Site-Specific Mutagenesis of Conserved Residues within Walker A and B Sequences of Escherichia coli UvrA Protein?

Transcription

1 3824 Biochemistry 1991, 30, Murphy, K. P., Privlov, P. L., & Gill, S. J. (1990) Science 247, Rshin, A. A. (1984) Biopolymers 23, Richrdson, J. S. (1981) Adv. Protein Chem. 34, Roder, H., Elove, G. A., & Englnder, S. W. (1988) Nture 335, Shfer, L., Klimkowski, V. J., Momny, F. A,, & Chumn, H. (1984) Biopolymers 23, Shoemker, K. R., Kim, P. S., Brems, D. N., Mrquee, S., York, E. J., Chikin, 1. M., & Bldwin, R. L. (1985) Proc. Ntl. Acd. Sci. US.A 82, Shrke, A., & Rupley, J. A. (1973) J. Mol. Biol. 79, Skolnick, J., & Kolinski, A. (1989) J. Mol. Biol. 212, Stnfield, R. L., Fieser, T. M., Lerner, R. A., & Wilson,. A. (1990) Science 248, Tketomi, H., Ued, Y., & G6, N. (1975) Znt. J. Pept. Protein Res. 7, Udgonkr, J. B., & Bldwin, R. L. (1988) Nture 335, Unger, R., Hrel, D., Wherlnd, S., & Sussmn, J. L. (1989) Proteins: Struct., Funct., Genet. 5, Vgsquez, M., & Scherg, H. A. (1985) Biopolymers 24, , Volwerk, J. J., & de Hs, G. H. (1982) in Lipid-Protein nterctions (Jost, P. C., & Griffith, 0. H., Eds.) Vol. 1, pp , Wiley, New York. Wetlufer, D. B. (1973) Proc. Ntl. Acd. Sci. U.S.A. 70, Wright, P. E., Dyson, H. J., & Lerner, R. A. (1988) Biochemistry 27, Site-Specific Mutgenesis of Conserved Residues within Wlker A nd B Sequences of Escherichi coli UvrA Protein? Gry M. Myles,t.l John E. Herst," nd Aziz Sncr*.t Deprtment of Biochemistry nd Biophysics, University of North Crolin School of Medicine. Chpel Hill, North Crolin , nd Deprtment of Chemistry nd Division of Chemicl Biodynmics, Lwrence Berkeley Lbortory, University of Cliforni. Berkeley, Berkeley, Cliforni Received November 8, 990; Revised Mnuscript Received Jnury S, 991 ABSTRACT: UvrA is the ATPse subunit of the DNA repir enzyme (A)BC excinuclese. The mino cid sequence of this protein hs reveled, in ddition to two zinc fingers, three pirs of nucleotide binding motifs ech consisting of Wlker A nd B sequence. We hve conducted site-specific mutgenesis, ATPse kinetic nlyses, nd nucleotide binding equilibrium mesurements to correlte these sequence motifs with ctivity. Replcement of the invrint Lys by Al in the puttive A sequences indicted tht K37 nd K646 but not K353 re involved in ATP hydrolysis. n contrst, substitution of the invrint Asp by Asn in the B sequences t positions D238, D5 13, or D857 hd little effect on the in vivo ctivity of the protein. Nucleotide binding studies reveled stoichiometry of 0.5 ADP/UvrA monomer while kinetic mesurements on wild-type nd mutnt proteins showed tht the ctive form of UvrA is dimer with 2 ctlytic sites which interct in positive coopertive mnner in the presence of ADP; mutgenesis of K37 but not of K646 ttenuted this coopertivity. Loss of ATPse ctivity ws bout 75% in the K37A, 86% in the K646A mutnt, nd 95% in the K37A-K646A double mutnt. These mino cid substitutions hd only mrginl effect on the specific binding of UvrA to dmged DNA but drsticlly reduced its bility to deliver UvrB to the dmge site. We find tht the deficient UvrB loding ctivity of these mutnt UvrA proteins results from their inbility to ssocite with UvrB in the form of (UvrA)2(UvrB)1 complexes. We conclude tht UvrA forms dimer with two ATPse domins involving K37 nd K646 nd tht the work performed by ATP hydrolysis is the delivery of UvrB to the dmge site on DNA. (A)BC excinuclese is the enzymtic ctivity resulting from the coordinted ction of UvrA, UvrB, nd UvrC proteins which excise wide vriety of modified nucleotides from DNA by hydrolyzing the eighth phosphodiester bond 5' nd the fifth phosphodiester bond 3' to the dmged bse (Sncr & Sncr, 1988; Grossmn & Yeung, 1990; Vn Houten, 1990). 'This work ws supported by Grnts GM32833 from the Ntionl nstitutes of Helth nd PCM from the Ntionl Science Foundtion nd in prt by grnt CTR1872 from the Council for Tobcco Reserch nc. nd by US. Deprtment of Energy Grnt DE-AC03-76SF * Correspondence should be ddressed to this uthor. *University of North Crolin School of Medicine. 1 Present ddress: Fred Hutchinson Cncer Reserch Center, 1124 Columbi St., Settle, WA University of Cliforni, Berkeley. A current model for the rection mechnism is s follows (Orren & Sncr, 1989, 1990). UvrA, which is n ATPse nd DNA binding protein with higher ffinity for dmged DNA thn for undmged DNA (Seeberg & Steinum, 1982), intercts with UvrB to form (UvrA),(UvrB), complex. UvrA delivers UvrB (which hs cryptic ATPse ctivity nd no ffinity for DNA) to the dmge site nd dissocites from the UvrB-dmged DNA complex. UvrC binds to this complex nd either directly or indirectly medites the dul single-strnd DNA incisions. ATP binding nd hydrolysis by UvrA nd UvrB (which becomes ctivted in the ternry UvrA-UvrB-DNA complex; Seeley & Grossmn, 1989,1990) re required during severl stges of the overll rection. This study is imed t chrcterizing the ATPse ctivity of UvrA nd its role in specific steps of the excision nuclese ctivity /91/ $02.50/ Americn Chemicl Society

