Mapping the protein-DNA interface and the metal-binding site of the major human apurinic/apyrimidinic endonuclease

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<ul><li><p>Mapping the Protein-DNA IntMetal-binding Site of the Ma</p><p>n</p><p>P</p><p>Livermore, CA 94551, USA</p><p>nicci</p><p>vesro</p><p>Here, site-directed mutagenesis, chemical footprinting techniques, andmolecular dynamics simulations were employed to gain insights into</p><p>*Corresponding author</p><p>ins</p><p>DNA. The base excision repair (BER) pathway major mammalian AP endonuclease, is an essential</p><p>doi:10.1006/jmbi.2000.3653 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 298, 447459typically involves the removal of a single damagednucleotide or baseless site from the DNA(reviewed by Mol et al., 1999). Apurinic/apyrimi-dinic (AP) sites are formed from spontaneoushydrolysis of the N-glycosyl bond, from attack of</p><p>component of the BER pathway (Xanthoudakiset al., 1996).</p><p>Ape1, in the presence of Mg2, cleaves the phos-phodiester bond immediately 50 to an AP site, gen-erating a 30 OH group and a 50 deoxyribose moiety(reviewed by Demple et al., 1994). The single resi-due gap can then be filled by DNA polymerasebeta (polb) (reviewed by Wilson, 1998), andthe remaining 50-deoxyribose phosphate groupexcised by its phosphodiesterase activity</p><p>Present address: J. P. Erzberger, Department ofMolecular and Cellular Biology University of California,Berkeley CA 94720, USA.</p><p>Abbreviations used: BER, base excision repair; AP,Introduction</p><p>In order to maintain genetichave developed various meanapurinic/apyrimidinic; OH , hydrotetrahydrofuran; WT, wild-type; D,</p><p>E-mail address of the correspondiwilson61@llnl.gov</p><p>0022-2836/00/03044713 $35.00/0how Ape1 interacts with its metal cation and AP DNA. It was found thatApe1 binds predominantly to the minor groove of AP DNA, and thatresidues R156 and Y128 contribute to protein-DNA complex stability.Furthermore, the Ape1-AP DNA footprint does not change along its reac-tion pathway upon active-site coordination of Mg2 or in the presence ofDNA polymerase beta (polb), an interactive protein partner in AP siterepair. The DNA region immediately 50 to the abasic residue was deter-mined to be in close proximity to the Ape1 metal-binding site. Exper-imental evidence is provided that amino acid residues E96, D70, andD308 of Ape1 are involved in metal coordination. Molecular dynamicssimulations, starting from the active site of the Ape1 crystal structure,suggest that D70 and E96 bind directly to the metal, while D308 coordi-nates the cation through the first hydration shell. These studies define theApe1-AP DNA interface, determine the effect of polb on the Ape1-DNAinteraction, and reveal new insights into the Ape1 active site and overallprotein dynamics.</p><p># 2000 Academic Press</p><p>Keywords: AP endonuclease; Ape1; base excision repair; DNA binding;metal coordination</p><p>tegrity, organismsto repair damaged</p><p>bases by free radicals, or by the action of repairenzymes called DNA N-glycosylases whichremove damaged or unconventional bases(reviewed by McCullough et al., 1999). Ape1, theApurinic/Apyrimidinic Endo</p><p>Lam H. Nguyen, Daniel Barsky, JanDavid M. Wilson III*</p><p>Molecular and StructuralBiology Division, LawrenceLivermore National LaboratoryP.O. Box 808, L-441</p><p>Apurinic/apyrimidimammalian base exgenome. Ape1 cleasites through a hydxyl radical; F,distance.ng author:erface and thejor Humanuclease</p><p>. Erzberger and</p><p>(AP) endonuclease Ape1 is a key enzyme in thesion repair pathway that corrects AP sites in the</p><p>the phosphodiester bond immediately 50 to APlytic reaction involving a divalent metal co-factor.(Matsumoto &amp; Kim, 1995). The final nick is sealedby DNA ligase I or an XRCC1-DNA ligase III com-plex (Prasad et al., 1996; Caldecott et al., 1994).