSM 19035. The B recombinasebinding site (six, shaded) and the origin of replication (on, hatched) are indicated. The direction of replication is shown by a honzontal arrow at the on region. Thegenes included (repS, or/a and gene B) are denoted by honzontal arrows. Numbers in parentheses indicate coordinates (according to 2). (B) Nucleotide sequence ofthe B recombinase binding region. B Recombinase binding sites I and II, as defined by chemical footprinting (this work), are boxed and in bold type. The invertedrepeat sequences recognized by B protein at site I are denoted by convergent arrows. Numbers denote the length of the indicated segments. Abbreviations: A, Ase\;Ac, Accl; B, BbrPl; E, EcoRl; F, Fsp\; H, Windlll; T, Taq\.

Hydroxyl radical footprinting

The end-labeled DNA fragments used for hydroxyl radicalfootprinting (20) were the same as those indicated for DNase Ifootprinting. Reactions contained, in a total volume of 20 \L\,2-8 nM end-labeled DNA, 25 mM Tris-HCl, pH 7.5, 10 mMMgCl2, 2 u_g poly(dldC) as non-specific competitor DNA and,where indicated, |3 protein (135 or 270 nM) and/or B.subtilis Hbsu(216 nM). After a 20 min incubation at 30°C the footprint wasstarted by addition of 3 (il of a freshly prepared solutioncontaining 4 mM EDTA, 2 mM ammonium iron(II) sulfatehexahydrate, 16 mM sodium ascorbate, 1.5% H2O2. After 4 minthe reaction was stopped by addition of 2 u.1 100 mM thiourea and2 |il 0.5 M EDTA. Samples were diluted 1:1 with water and theDNA precipitated, resuspended in sequencing loading buffer andanalyzed in 6% urea—polyacrylamide gels.

Methylation interference assays

The end-labeled DNA fragments used for methylation interfer-ence experiments (21) were the same as those indicated for theDNase I footprinting. End-labeled DNA was partially methylatedwith 50 mM dimethyl sulfate in 50 mM sodium cacodylate, pH 8,25 mM MgCh. 0.1 mM EDTA. After 10 min at room temperaturethe reaction was stopped with 0.2 M p-mercaptoethanol. TheDNA was precipitated twice with ethanol. Methylated DNA wasincubated with 52-210 nM fj protein in 25 mM Tris-HCl, pH 7.5,10 mM MgCl2, 30 mM NaCl, 5% (v/v) glycerol, in the presenceof 2 (ig poly(dldC) as non-specific competitor DNA. The totalvolume was 20 u.1. Free and protein-bound DNA were resolvedin a band shift gel as indicated above, eluted from the gel and

precipitated with ethanol. DNA was resuspended in 100 (il 1 Mpiperidine and incubated for 30 min at 90°C to generate breaks atmethylated positions. Piperidine was evaporated under vacuumwith 1 vol ethanol and the DNA analyzed in 6% urea-polyacryl-amide gels.

Missing nucleoside assays

The end-labeled DNA fragments used, containing the p proteinbinding site, were the same as those indicated for DNase Ifootprinting. DNA was treated with hydroxyl radicals as indi-cated for hydroxyl radical footprinting to obtain DNA fragmentswhich contained, on average, no more than one gap per molecule(22). DNA was precipitated and resuspended in 20 (il 25 mMTris-HCl, pH 7.5, 10 mM MgCl2 and 2.5 u.g poly(dldC). The Pprotein (52-210 nM) was added and the mixture incubated for 20min at room temperature. Bound and unbound DNA wereseparated in a band shift gel, excised from the gel and purified byan overnight diffusion in 0.5 M ammonium acetate, 0.1% SDS,1 mM EDTA, precipitated with ethanol and analyzed in 6%urea-polyacrylamide gels.

RESULTS

The fj recombinase binds sequentially to two adjacent sites

A study of the binding of P protein to its target site (the six site)by band shift and DNase I footprinting assays, using a 312 bpAsel-Fspl DNA fragment from plasmid pSM 19035 (Fig. 2),indicated that it binds to two discrete segments, named I and II (6;see Fig. 2). Band shift assays suggested that low proteinconcentrations led to the binding of just one dimer of P protein to



Pu F C1C2

C2

C l - 1Cl -II

FREE

Figure 3. Binding of p recombinase to the six site: band shift assay. Theend-labeled 194 bp DNA fragment contaning the 172 bp Taq\ DNA segmentthat includes the p" protein binding site (see Frg. 2) was used. DNA wasincubated with increasing amounts of P protein (26, 52, 105, 210, 420 or 840nM). P Protein-.su: DNA complexes were resolved in a 4% polyacrylamide gel.The complexes indicated in the figure are Cl -I, Cl -IT and C2. Unbound DNAis denoted as free DNA.