2 Mutgenesis of UvrA Protein 1 UvrA D238N D513N D857N Asp513 1 Asp857 FGURE 1: Point muttions in UvrA's Wlker A nd B homologous regions. (M denotes metl binding Zn finger motif.) Sequence nlyses of the uvra gene nd protein reveled three nucleotide binding motifs (ech comprised of single Wlker A nd B sequence; Wlker et l., 1982) interspersed with two intct nd one prtil zinc finger (Husin et l., 1986; Doolittle et l., 1986; Figure 1). This hs led to the proposl tht the uura gene evolved from the fusion of primordil ATPse nd zinc finger gene followed by severl gene dupliction events. Furthermore, comprison of UvrA's sequence with the sequences of severl other ATPses reveled tht the internl homology extends fr beyond the Wlker A (GXGXXGKS) nd B (CYCYCYCYD, = hydrophobic) sequences to include bout 125 mino cids surrounding ech of the Wlker A nd B sequences. Thus, it ws proposed tht the ATPse functionl unit is comprised of two mino cid stretches, i.e., the A nd B segments which contin the Wlker A nd B sequences, respectively. t ws lso suggested tht these segments must fold independently nd could, therefore, be referred to s domins (Doolittle et l., 1986; Higgins et l., 1986). While the sequence comprison provided compelling evidence for the presence of multiple ATPse sites in UvrA, direct experimentl evidence hs been lcking. n this study, we hve conducted kinetic nd equilibrium mesurements to investigte the nucleotide binding sites in UvrA. We hve mutgenized invrint residues in ll six Wlker A nd B sequences in n effort to correlte the sequence of this protein with its structure nd function. Our dt suggest tht UvrA hs two ATP binding/ hydrolysis functionl domins which interct coopertively in the presence of ADP nd tht ech of these domins plys criticl role in loding UvrB onto dmged DNA by promoting the formtion of ( UV~A)~( Uvr B), complexes. MATERALS AND METHODS Mterils. UvrB nd UvrC proteins were purified essentilly s described by Thoms et l. (1985). Restriction enzymes, T4 kinse, nd bcteril lkline phosphtse were purchsed from Bethesd Reserch Lbortories. T4 DNA polymerse nd T4 ligse were from Bio-Rd, nd Sequense ws from United Sttes Biochemicls. The rdiolbeled nucleotides [2,8-3H]ATP(30 Ci/mmol), [2,8-3H]ADP (25.4 Ci/mmol), nd [T-~~PATP (7000 Ci/ mmol) were from CN Biomedicls, nc. [CY-~~P~ATP (800 Ci/mmol) ws from New Englnd Nucler. The chromtogrphy resins were obtined from the following sources: AcA-22 nd AcA-34 from BF Biotechniques, BioGel P-10 nd DEAE-BioGel from Bio-Rd, nd singlestrnd DNA-cellulose nd Blue Sephrose from Sigm. The Biochemistry, Vol. 30, No. 16, thin-lyer chromtogrphy pltes, Polygrm ce1300 poly- (ethylenimine) (non-uv), were purchsed from Brinkmnn nstruments. The plsmid nd phge vectors pb24, pkk233-2, nd M13K07 were obtined from B, Phrmci, nd Dr. C. Hutchison (University of North Crolin), respectively. The Escherichi coli K-12 derivtives used in this work were CJ236 (Kunkel et l., 1987), NM522 (Phrmci), nd UNC522 (Nvrtnm et l., 1989). Buffers. The following buffers were used: TEN 7.4, 10 mm Tris-HC1, ph 7.4, 1 mm EDTA, nd 10 mm NC; lysis buffer, 50 mm Tris-HC1, ph 8.0, 100 mm NC, 1 mm EDTA, 10 mm 2-mercptoethnol, nd 10% sucrose; core buffer, 50 mm Tris-HC1, ph 7.5, 1 mm EDTA, nd 20% glycerol, storge buffer, 50 mm Tris-HC, ph 7.5, 100 mm KC, 1 mm EDTA, 2 mm dithiothreitol, nd 50% glycerol; ATPse buffer, 50 mm Tris, ph 7.5, 100 mm KC, 10 mm MgC,, 5 mm dithiothreitol, nd 100 pg/ml bovine serum lbumin; (A)BC excinuclese buffer, 50 mm Tris-HC, ph 7.5, 50 mm KC, 10 mm MgC,, 5 mm dithiothreitol, nd 50 pg/ml bovine serum lbumin. Construction of Point Mutnts. Templte for site-specific mutgenesis ws prepred from CJ236/pUNC 1940; this plsmid crries the uura gene on n Nco-Pst cssette inserted into the polylinker region of pb24n ( pb24 derivtive with n Nco site in the Sm site of the polylinker). The site-specific muttions were introduced by the method of Zoller nd Smith (1983) s modified by Kunkel et l. (1987). The mutnts were identified by dideoxy sequencing (Snger et l., 1977) using Sequense (Tbor & Richrdson, 1987) in plce of Klenow frgment nd were expressed by subcloning the mutnt genes into the Nco-Pst site of pkk n Vivo Complementtion. The bility of mutnt UvrA proteins to complement uura- muttion ws tested by introducing the plsmids crrying the mutnt genes into UNC522 (uura::tnlo) nd mesuring the UV survivl of the trnsformnts fter exposure to 15 J m-2 of 254-nm light from germicidl lmp. Subtle differences in UV sensitivity between the wild type nd mutnts were detected by UV survivl conducted over dose rnge of erg/",. Purifiction of UvrA Point Mutnts. The wild-type protein, which we will refer to s UvrA-000, nd the two mutnts UvrA-100 (K37A) nd UvrA-010 (K353A) were purified from UNC522/pUNC 1940, UNC522/pUNC , nd UNC522/pUNC , respectively, with minor modifictions of the method of Thoms et l. (1 985). Preliminry chrcteriztion of UvrA-001 (K646A) nd UvrA-1 01 (K37A-K646A) overproducers reveled tht these mutnts hve strong propensity towrd inclusion body formtion nd tht soluble protein ws undetectble when cultures were grown t 37 "C. Consequently, these mutnts were purified by different procedure. Cells crrying the mutnt plsmids were grown t 26 "C to minimize inclusion body formtion. Cell-free extrct ws prepred by freeze-thw nd soniction followed by two successive centrifugtions t 20000g nd 1 OOOOOg. During these centrifugtions, -95% of the mutnt proteins sedimented with the cellulr debris. The clrified superntnt ws pplied to 5-g single-strnd DNA-cellulose column equilibrted with 0.1 M KCl/core. The column ws wshed extensively with column buffer, nd the mutnt UvrA proteins were eluted with 2 M KCl/core. The pek frctions were combined nd dilyzed overnight ginst 0.1 M KCl/core to selectively precipitte mutnt UvrA. The precipitte ws collected by centrifugtion, resuspended in 0.5 M KCl/core, nd dilyzed into storge buffer. Purity of the mutnt proteins