</p><p># 2000 Academic Press</p></li><li><p>Although Ape1 and polb do not form a stable pro- was detected for the complementary strand,</p><p>448 AP DNA Interactions and Divalent Metal Coordination of Ape1tein-protein complex (Dimitriadis et al., 1998), polbdoes bind Ape1-AP DNA binary complexes toform a higher-order ternary complex, and thephosphodiesterase activity of polb is accelerated bythe presence of Ape1 (Bennett et al., 1997). polbalso forms a complex with XRCC1 (Caldecott et al.,1996; Kubota et al., 1996) or mammalian DNAligase I (Prasad et al., 1996). In addition, polbb, theXRCC1 N-terminal domain, and a gapped DNAsubstrate can form a higher-ordered ternary com-plex in vitro (Marintchev et al., 1999). These datasuggest coordination of various enzymatic steps inthe BER pathway. Besides AP endonucleaseactivity, Ape1 acts as a 30-phosphodiesterase,removing lesions resulting from oxidative damageof DNA such as 30-phosphoglycolates (Suh et al.,1997), and has both 30 to 50 exonuclease andRNAseH activities (reviewed by Demple &amp;Harrison, 1994; Rothwell &amp; Hickson, 1997). It isnoteworthy that these activities of Ape1 are rela-tively poor in comparison to those exhibited by itsbacterial counterpart, exonuclease III (ExoIII).</p><p>AP endonucleases are classified into two familiesbased on amino acid sequence homology to Escher-ichia coli ExoIII or endonuclease IV (EndoIV)(reviewed by Demple &amp; Harrison, 1994). Ape1 ishomologous to ExoIII. The three-dimensional struc-tures of Ape1, ExoIII, EndoIV, and the EndoIV-APDNA complex have been determined by X-raycrystallography (Mol et al., 1995; Gorman et al.,1997; Hosfield et al., 1999). EndoIV inserts side-chains into the DNA base stack through the minorgroove, compresses the DNA backbone, bends theDNA 90 , and promotes double-nucleotide flip-ping to sequester the extrahelical AP site into theenzyme catalytic pocket (Hosfield et al., 1999). Themolecular details of the Ape1-AP DNA interactionsare not fully known, yet biochemical studies haveshed significant light on its repair reaction.</p><p>Based on kinetic and binding studies of Ape1and its mutants, the reaction pathway, with a mini-mal number of complexes, is as follows: Ape1binds specifically to AP DNA in the absence ofMg2 to form a stable intermediate complex(Wilson et al., 1997). This complex is thenconverted to a catalytically competent complex inthe presence of Mg2. Catalysis subsequentlyoccurs, resulting in cleavage of the phosphodiesterbond immediately 50 to the AP site and the for-mation of a protein-product complex. This complexthen dissociates, releasing Ape1 from nicked APDNA (Lucas et al., 1999). Product dissociationappears to be Mg2 concentration-dependent(Masuda et al., 1998b).</p><p>Ape1 requires at least four base-pairs 50 andthree base-pairs 30 of an AP site for incision activity(Wilson et al., 1995). For the AP strand, methyl-ation of guanine residues located one or threebase-pairs 50 of the AP site, or ethylation of phos-phate groups two or three positions 30 of the APsite prevented Ape1-AP DNA binary complex for-mation. While no phosphate ethylation interferencemethylation at two base-pairs 50, or one or threebase-pairs 30 of the AP site impaired Ape1 binding(Wilson et al., 1997). These data provided thefirst information of how Ape1 engages its targetsubstrate.</p><p>Here, we refine our understanding of the mol-ecular interactions of Ape1 and AP DNA, examinethe effect of polb and Mg2 on the DNA structureof Ape1-AP DNA binary complexes, map the Ape1metal-binding site in terms of proximity to theDNA substrate, and provide direct evidence of theamino acid residues involved in metal coordi-nation.