DNA, while at higher protein concentrations two P protein dimersbound to DNA (6). From these experiments it was not possible todeduce whether the protein has any binding preference for eithersite and, if it does, which is the preferred site. The complexcontaining one (3 protein dimer was named Cl, while thatcontaining two dimers, occupying sites I and II simultaneously,was named C2 (6). A band shift assay performed with a smallerDNA fragment (194 bp, containing the Taq\ segment; see Fig. 2)shows that complex Cl is indeed composed of two complexes ofslightly different electrophoretic mobilities (Fig. 3). The com-plexes have been named C1 -I and C1 -II, the former being clearlymore abundant than the latter. It is likely that these two complexescorrespond to a single p protein dimer bound to either site I or tosite II.

To determine the nature of complexes C1 -I and C1 -II they wereexcised from the gel and subjected to in situ DNase I footprinting.The same analysis was performed with free DNA and with the C2complex. The footprints (Fig. 4) show that in the Cl-I complex(labeled C1 in Fig. 4) site I is occupied by (3 protein (presumablyby a single dimer), while site II remains free. Due to its lowabundance, analysis of complex Cl-II did not give a clear result(not shown), though it is likely that in this complex site FI isoccupied, whereas site I remains free. Both site I and site II areoccupied in the C2 complex. Since the Cl-I complex wassignificantly more abundant than Cl-II, we conclude that pprotein has a higher binding affinity for site I than for site II. Theseresults indicate that P protein binds sequentially to the adjacentsites I and II.

Hydroxyl radical footprinting of the f> recombinase-DNAcomplex

The P protein—DNA complex was analyzed by hydroxyl radicalfootprinting. As revealed in Figure 5, P protein protected asubstantial length of its binding site from the cleaving agent.Protected positions cluster in two regions, which correspond tothe two sites (I and II) defined by DNase I footprinting (6). Thedistribution of contacts (or protected positions) at site I is clearly

-si

Figure 4. Binding of the p recombinase to the six site: in situ DNase Ifootprinting. Complexes labeled Cl-I and C2 in Figure 3, as well as the bandcorresponding to free DNA, were excised from the gel and subjected to in situDNase I footprinting. The DNA fragments used contained the 172 bp TaqlDNA segment that includes the p protein binding site (Fig. 2) and were obtainedfrom plasmid pREST either as a 194 bp BamHl-Pstl fragment to label thebottom strand (A) or as a 211 bp Kpn\—HmA\\\ fragment to label the top strand(B) (see Materials and Methods). Abbreviations: F, free DNA; Cl, Cl-1complex; C2, C2 complex; Pu, chemical sequencing reaction for punnes usedas a size standard.

different from that observed at site II, which suggests that pprotein binds differently to each site (see below). Protection wasobserved at both the major and minor groves at sites I and II (Fig.5C). Some protection was strong and reproducible, while otherswere weak and could not be detected in all assays (Fig. 5C).

Hbsu protein does not modify the interaction of Pprotein with its target site

P Protein requires a chromatin-associated protein, such as Hbsu,to mediate DNA recombination both in vivo and in vitro (5,6). Toinvestigate whether Hbsu binds to the six site or if it has a role inthe interaction of P recombinase with its target site, P protein—DNA complexes formed in the presence or absence of Hbsu wereanalyzed by chemical footprinting. The results indicate that Hbsuby itself gives no detectable footprint on the linear DNA templateused, neither in the presence nor absence of p protein (Fig. 5).Hbsu is thought to bind in a specific manner to the p proteiiwixDNA complex to help in formation of the synaptic complex, sinceonly one to two Hbsu dimers per DNA molecule are required (5)



- H HP p p - Pu Py Pu Py - 3 3 3H H -

j

l

• i

B

Figure 5. Binding of P recombinase to DNA: hydroxyl radical footprinting. TheDNA fragments used contained the 172 bp Taql DNA segment that includes thep protein binding site (Fig. 2), obtained either as a 194 bp Bam\\\-Pst\ fragmentto label the bottom strand (A) or as a 211 bp Kpn\-Hindlll fragment to label thetop strand (B) (see Materials and Methods). The DNA was incubated with eitherP protein (136 or 273 nM, indicated as P), Hbsu protein (216 nM, indicated asH) or both (indicated as HP) prior to hydroxyl radical footprinting. The regionsprotected from attack by hydroxyl radicals by P protein are indicated by solidbars. Sites I and II are marked. A scheme of the footprint is shown in (C), wherethe DNA is represented in a planar projection. F, front of the helix; B, back ofthe helix. Each dot corresponds to an individual base; those protected bybinding of P protein are indicated by the identity of the base. Strong protectionis shown as black dots. Weak protection (open dots with the initial of the baseon one side) was not clear in all assays. The scheme was inferred from severalindependent footprints and from gels run to different extents to resolve large orsmall DNA fragments.