3 3826 Biochemistry, Vol. 30, No. 16, 1991 prepred in this mnner ws usully greter thn 85%. Becuse of the decresed solubility of the K646A mutnt, dditionl muttions t K646 were constructed. The rtionle for mking the two mutnts UvrA-002 (K646R) nd UvrA- 003 (K646M) ws tht Arg my be isofunctionl nd Met isosteric with Lys nd, therefore, it ws thought tht one or both of these substitutions would not perturb the folding nd/or tertiry structure of UvrA nd consequently not promote inclusion body formtion. These proteins, however, were indistinguishble from UvrA-001 both in terms of their in vivo ctivities nd lso in terms of their low solubility (dt not shown) nd, therefore, were not studied further. Protein concentrtion ws determined by the method of Brdford (1 976) using the Bio-Rd kit, nd confirmed by densitometric scnning of Coomssie blue stined SDSpolycrylmide gel contining ech mutnt protein nd highly purified UvrA stndrd. ATPse Assy. The ATPse ctivity of UvrA ws ssyed by the method of Scott et l. (1977) with minor modifictions. Rection mixtures (50 pl) contined 2 pci of [3H]ATP (1.3 pm), nonrdioctive ATP from 10 to 500 pm, nd the indicted concentrtions of UvrA. Rections were incubted t room temperture such tht no greter thn 15% of the substrte ws hydrolyzed. ATP hydrolysis ws quntified by spotting 2 pl of the rection mixture on TLC plte spotted with 20 nmol ech of nonrdioctive ATP nd ADP. The pltes were developed nd nlyzed s described previously (Scott et l., 1977). Nucleotide Binding. Binding of nucleotides to UvrA ws mesured by two methods: the equilibrium gel filtrtion technique of Hummel nd Dreyer (1962) nd the rte of dilysis technique of Colowick nd Womck (1 969). Equilibrium gel filtrtion ws conducted s follows. A 30-mL column of P-10 ws equilibrted t room temperture with buffer contining 50 mm Tris-HC1, ph 7.5, 300 mm KC1, 10 mm MgCl,, 1 mm dithiothreitol, 6.5% glycerol, 20 pci of [3H]ADP (25.4 Ci/mmol), nd non-rdioctive ADP t the indicted concentrtions. UvrA (-3 nmol) ws incubted t 23 OC for O min in 500 pl of the sme buffer nd then loded onto the column. The column ws developed with the sme buffer t rte of 10 ml/h. Frctions of 420 pl were collected, 400 pl of which ws mixed with 5 ml of Scintiverse (Fisher), nd the rdioctivity ws determined by scintilltion counting. n ddition, the protein concentrtion in ech frction ws determined by the Brdford method. The picomoles of nucleotide nd protein ws mesured over the whole pek, nd the mole rtio ws used to determine the binding stoichiometry. The Hummel-Dreyer method ws lso used to mesure binding to ATP. However, becuse of the reltively low ffinity of UvrA to ATP s compred to ADP, it ws not possible to use this technique to determine the ATP binding stoichiometry. nsted, binding with UvrA-000 nd UvrA-100 ws conducted t 0.1 pm ATP with 20 pci of [3H]ATP (30 Ci/mmol) s trcer. n ddition, to prevent complictions tht would rise from ATP hydrolysis, we conducted these binding experiments t 4 OC. The rte of dilysis technique (Colowick & Womck, 1969) utilizes specilly designed chmber consisting of two cylindricl reservoirs seprted by semipermeble membrne. A 550-pL rection mixture contining 50 mm Tris-HC1, ph 7.5, 300 mm KC, 10 mm MgC12, 1 mm dithiothreitol, 100 pg/ml bovine serum lbumin plus 3.8 pm UvrA, nd 0.2 pm [3H]ADP ws incubted for 10 min t 23 OC nd then ws loded into the top reservoir of the dilysis chmber. Buffer contining 50 mm Tris-HC, ph 7.5, 300 mm KC, 10 mm Myles et l. Tble : n Vivo Complementtion by UvrA Wlker A nd B Mutnts t 150 erg/mm2 protein reltive survivl protein reltive survivl x 10-1 D238N 4.4 x o x 10-3 D5 13N 6.3 X X lo- D857N 2.6 X O X O- uura:tn X O X Seril dilutions of sttionry culture of UNC522 (uura:tnlo) expressing ech of the mutnt uura genes were plted nd irrdited with 15 J m-2 of 254-nm light. After h t 37 OC, the number of colonies ws determined nd multiplied by the dilution fctor to yield the number of survivors. MgCl,, 1 mm dithiothreitol, nd 10% glycerol ws pssed through the bottom chmber by grvity, nd 2-mL frctions were collected. At 15-frction intervls, the nonrdioctive ADP concentrtion ws incresed to compete off bound [3H]ADP. Rdioctivity in ech frction ws determined by scintilltion counting. The ADP bound for given totl ADP concentrtion ws clculted by tking the rtio of rdioctivity in substurting frctions nd dividing by the rdioctivity in frctions obtined with sturting cold ADP. (A)BC Excinuclese Assys. Binding of UvrA nd UvrB ws probed by DNse footprinting using 137 bp psorlen monodducted substrte (Vn Houten et l., 1987) nd by loding of UvrB onto UV-irrdited pbr322 mesured by gel exclusion chromtogrphy on 12-mL AcA22 column (Orren & Sncr, 1989). Nicking of 5 terminlly 32P-lbeled UVirrdited DNA by (A)BC excinuclese ws ssyed by seprting the rection products on 8% polycrylmide sequencing gels (Sncr & Rupp, 1983). Substrte for this nicking ssy ws 107 bp DNA frgment gel purified from He digest of 1.2 pg of pb24n end-lbeled with [y-32p]atp nd T4 polynucleotide kinse. Gel filtrtion chromtogrphy on AcA-34 resin to detect UvrA-UvrB interctions ws crried out s described previously (Orren & Sncr, 1989). RESULTS Point Muttions in Conserved Residues of Wlker A nd B Sequences. Figure 1 shows the segmentl orgniztion of UvrA s proposed by Doolittle et l. (1986). According to this scheme, UvrA is mde up of three ATPse A segments nd three B segments interspersed with zinc fingers. An A segment nd B segment which contin the Wlker A nd B sequences, respectively, re proposed to fold into functionl ATPse domin. To test whether UvrA hs three ATP binding/hydrolysis domins, s predicted from the sequence, the most conserved residues in Wlker s A nd B sequences, lysine nd sprtic cid, respectively, were replced with nonconservtive residues by site-specific mutgenesis. As shown in Figure 1, Lys ws substituted with Al, nd Asp ws replced by Asn. n ddition to these substitutions, K646M nd K646R constructs were mde nd prtilly chrcterized. n Vivo Repir Activity of UvrA Point Mutnts. The effect of the muttions on UvrA function ws investigted by inserting plsmid crrying the mutnt gene into UNC522 (uora::tnlo) nd testing for complementtion of the UvrA deficiency by mesuring the UV sensitivity of the resulting mutnt. The results re summrized in Tble. Surprisingly, substitution of ny of the Asp residues with Asn hs only subtle effect on the complementing ctivity of the mutnt proteins. n contrst, chnging the invrint lysines to Al hd striking effect in two out of three mutnts; K37A nd K646A re noticebly defective in complementing ctivity while K353A hs wild-type phenotype. The double-mutnt UvrA-1 01 (K37A-K646A) hs only minor, but reproducible,