</p><p>Results</p><p>Ape1 protects six to seven bases on eitherDNA strand around the AP site and thepresence of polbbb does not change the footprint</p><p>The purity of the Ape1 proteins used in thiswork is shown in Figure 1(a). To determine howApe1 interacts with AP DNA, we employed thechemical footprinting reagent hydroxyl radical(OH ). Due to their small size, OH are usefulprobes for studying DNA contacts at high resol-ution (Dixon et al., 1991). OH cleave the DNAdirectly by attacking the deoxyribose ring(Hertzberg &amp; Dervan, 1984; Balasubramanian et al.,1998). The double-stranded DNA used in thisstudy was a 26 bp duplex with tetrahydrofuran(F), an abasic site analog (Wilson et al., 1995), nearthe center (Figure 1(b)).</p><p>As shown in Figure 2, Ape1 protects seven baseson the strand containing F and six bases on thecomplementary strand. The F residue is located inthe center of the protected region on the abasicstrand. The guanine base opposite the F residue isstrongly protected, whereas the surrounding basesare less protected by Ape1 from OH -mediatedcleavage. There is a hypersensitive band within thefootprint that migrates at the same position on thegel as the Ape1 cleavage product. A simpleinterpretation is that this hypersensitivity is theresult of Ape1 incision due to residual enzymaticactivity even in the presence of EDTA. Consistentwith this interpretation, at the necessarily highlevels of Ape1 used in the footprinting assays,Ape1 incised an amount of labeled AP DNA(1 %) similar to that present in the hypersensitiveband. In addition, as shown below, such hypersen-sitivity is not observed within the footprint of thecatalytically inactive Ape1 mutant D210N. AD210N mutation reduces incision activity by25,000-fold, without affecting the specific DNA-binding activity of Ape1, indicating a critical rolefor this residue in the catalytic reaction (Erzberger&amp; Wilson, 1999).</p><p>Using the OH footprinting approach, we exam-ined whether the presence of polb, which has beenshown to form a ternary complex with Ape1 andAP DNA (Bennett et al., 1997), causes any change</p></li><li><p>AP DNA Interactions and Divalent Metal Coordination of Ape1 449in the footprint. As shown in Figure 2, we cannotdetect any difference in the footprint pattern ofApe1-AP DNA complexes in the presence of polb,indicating that polb does not bind elsewhere to theDNA, and that either polb binds directly to Ape1without changing the Ape1-DNA interactions orreplaces exactly some of the Ape1-DNA contacts.Ternary complexes were observed by gel retar-dation assays (Bennett et al., 1997; data not shown).</p><p>Ape1 mutants Y128A and R156Q have reducedAP DNA binding activity</p><p>The non-specific nuclease DNaseI displays struc-tural similarity to the ExoIII family of proteins(Mol et al., 1995). Comparison of the crystal struc-tures of Ape1 and the DNaseI-DNA complex led toa proposed model for the Ape1-AP DNA binarycomplex (Gorman et al., 1997). In this model, Y128and R156 residues of Ape1 are implicated in DNAcontacts. To test this prediction and to better definethe DNA-binding interface of Ape1, we con-</p><p>Figure 1. Protein and AP DNA substrate reagentsused in this work. (a) Purified Ape1 mutant proteins.Ape1 proteins (1.0 mg) used in this work were fractio-nated on an SDS/12 % polyacrylamide gel and stainedwith Coomassie blue dye. Lane 1, wild-type Ape1; lane2, D210N mutant; lane 3, D308A; lane 4, D70R, lane 5,D210N/D308A double mutant; lane 6, E96Q; lane 7,H309S; lane 8, N68A; lane 9, R156Q; lane 10, Y128A,and lane 11, D210N/D70A double mutant. The proteinmolecular mass standards (in kDa) are indicated on theright. (b) The duplex DNA substrate. 26F is 50-AATT-CACCGGTACCFTCTAGAATTCG-30, 26G is the comp-lementary strand where a G is positioned directlyopposite F. F is the tetrahydrofuran residue, a syntheticabasic site analog (Wilson et al., 1995). The arrow indi-cates the phosphodiester linkage incised by Ape1.structed Y128A and R156Q Ape1 mutants andasked if there was a loss in DNA-binding affinityby gel retardation assays. As shown in Figure 3,both the Y128A and R156Q mutations resulted in a&gt;100-fold reduced DNA-binding capacity (with acorresponding reduction in specific incision activityof fourfold and 70-fold, respectively), consistentwith the involvement of these residues in AP DNAcomplex stability.