and it cannot be competed for by excess supercoiled DNA lackingthe six site (13). It is possible that Hbsu recognizes a structure thatis present only in supercoiled DNA or in a transiently formedsynaptic complex on supercoUed DNA, but not in linear six DNA.Nevertheless, it should be noted that the same lack of footprint has

been found when analyzing the binding of HU protein (two to fivedimers) to the recombination complex formed at the phage Mutranspososome (23) by the same technique. In this case the regionto which HU binds was localized by covalently linking a DNAcleaving system to the protein, which clearly demonstrated thespecific location of HU in the recombination complex (24).

It is interesting to note that the presence of Hbsu does notmodify the P protein—DNA complex, as deduced from theprotection pattern with hydroxyl radicals. It is likely, therefore,that the role played by Hbsu in ^-mediated DNA recombinationdoes not imply a direct interaction with P protein that modifies theP protein-DNA complex.

Nucleosides critical for p recombinase binding to DNA

Treatment of DNA with dimethylsulfate leads to methylation ofguanine residues at the N7 position (accessible through the majorgroove) and, with reduced efficiency, of adenine residues at theN3 position (protruding through the minor groove). Methylationinterference experiments (21,25) allow identification of thoseadenine or guanine residues that when methylated interfere withprotein binding. As revealed in Figure 6, the residues whosemethylation impaired binding of P protein to the six site areclustered in sites I and II. The residues located at site I are atdifferent positions and show a totally different distribution tothose that interfere at site II. These residues include both guaninesand adenines, suggesting that the protein contacts the DNAthrough both the major and minor grooves.

The missing nucleoside assay allows identification of thenucleosides that are important for binding of a protein to DNA(26). The hydroxyl radicals initially attack the DNA sugar-phos-phate backbone, but the reaction leads to loss of a completenucleoside, so that the final product is a DNA molecule with a gap(22). The assay identifies which gaps allow or impair proteinbinding. As revealed in Figure 7, the positions critical for Pprotein binding have different distributions at sites I and II. Thosecritical at site I include a 5'-TTG-3' sequence on the top strandand, 14 bp away, another 5'-TTG-3' stretch on the bottom strand.Positions critical for binding to site II are 5'-GTT-3' on the topstrand and a 5'-ATG-N3-A-N2-TTAAATG-3' sequence on thebottom strand.

The missing nucleosides may interfere with protein bindingeither by loss of specific amino acid-nucleoside contacts or byaltering a particular DNA conformation required for binding (26).The symmetry of the critical nucleosides at site I and the fact thatP protein is known to be a dimer in solution (15) suggest that Pprotein probably recognizes the sequence 5'-CAA-Ni4-TTG-3at site I, each P protomer interacting with the 5'-TTG-3 residuesof each strand. The sequence recognized at site II is notsymmetrical and has no evident similarity with the targetrecognized at site I. Therefore, p recombinase may recognizeeither different sequences at sites I and II or a degenerate targetsequence at site II.

DISCUSSION

The P recombinase binding region (the six site) is composed oftwo sites, I and II (6,15). A similar observation has been reportedfor the ResP resolvase of plasmid pAMpi (7). The use of a DNAfragment smaller in size than that used previously has allowedvisualization of a P protein-DNA complex in which only one siteis occupied, presumably by a single P dimer. In situ DNase I



BS TS

Pu F F B F F B Pu

BS TS

F F B F F j Pu Py

•HHr**a

i M «

G —

Figure 6. Nucleosides whose methylation interferes with B recombinase binding to DNA. (A) The DNA fragments used, labeled either on the bottom strand (BS) oron the top strand (TS), were those described in Figure 5. The DNA was partially methylated and incubated with B protein (210 nM). Protein-bound DNA (B), whichincludes DNA fragments methylated at positions that do not interfere with protein binding, was separated by non-denaturing polyacrylamide electrophoresis from freeDNA (F), which is enriched in DNA fragments methylated at positions critical for protein binding. Free and bound DNA were purified, treated with pipendine toproduce breaks at methylated positions and analyzed by denaturing polyacrylamide gel electrophoresis. Adenine or guanine residues whose methylation impairsbinding of B protein to DNA are indicated with dashes; the neighbor sequences are included for clarity. Sites I and II are also indicated. Chemical sequencing reactionsfor purines (Pu) or pyrimidines (Py) were used as size standards. (B) Samples as (A), but the electrophoresis was run for a longer time to resolve the larger DNAfragments.

footprinting of the complexes obtained at different proteinconcentrations has indicated that P protein binds preferentially tosite I. Given the poor affinity for site II and that simultaneousbinding of the two dimers is a cooperative process (6), it is likelythat stable binding to site II requires a protein-protein interactionwith the P dimer bound at site I.