4 1.25 Mutgenesis of UvrA Protein Biochemistry, Vol. 30, No. 16, UvrA UvrA A UvrA UvrA-001 ~ 4 UvrA-101 OUY ADP A 50uY ADP + 75uY ADP 4 1OOuM ADP / l/[atp] (mh4-l) FGURE 2: Linewever-Burk nlysis of UvrA's Wlker A point mutnt ATPse ctivity. Fifty-microliter rections in ATPse buffer (see Mterils nd Methods) contin 67 pmol (2 pci) of [3H]ATP, 0.7 pmol of UvrA, nd from O to 500 pm cold ATP. Rections were strted by the ddition of UvrA nd were incubted t room temperture for the times required to chieve 10-15% mximl ATP hydrolysis (Le., 30 min-3 h). Rections were quenched by spotting on TLC pltes. Tble : ATPse Activity of Wlker A Point Mutnts: Summry of Kinetic Constnts V,, UvrA. protein Vm" K, (rm) VmxKm O = picomoles of ATP hydrolyzed per minute per picomole of complementing ctivity. These dt demonstrte the importnce of the A1 nd A3 segments nd my be tken s evidence tht UvrA hs two ATP ctive sites, one involving K37 nd one involving K646. Also, the lysine residues in two of the Wlker A sequences re of vitl importnce for the in vivo functions of UvrA while the Asp residues in the Wlker B sequences re pprently lrgely replceble. Kinetic Studies with Wild-Type nd Mutnt UurAs. Since replcement of Asp residues in Wlker B sequences did not drmticlly ffect the phenotype, we concentrted our in vitro work on mutnts of the invrint lysines in the Wlker A sequences. Wild-type UvrA s well s UvrA-100, UvrA-001, UvrA-010, nd UvrA-101 ws purified nd tested for ctivity. For quntittive comprison of the ctivities of wild-type nd mutnt proteins, it is importnt tht protein preprtions of ner-identicl purity be tested. Wild-type UvrA, UvrA- 100, nd UvrA-0 O displyed the sme solubility nd chromtogrphic properties nd were purified by the sme procedure. However, preliminry chrcteriztion of UvrA-001 nd UvrA- 101 overproducers reveled tht these mutnts hve strong tendency to form inclusion bodies nd tht only smll frction of the overproduced protein ws soluble when cultures were grown t 37 "C (dt not shown); therefore, cells were grown t 26 OC to minimize inclusion body formtion. The proteins were isolted by different procedure from UvrA-000, UvrA-100, nd UvrA-010 s described under Mterils nd Methods. The mutnt proteins purified in this mnner were comprble to wild type in purity, llowing for quntittive comprison of their ctivities. Figure 2 shows the Linewever-Burk plots for the ATPse ctivity of UvrA nd its mutnt derivtives, nd Tble 1 summrizes the kinetic constnts obtined from these plots /[ATP] (mu-') FGURE 3: ADP inhibition of UvrA's ATPse ctivity. Ech 50-pL rection ws in ATPse buffer including 67 pmol(2 pci) of ["]ATP, 2.8 pmol of UvrA, nd pm ATP. ADP inhibition ws mesured by including 20, 50, 75, or 100 pm ADP in the rection mixtures s indicted. Rections were performed t room tem rture for the times required to hydrolyze t most 10-15% of the [ p" HATP ( min) nd were quenched by spotting 2 pl on TLC pltes. The dt re nlyzed on Linewever-Burk plot. Tble 111: Hill Coefficients for Wlker A Point Mutnts protein "(M cffstor) ~(ADP? f i f i f 0.04 n(adp) is the men Hill coefficient obtined by verging the n vlue derived for ech concentrtion of ADP tested. The error expressed here is one stndrd devition. The slight nonlinerity suggests negtive coopertivity; however, nlysis of these dt on Hill plot does not support this (see below). nspection of the kinetic constnts shows tht while UvrA-010 is comprble in ctivity to wild type, both UvrA-100 nd UrvA-001 show decrese in their V,,, nd n increse in their K, vlues. As result, the specificity constnt (V,/K,) for these mutnts shows decrese of 75% nd 86% for UvrA-100 nd UvrA-001, respectively. Likewise, UvrA-101 shows drstic increse in K, nd decrese in V,, such tht the specificity constnt is only 5% of the vlue of the wild-type protein. These results suggest tht both Lys37 nd Lys646, but not Lys353, re involved in the ATPse functions of UvrA. To better define the individul roles of Lys37 nd Lys646 in the ATPse ctivity, we conducted inhibition studies with ADP. Seeberg nd Steinum (1982) hve previously reported tht ADP is competitive inhibitor of UvrA's ATPse ctivity. Our results, shown in Figure 3 re consistent with this report but differ in tht our dt indicte tht ADP promotes positive coopertivity. Becuse of the nonlinerity of the Linewever-Burk plots, the vlues of Ki cnnot be extrpolted directly from the slopes in Figure 3. nsted, Ki ws determined from the x xis of grph of Km,pp vs [ADP] (Hill replot; Figure 4). Vlues for Kmpp for this grph were obtined from plots of log (u/vmx- u) vs log [ATP] for ech [ADP] nd were tken to be the ATP concentrtions where u/( V,, - u) = 1. As is pprent from Figure 4, the Hill replot is biphsic with the two symptotes intercepting the x xis t Ki = 5 nd 50 PM. Vlues for Hill coefficients of wild-type nd mutnt UvrA proteins re presented in Tble 111. For UvrA-000, the Hill coefficient t [ADP] = 0 pm is n = 0.94 wheres the verge