</p><p>Sites of AP DNA in proximity to themetal-binding site of Ape1</p><p>To gain additional information regarding thetopography of Ape1-AP DNA binary complex, wedetermined which bases of the AP DNA substrateare located near the metal-binding site of Ape1 byemploying an Fe2-cleavage assay (Mustaev et al.,</p><p>Figure 2. Ape1 binds in the minor groove and DNApolymerase b does not cause a change in the Ape1-specific footprint. (a) The OH footprint of Ape1-APDNA complex. Lanes 1 to 7 are samples with labeled26F strand; lanes 8 to 14 are with labeled 26G strand.Lanes 1 and 14 are the no cleavage agent controls.Lanes 2 and 13 are the no protein controls with 10 mMFe(AS)2. Lanes 3 and 12 are the no protein controls with5 mM Fe(AS)2. Lanes 4 to 11 are with 10 mM Fe(AS)2.Lanes 4 and 11 are reactions with 160 nM Ape1; lanes 5and 10, 160 nM Ape1 and 580 nM polb; lanes 6 and 9,160 nM Ape1 and 1.74 mM polb; lanes 7 and 8, 1.74 mMpolb. (b) Summary of the OH footprint data. The filledvertical bars above each base indicate protection fromcleavage by OH in solution. The height indicates therelative strength of footprint protection as determinedby Phosphorimager scans of three independent exper-iments.</p></li><li><p>were obtained with D210N as observed with wild-type (WT) Ape1 protein and 26G-labeled duplexAP DNA substrates (data not shown).</p><p>As shown in Figure 4, the strongest iron-mediated cleavage signals on the F-containingstrand or complementary strand are all 50 to the Fresidue. The addition of Mg2 reduced these iron-promoted cleavages, indicating that Mg2 and Fe2</p><p>are competing for the same metal-binding site inApe1 (Figure 4). The iron cleavage signals alsodecreased in the absence of the reducing agentdithiothreitol (DTT; data not shown), consistentwith the DNA being cleaved by a OH mechanism(Zaychikov et al., 1996). We conclude that themetal is located immediately upstream of the APresidue prior to catalysis, consistent with the metal</p><p>450 AP DNA Interactions and Divalent Metal Coordination of Ape11997). OH generated by the liganded iron underaerobic conditions degrade biopolymers (such asDNA or protein) with a diffusion-limited ratewithin an estimated range of 1 nm.</p><p>Since Fe2 was able to support the incisionactivity of Ape1 at the excess protein to DNAratios (at least 6:1) used in our binding and foot-printing assays (data not shown), we performedthe metal-cleavage studies with the catalyticallyinactive D210N mutant (Erzberger &amp; Wilson,1999). This both reduced the amount of incisedbackground product generated and allowed us todetermine the DNA bases in close proximity to themetal in the ternary complex (Ape1-AP DNA-Fe2)prior to incision. Similar iron cleavage patterns</p><p>ion being involved in cleavage of the phosphodie-ster bond immediately 50 to the AP site.</p><p>Figure 3. Ape1 mutants Y128A and R156Q havereduced repair activity. (a) DNA-binding activity of WTand mutant Ape1 proteins. Positions of protein-DNAcomplexes (C) and unbound AP DNA duplex substrates(S) are indicated on the left: 5 nM 26F duplex DNAprobe was incubated with increasing amounts of Ape1protein. Lane 1 is the no protein control. Lanes 2 to 5are reactions with WT Ape 1. Lanes 6 to 10 are withR156Q Ape1 mutant. Lanes 11 to 15 are with Y128AApe1 mutant. Lanes 2, 6, and 11, 5 nM respective Ape1protein; lanes 3, 7, and 12, 15 nM; lanes 4, 8, and 13,45 nM; lanes 5, 9, and 14, 135 nM; lanes 10 and 15,405 nM. (b) Incision activity assay of WT and mutantApe1 proteins. Lane 1 is the no protein control. Lanes 2to 7 are reactions wit...</p></li></ul>

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