The complex formed at site I (named Cl-I) showed a slowerelectrophoretic mobility than complex C1 -II, presumably formedat site n. When a protein bends the DNA the magnitude and theposition of the bend relative to the fragment ends greatly affectsthe mobility of the protein-DNA complex (27, reviewed in 28).The P protein seems to bend DNA upon binding (6). It is likely,therefore, that the reduced mobility of complex Cl-I relative toCl-II reflects the fact that the Cl-I complex is either more bentthan Cl-II and/or that the p protein' binding site is located at amore central position in the fragment.

The sequences protected from attack by hydroxyl radicals byP recombinase cluster in two regions which coincide with thepreviously defined sites I and II. The area protected at each siteindicates that the protein contacts, or at least covers, a significantportion of both sides of the DNA helix in a region spanning ~2.5helix turns at each site. At site I the major groove of the DNA isprotected over a complete helix turn and the footprint also extendsto the adjacent minor grooves. The protection pattern at site II

suggests that the protein also covers sequences located in both themajor and minor grooves of the DNA. The hydroxyl radicalfootprinting, the methylation interference and the missingnucleoside assays clearly show that P recombinase makes verydifferent contacts at sites I and II. A summary of these results isshown in Figure 8. The positions protected from hydroxyl radicalsand those critical for protein binding display a symmetrical patternat site I, but not at site II. The missing nucleoside assay shows thatthe sequences critical for protein binding at site I are 5'-CAA-N14-TTG-3'. The p protein probably interacts with the 5'-TTG-3'residues located on each strand. Since P recombinase is known tobe a dimer in solution (15), the symmetry of these recognitionsequences suggests that it binds to site I as a dimer. The positionscritical for binding at site II are 5'-CATTTAAGTT-N3-CAT-3'.Therefore, it is likely that the protein binds to sites 1 and II in adifferent manner. An alternative possibility is that site II containsa degenerate half-site that can be recognized with poor affinity byone of the protomers of the P protein dimer, since a 5'-TTG-3'sequence is present at the site II region important for proteinbinding. Although the number of residues critical for binding atsite II is unusually large, the affinity of P protein for this site is 5-to 8-fold lower than for site I. The protein may make specificcontacts with just a few of these nucleosides, but the rest of them



F B F B F B F B

1/

"S.

• * «

i. —G I

Figure 7. Nucleosides required for p recombinase binding to DNA. The DNA fragments used, labeled either on the bottom strand (C and D) or on the top strand (Aand B), were those described in Figure 5. The DNA was treated with hydroxyl radicals so as to generate, on average, one gap per DNA molecule. This DNA wasincubated with p protein (210 nM). Protein-bound DNA (B), which includes DNA fragments gapped at positions that do not interfere with protein binding, wasseparated in a band shift gel from free DNA (F). Free and bound DNA were purified and analyzed by denaturing polyacrylarrude gel electrophoresis. Residues whoseelimination impairs binding of p protein to DNA are indicated, as well as the location of sites I and II. (B) and (D) show the same samples as (A) and (C), but theelectrophoresis was run for a longer time to resolve the larger DNA fragments.

are likely to be necessary for maintenance of a particular DNAconformation important for protein binding.

A computer analysis of the six site indicates that sites I and IIcontain two imperfectly conserved sequences 13 bp in length withdyad axis symmetry (15; see Fig. 8). Binding of (3 protein to siteI, as deduced from the chemical footprinting assays, defines adifferent and smaller inverted repeat at site I. This repeat containstwo 10 bp long inversely oriented sequences separated by 14 bpand is displaced 4 bp relative to that proposed previously (Fig. 8).The chemical footprinting results at site II show that there is nobinding symmetry in this case. Therefore, the imperfect invertedrepeat present at site II is probably not related to protein binding.The intervening sequence between sites I and II is expanded by8 bp with respect to that proposed previously (Fig. 8).