5 3828 Biochemistry, Vol. 30, No. 16, 1991 Myles et l bop] (4 FGURE 4: Hill replot of UvrA's ATPse ctivity. Dt from Figure 3 re nlyzed on Hill plots. The K,,, vlues were extrpolted s the [ATP] corresponding to u/( V,,, - 3 = 1.O for ech [ADP]. The Km,pp vlues for UvrA-000 were plotted vs [ADP] to yield the Hill replot shown here. Asymptotes re constructed for the liner portions of the curve (Le., t [ADP] = 0 pm nd t [ADP] = 100 pm), nd the Ki vlues re extrpolted from the intersection of the x xis by ech symptote. vlue for the Hill coefficient is n = 1.51 in the presence of ADP, consistent with the observed nonlinerity in the Linewever-Burk plots. These dt show tht while UvrA-000, UvrA-010, nd UvrA-001 exhibit positive coopertivity with n - 1.5, this coopertivity is viturlly eliminted by mutgenizing K37; Le., n = 1.06 for UvrA-100 nd n = 1.10 for UvrA-101. These dt indicte tht UvrA hs two nucleotide binding sites, one involving K37 nd one tht includes K646, nd furthermore tht the two sites re not equivlent s muttion of K37 elimintes coopertivity while muttion of K646 does not. Nucleotide Binding. By conducting nucleotide binding studies, we wished to provide supporting evidence for the conclusions of the kinetic experiments, Le., tht UvrA possesses two ATP binding sites. To void the complictions tht would rise from hydrolysis of ATP, we used ADP for these experiments. ADP nd ATPyS inhibit the ATPse ctivity of UvrA competitively nd consequently shre the sme binding sites with ATP. t is resonble, therefore, to expect tht binding mesurements with these nucleotides will ccurtely reflect the stoichiometry nd reltive binding ffinities of the ctive sites. Becuse ADP hs -5-fold higher binding ffinity for UvrA thn ATPyS (Seeberg & Steinum, 1982), most of our binding mesurements were performed with ADP. Binding experiments were done exclusively with UvrA-000 nd UvrA- 100; UvrA-01 0 ws not studied becuse in ll spects it behves like the wild-type protein. Binding to UvrA- 001 nd UvrA-101 ws not mesured becuse of the unvilbility of quntities of these proteins sufficient for binding experiments. Figure 5A shows typicl elution profile for UvrA-000 in Hummel-Dreyer-type experiment, nd Figure 5B represents hyperbolic sturtion curve for UvrA-000 nd UvrA-100, which ws generted from series of Hummel- Dreyer profiles such s shown in Figure 5A. The Sctchrd plot of the sturtion curve (Figure 5C) yields stright line with slope nd x-xis intercept which yield KD of 0.25 pm nd binding stoichiometry [r(st)] of 0.57 ADP/UvrA monomer, respectively. Further, both UvrA-000 nd UvrA- 100 hve indistinguishble ADP binding ffinities nd stoichiomet r i es. Becuse muttion of Lys37 hs no effect on UvrA's ADP binding ffinity or stoichiometry, it is uncler whether ADP is binding to the crboxy-terminl domin (Le., A3) or, lterntively, whether ADP binds to the mino-terminl domin (Le., Al) nd Lys37 hs no role in this ctivity. To differentite these possibilities, we mesured binding to ATP t 0.1 pm (t 4 "C to minimize ATP hydrolysis). Figure 5D shows tht UvrA- 100 is clerly defective in binding ATP s compred to UvrA-000. Becuse the ADP binding stoichiometry is l/dimer (see below) nd becuse ADP inhibits UvrA's AT- Pse ctivity competitively, we conclude tht under conditions of binding only, ADP nd/or ATP bind exclusively to the mino-terminl domin. Further, the role of Lys37 is to provide the y-phosphte contct with ATP. Becuse kinetic dt indicted two binding sites per UvrA functionl unit, we resoned tht low-ffinity binding site could hve been missed becuse of limittions in the sensitivity of the Hummel-Dreyer nlysis. Therefore, we lso mesured binding with the rte of dilysis technique of Colowick nd Womck (1969) which cn detect binding to low-ffinity sites. A representtive run for UvrA-000 is shown in Figure 6A nd Sctchrd plot of the dt in Figure 6B. Agin, the binding is hyperbolic with no evidence for multiple nucleotide binding sites; tht is, incresing mounts of low concentrtions of competing ADP incrementlly pproch sturtion without reching plteu t n intermedite off rte. The Sctchrd plot is liner nd yields KD = 0.7 pm nd = 0.45, in resonble greement with the vlues obtined by the Hummel-Dreyer method. t is importnt to emphsize tht high concentrtions of ADP (i.e., [ADP] > 20 pm) do not lter the vlue for the binding stoichiometry; tht is, binding never exceeds 0.5 ADP/UvrA monomer even t [ADP] s high s 50 pm. Thus, considering ll vilble dt, we conclude tht in the bsence of ATP hydrolysis ADP sturtes UvrA t stoichiometry of O.S/monomer. Since the ntive protein is dimer (Orren & Sncr, 1988, 1989; Oh et l., 1989) with n ssocition constnt of KA - lo8 M-' (Orren & Sncr, 1988) under the conditions of our binding studies (6 pm UvrA), essentilly ll of the protein is in the dimer form, nd, therefore, it is more ccurte to consider the binding stoichiometry s 1 mol of ADP/mol of UvrA dimer. ndeed, when the specific ctivity of UvrA is mesured s function of protein concentrtion (Figure 7), there is striking dependence of ATPse ctivity on concentrtion over the rnge nm with n inflection point t round 10 nm. These dt re consistent with the reported monomer-dimer equilibrium ssocition constnt (Orren & Sncr, 1988) nd, furthermore, suggest tht the ATPse ctivity is either strictly dependent upon or gretly stimulted by its dimeriztion. Significntly, theoreticl ATPse ctivity versus [UvrA] curve bsed on dimeriztion constnt of lo8 M-' nd ssuming tht the UvrA monomer lcks ATPse ctivity is in excellent greement with the experimentl dt. We, therefore, fvor the view tht dimeriztion is essentil for the protein's ATPse ctivity. DNA ncision nd Binding. When the mutnt UvrA proteins were used to reconstitute (A)BC excinuclese, we found tht the nuclese reconstituted with UvrA-010 hd ctivity identicl with tht reconstituted with the wild-type protein. n contrst, UvrA- 100 hd gretly diminished nd UvrA-001 nd UvrA- 101 hd brely detectble in vitro complementtion ctivities (Figure 8). According to the model of Orren nd Sncr (1989, 1990), UvrA mkes (UvrA)i(UvrB), complex, nd this complex delivers UvrB to the dmge site guided by UvrA's ffinity for dmged DNA. Therefore, diminished overll nuclese ctivity could be due to either decresed ffinity of UvrA for dmged DNA or the inbility of UvrA to deliver UvrB to the dmge site. To distinguish between

6 Mutgenesis of UvrA Protein A Biochemistry, Vol. 30, No. 16, = u A j v > L_ \ - Y Y 0.2 Column Froclion UvrA UvrA-100 ' D ,....,....,.,, UvrA UvrA-100 h = w * pmol ADP/pmol UvrA Column Froclion FGURE 5: Nucleotide binding of UvrA by equilibrium gel filtrtion. (A) A representtive column profile. The column ws equilibrted with buffer contining 1.75 pm ADP (see Mterils nd Methods for column specifictions). 3.2 nmol (330 pg) of UvrA ws equilibrted with the column buffer t room temperture for 15 min in 500-pL volume prior to being loded onto the column. The totl micromolr ADP bound in ech frction ws determined by subtrcting the bckground cpm (which corresponds to 1.75 /LM ADP) from the cpm in ech frction comprising the protein pek nd summing ech net cpm vlue. The protein concentrtion in ech frction comprising the pek ws determined by the Brdford ssy using commercil preprtion of BSA s stndrd. Ech column run such s this constitutes one point on the sturtion curve B. (B) ADP binding sturtion curve. Dt shown in (A) re plotted s the [ADP]/[UvrA] for ech ADP concentrtion. Points were determined from 0.07 to 50 pm; the points to 5 /LM[ADP] re shown here. UvrA-000 points were obtined with 3.2 nmol of protein while UvrA-100 points re from mesurements with 3.0 nmol of protein. (C) Sctchrd plot of equilibrium gel filtrtion binding dt. The dt from (B) plotted s the binding stoichiometry (r) divided by the free nucleotide concentrtion vs r. The free nucleotide concentrtion in frction is tken s the strting ADP concentrtion for given column run. The dt shown here include ll the points in (B) nd include those obtined t higher ADP concentrtions (up to 35.8 pm). Higher concentrtions of ADP do not result in nucleotide pek which cn be quntitted ccurtely ginst the bckground. (D) Equilibrium gel filtrtion profile for ATP binding. Binding nd column conditions were essentilly s described in (A) except tht here binding ws mesured to 0.1 pm ATP. these two possibilities, we conducted DNse footprinting experiments (Figure 9). n contrst to n erlier report (Vn Houten et l., 1988), ATP does not pper to increse the ffinity of UvrA for the DNA substrte; rther, it results in the "tightening" of the footprint by reducing nonspecific binding (Figure 9A). UvrA-100 nd UvrA-001 differ from wild-type UvrA in tht both re deficient in this ATP-dependent "tightening" of the footprints (more obvious in UvrA-001 in the figure but reproducibly seen with both mutnts in severl repets). The specific binding ffinity of UvrA-100 is indistinguishble from wild type, nd tht of UvrA-001 is lower by bout fctor of 2. (n compring the footprints, llownce should be mde for the unequl digestion by DNse 1 nd/or different level of exposure of the utordiogrms.) Dmge specificity for these mutnts ws confirmed by gel retrdtion experiments using rndomly dmged 107 bp DNA frgment (dt not shown). Thus, while there re some differences in the ffinity of wild-type nd UvrA-001 proteins for substrte DNA, the differences re not sufficient to ccount for the drstic decrese in incision ctivity of the excinuclese, nd, therefore, the ltter must be due to more pronounced effect on the delivery of UvrB to the dmge site. Loding of UvrB onto Dmged DNA. The loding of UvrB onto DNA by wild-type nd mutnt UvrAs ws tested by two methods: DNse footprinting nd gel exclusion chromtogrphy. DNse footprints of UvrA obtined in the presence of ATP nd UvrB revel hypersensitive site t the 1 1 th phosphodiester bond 5' to the dduct (Vn Houten et l., 1987). Since this site does not form in the bsence of UvrB, it hs been suggested tht it signls the presence of UvrB t the dduct site. This is clerly seen in Figure 9A with wildtype UvrA nd to much lesser degree in Figure 9B with UvrA-100; there is no hypersensitive site (UvrB footprint) detectble with UvrA-001 (Figure 9C). n fct, comprison of the level of incision observed in Figure 8 nd the intensity of the hypersensitive bnd nd UvrA footprint in Figure 9 shows close correltion between the intensity of incision nd tht of the hypersensitive bnd, but no such correltion exists between the specific ffinity of UvrA nd incision, suggesting

7 3830 Biochemistry, Vol. 30, No. 16, 1991 Myles et l. A (..., P V B 0. 8 ~ \ Frction Number , KO - 0.7uU r(rot) = 0.45 : LlJ pmol AOP/pmol UvrA FGURE 6: ADP binding by the rte of dilysis. (A) ADP binding profile from single run. 300 pg of UvrA ws dilyzed ginst buffer contining 50 mm Tris, ph 7.5, 300 mm KC, 10 mm MgC2, 1 mm DTT, 100 wg/ml SA, nd 10% glycerol. After equilibrtion of UvrA with the buffer, ["]ADP ws dded to 0.2 pm. The protein-adp rection mixture ws loded into the top chmber s described under Mterils nd Methods, nd buffer ws pssed through the bottom chmber. Cold ADP ws dded to the top chmber t the concentrtions nd frctions indicted. (B) Dt from two seprte rte of dilysis runs s shown in (A) re plotted s described by Sctchrd ( 1949). tht the defective step with (A)BC excinuclese with mutnt UvrA is the delivery of UvrB to the dmge site. This conclusion is supported by direct mesurements of UvrB loding by wild-type nd mutnt UvrAs using gel exclusion chromtogrphy. The results re shown in Figure 10. n the presence of ctlytic mounts of UvrA, nerly stoichiometric [see Orren nd Sncr (1989)l levels of UvrB re loded onto UV-dmged pbr322 by wild-type protein nd UvrA-010. The loding is drsticlly decresed with UvrA- 100 nd brely bove bckground level with UvrA-001 nd UvrA-101. Thus, we conclude