The fact that both methylated guanines (methyl group protrud-ing through the major groove) and methylated adenines (methylgroup accessible through the minor groove) interfere with Pprotein binding to its target site suggests that the protein contactsDNA through both the major and minor grooves. In principle ourdata cannot distinguish whether methylated adenines inhibit Precombinase binding by preventing interactions between theprotein and base pairs in the minor groove or by impeding abending that requires a narrowing of the minor groove. Thisproblem has been solved for other proteins of the Tni family, towhich P protein belongs. The y5 resolvase is believed to interactwith both the major groove and the adjacent minor groove(29-31). The Hin recombinase, an invertase of the Tni family forwhich crystallographic data are available, also makes sequence-

specific contacts in both the major and minor grooves of DNA(32,33 and references therein). The y5 and Hin recombinases bindto DNA through a helix-turn-helix domain located at theC-terminus, a domain that seems to be present in all recombinasesof the Tni family, including the P protein. In particular, some ofthe amino acid residues of Hin recombinase known to makespecific contacts either in the major or in the minor groove (33)are highly conserved in all recombinases of the family and arealso present in the P protein. Since the distribution of nucleosideswhose methylation interferes with binding of y5 resolvase toDNA (34) is very similar to that observed for binding of P proteinto site I, it is likely that the P protein-site I complex is not verydifferent from the y&-res DNA complex. Therefore, it is likelythat P recombinase docks to its binding sequences at site I makingcontacts through both the major and minor grooves of DNA.Since its binding to site II seems to generate a differentprotein-DNA structure, the interference results could have twointerpretations. In principle the methylation interference patternsuggests that the P protein makes contacts in both the major andminor grooves of DNA also at site II. Nevertheless, the largenumber of residues whose methylation interferes with proteinbinding, together with the poor affinity of P protein for site II, mayindicate that a stable binding at site II requires a particular DNAconformation that cannot be properly formed when certainresidues are methylated (see above).

It is interesting to compare the sequences involved in therecombination reactions catalyzed by the different DNA re-combinases of the Tni family (see Fig. 1). These sequences can



Site I

CTTTTATAGQTCAATjKAiOTATACTOATTTGTCCTATTaATTAGATAGCA

QAAAATATCCJUTrTATCTCATATOAATAAACAaaATAACTAATCTATCaT. • • • • • • • • • • • • • • •ZAA

ACKNOWLEDGEMENTS

This work was supported by grants PB93-0116 from theDGICYT and SBF B5 to JCA. We are grateful to Udo Heinemannfor his generous gift of Hbsu protein, to Marie-Agns Petit forcommunicating her results prior to publication and to A. C. Stiegeand L. Yuste for excellent technical assistance. The comments ofone of the referees are greatly acknowledged.

vvv« »ooooo—o oooooo»»o« • • • •(TrATAATAGCTTTATAaAQTAGGTCATTTAAGTTGAGCATAATAGaAGQA

-I h + + +CATATTATCGAAATATCTCATCaWCTAAATTCAACTCOTATTA'TCCTCCT

Site II

Figure 8. Interaction of p" protein with sites I and n. The figure summarizes theresults obtained by chemical footprinting and interference assays. Positionsprotected by (3 recombinase from attack by hydroxyl radicals are indicated withdots (filled dots for strong protection, open dots for weak protection). Guanineor adenine residues whose methylatjon interferes with protein binding aremarked with stars. Nucleosides whose elimination impairs protein binding areindicated by triangles. The horizontal arrows denote sequences with imperfectdyad symmetry. Those located below the DNA sequence (broken line)correspond to the largest inverted repeats found by a computer search (15),while the inverted sequence depicted inside the sequence at site I (thincontinuous line) is that defined by binding of p" protein, as deduced fromfootprinting data. Binding sites I and II are shown one on top of one another toallow better comparison, though they are contiguous.

be divided into two regions. One of them would be the crossoverpoint, which corresponds to site I of 76 resolvase (the archetypefor DNA resolvases), site I of P recombinase (a resolvase-4nvertase)and the hix site of Hin recombinase (an example of DNAinvertases). As discussed above, the way of binding of 76resolvase, p recombinase and Hin invertase to their crossoversites is very similar. The rest of the DNA sequences required toassemble a recombination complex (sites II and ITJ for y5resolvase, site II for P recombinase plus the Hbsu target site andthe enhancer of Hin recombinase, to which Fis protein binds),differ in all three cases. These sites seem to have an architecturalrole in alignment of the crossover sites so that recombination canoccur. Each system would achieve the proper orientation of therecombination sites by a different, but equivalent, mechanism.The differences in the way of assembling a functional recombina-tion complex could explain why each system leads to a particularrecombination event: DNA resolution (y6 resolvase), inversion(Hin and related invertases) or both (p recombinase).

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3 recombinase of plasmid psm 19035 binds to two adjacent sites

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