tht muttions t either K37 or K646 ffect the ATPse ctivity of UvrA nd tht this decresed ctivity drsticlly reduces its bility to deliver UvrB to the dmge site. There re two possibilities for the role of ATP hydrolysis in the UvrA-medited loding of UvrB: (1) ATP hydrolysis is essentil for formtion of A2B, complexes (Orren & Sncr, 1989) or (2) ATP hydrolysis is required for the trnsfer of UvrB from A2B, complexes onto dmged DNA. To test the former possibility, we ssyed the ssocition of UvrA with UvrB by gel filtrtion chromtogrphy. We found tht ssocition of the mutnt UvrAs, 100 nd 001, with UvrB ws drsticlly reduced s compred to the wild-type protein (dt not shown) '...' [UvrA] (nu) FGURE 7: ATPse ctivity s function of UvrA concentrtion. Fifty-microliter rections were performed t room temperture in ATPse buffer (see Mterils nd Methods for its composition) contining 1.25 pm ATP, 2 pci of ["]ATP (1.18 pm), nd UvrA from 270 pm to 216 nm, s indicted, nd for times such tht the picomoles of ATP hydrolyzed per UvrA per minute is held constnt (Le., from 200 min to 15 s). Protein ws prewrmed in rection buffer for 15 min t room temperture, nd rections were strted by dding the ATP (3H nd cold together). Rections were quenched by spotting 2 pl on TLC plte. The symbols re ctul dt points while the curve is theoreticl nd is bsed on the ssumption tht dimers form with KD = 10 nm nd hve full ATPse ctivity while monomers hve no ATPse ctivity. DSCUSSON By compring the sequences of number of ATPses known to hve Wlker A nd B sequence motifs, Doolittle et l. (1 986) nd Higgins et l. (1986) discovered tht the sequence homology extended well beyond the A nd B motifs to cover mino cids ech round the A nd B sequences. These were termed the A nd B segments, nd it ws proposed tht prototype ATPse would be mde of one A nd one B segment fused together to fold into n ATPse domin. Ech ATPse ws presumed to contin dditionl components to link the ATP hydrolysis to prticulr function (e.g., trnsport). Although the originl lists contined, in ddition to UvrA, only few membrne trnsport proteins, very interesting proteins such s Mdr (multidrug resistnce; Gros et l., 1986) nd CFTR (cystic fibrosis trnsmembrne regultor; Foote et l., 1989) hve been dded to the list. More recently, more restrictive UvrA superfmily hs been proposed (Gorbleny & Koonin, 1990). UvrA contins three A -(Al-A3) nd three B (Bl-B3) segments set prt by three zinc finger motifs. The second of these zinc finger sequences contins only two of the four cysteines required for zinc cheltion nd, therefore, is presumed to be nonfunctionl. The B1 nd A2 sequences re severely truncted, nd, therefore, it hs been proposed tht A1-B2 nd A3-B3 re folded into two functionl domins, ech incorporting its own zinc finger. We hve conducted site-specific mutgenesis on the two invrint residues of the Wlker A nd B sequences, Lys nd Asp, respectively, to test the predictions from the mino cid sequence. t hs been proposed on the bsis of structurl (Pi et l., 1977; Fry et l., 1985, 1988) nd ffinity lbeling studies tht the conserved Lys in the Wlker A sequence intercts with either the - or the y-phosphte of ATP. Similrly, it hs been proposed tht the Asp in the Wlker B sequence coordintes Mg2+, thus fcilitting the positionl chnge in coordintion of Mg2+ s the rection proceeds from Mg-ATP to Mg-ADP (Fry et l., 1986). Sitespecific mutgenesis studies, in generl, hve confirmed the importnce of these residues for ATPse

8 Mutgenesis of UvrA Protein [JvrA] (nm) [UvrB] (nm) [UvrC] (nm) ATP (mm) UV (KJ) scut -m 5 CUt -D FGURE 8: UvrA Wlker A point mutnt medited incision of UVirrdited 107 bp DNA frgment. Twenty-microliter incision rections contined cpm of the DNA frgment (UV or non-uv s indicted), 6.7 nm UvrA, 80 nm UvrB, 60 nm UvrC, nd 2 mm ATP in (A)BC excinuclese buffer supplemented with 10% glycerol. Rections were incubted t room temperture for 30 min, quenched by precipitting the DNA with EtOH t -80 C. DNA ws suspended in formmide plus dyes nd electrophoresed on n 8% sequencing polycrylmide gel. Bnds were visulized by utordiogrphy. KJ, kilojoules per squre meter. Since the frgment is 5 lbeled, of the two frgmcnts resulting from incisions on the 5 nd 3 sides of UV photoproducts, only the one resulting from the 5 incision woud be detectble by utordiogrphy. The doublet ner the top of the gel in ll of the lnes is due to minor contminnt of the 107 bp substrte. function. Substitution of Lys by nother mino cid in E. coli F, ATPse (Prsonge et l., 1987) nd yest RAD3 protein (Sung et l., 1988) resulted in complete loss of complementing ctivity in vivo nd of ATPse ctivity in vitro. Substitution of Asp of the Wlker B sequence in the,b subunit of F, ATPse of E. coli (A-Shwi et l., 1988) nd of thermophilic AT- Pse (Yohd et l., 1988) lso resulted in >90% loss of ctivity. t ws, therefore, surprising tht our K37A nd K646A mutnts hd significnt complementing ctivities in vivo nd tht D5 13N nd D857N muttions hd little effect on ctivity. t is likely tht muttion of either Lys37 or Lys646 does not totlly eliminte the respective ctivities. Alterntively, it is possible tht complete elimintion of one ctivity still yields protein of prtil ctivity. Our site-specific mutgenesis dt re entirely consistent with the ntive form of UvrA possessing two ATPse sites; tht is, kinetic dt with ADP s competitive inhibitor yield n = 1.5 for the Hill coefficient. This positive coopertivity between ctive sites is eliminted by mutgenesis of K37 (i.e., for UvrA-100, n = 1.06, nd for UvrA-101, n = 1.10). However, in the K646A mutnt, n = 1.36, indicting tht only the ATPse ctivity ssocited with the A1 sequence is responsible for the positive coopertivity. n contrst to site-specific mutgenesis, nucleotide binding mesurements by equilibrium methods yielded 0.5 binding site Biochemistry, Vol. 30, No. 16, per UvrA monomer. This, combined with hydrodynmic mesurements indicting tht the ntive stte of UvrA is dimer with n ssocition constnt KA - lo8 M-l, suggests tht the ctive form of UvrA is dimer possessing one nucleotide binding site. ndeed, dilution of UvrA so s to shift the monomer-dimer equilibrium in the direction of the monomer resulted in drstic decrese in the protein s specific ctivity, indicting tht dimeriztion is essentil for the protein s ATPse function. We do not consider the kinetics nd binding dt to be contrdictory, however, s binding is mesure of the rtio of nucleotide to protein while kinetics mesure the types (with regrd to ffinity nd efficiency) of ctive sites in the ctive form of n enzyme, in this cse UvrA dimer. Therefore, model consistent with both kinetic nd binding dt would be tht UvrA dimer binds one ATP molecule t site which involves K37 nd tht hydrolysis of nucleotide t this site ctivtes the second lower ffinity ATPse site in the dimer which involves K646. Alterntively, s reported by Orren nd Sncr (1 988), ADP stbilizes UvrA dimeriztion; therefore, it is possible tht n ADP-dependent shift in the monomer-dimer equilibrium my be responsible for this positive coopertivity. Replcement of K37 by sitespecific mutgenesis elimintes the positive coopertivity, indicting tht this site hs n llosteric function. However, the mutnt protein still binds ADP with the sme ffinity s the wild-type protein, indicting tht K37 is not essentil for binding to this nucleotide. n contrst, ATP binding is significntly reduced in this mutnt, nd, therefore, we conclude tht this mino cid is responsible for y-phosphte contct with ATP. The site involving K646 is importnt for ctlysis but pprently hs no llosteric effect. Tht UvrA dimers hve mximl ATPse ctivity nd tht UvrA binds nucleotide with stoichiometry of 1 per dimer imply tht nucleotide binding sites form t the interfce of the monomers or lterntively t single site on UvrA monomer (Le., hlf-of-the-sites rectivity). The bsence of negtive coopertivity, by Hill nlysis of ATPse dt s well s the hyperbolic ADP binding curve, suggests tht the ctive site forms cross the interfce of the two monomers. Further, these dt lso strongly suggest tht dimeriztion occurs in hed-to-hed, til-to-til rrngement to crete two nonequivlent ctive sites. This hs been verified by the demonstrtion tht the purified mino-terminl domin (monomer M, = ) hs ntive moleculr weight of by gel filtrtion chromtogrphy nd tht the mino- nd crboxyterminl domins do not interct noncovlently (Myles & Sncr, 1990). Our dt lso shed some light on the role of the ATPse function of UvrA in the overll rection ctlyzed by (A)BC excinuclese. Previous work hs shown tht UvrA hs two functions; recognition of DNA dmge nd delivery of the UvrB subunit to tht site. Our results show tht neither ATP binding nor hydrolysis is essentil for binding to the dmge site but tht ATP binding/hydrolysis is necessry for delivering UvrB to tht site. n this regrd, the two ATPse sites (s defined by K37 nd K646 muttions) behved differently. UvrA-100 (K37A) hd bout 10% of the UvrB loding ctivity s evidenced by the intensity of the UvrB hypersensitive site with DNse footprinting nd direct mesurement by gel exclusion while UvrA-001 (K646A) hd bout 1% of this ctivity. Further, (A) BC excinuclese reconstituted with these mutnts hd incision ctivities comprble to the loding. Recently, Seeley nd Grossmn (1 989) presented evidence which indicted tht the cryptic ATPse of UvrB, which becomes overt in the UvrA-UvrB-DNA complex, ws essentil

9 3832 Biochemistry, Vol. 30, No. 16, 1991 Myles et l. A UvrA-000 B UvrA- 00 [UvrA] (nm) [UvrB] (nm) UvrAJ(nM) io [UvrB] (nm) [ATP (mm) [ATP](mM) UvrA t C UvrA-001 UvrA [ FGURE 9: DNse footprinting of UvrA Wlker A point mutnts. Footprinting rections re 50 pl nd were performed in (A)BC excinuclese buffer contining cpm of DNA nd the concentrtions of UvrA indicted. Where indicted, 4 nm UvrB nd/or 2 mm ATP were lso included. Rections were incubted t room temperture for 25 min prior to the ddition of 2.5 pl of DNse (150 pg/ml in 0.1 M CC,); thc nuclese digestion ws incubted t room temperture for 5 min nd quenched by precipitting the DNA with EtOH using 2 pg of clf thymus DNA s crrier. The precipitted DNA ws resuspended in 25 pl of 95% formmide dyes (Bromphenol blue, 0.1% w/v, nd Xylene cyno1 0.1% w/v) nd heted t 90 "C for 5 min. Five microliters ws electrophoresed on n 8% sequencing polycrylmide gel; bnds re visulized by utordiogrphy. (A) UvrA-000; (B) UvrA-100; (C) UvrA-001. The sterisks indicte the position of the DNse hypersensitive site in the UvrA-UvrB-DNA or UvrB-DNA complexes.

10 Mutgenesis of UvrA Protein Biochemistry, Vol. 30, No. 16, * - UV~A b- UvrB FGURE 10: Loding of UvrB by UvrA Wlker A point mutnts. Rections were performed s described under Mterils nd Methods. Four hundred microliters of the plsmid-contining frction from series of five column runs were heted t 95 OC for 30 min nd electrophoresed on 10% SDS-polycrylmide gel stined with silver (Bio-Rd kit). Twenty microliters of ech lod ws mixed with 400 pl of column buffer, heted s described for the plsmid frctions, nd electrophoresed on the sme gel. These smples serve s control for equivlent protein loding nd recovery nd lso demonstrte tht the pprent bsence of UvrA in the plsmid frctions is not the result of n electrophoresis rtifct. for loding UvrB onto the dmge site lthough muttion of this ATPse ctivity pprently hs no effect on the UvrA- UvrB interction. Our results show tht UvrA s ATPse ctivity is essentil for this step. However, the two conclusions re not contrdictory. The loding of UvrB onto the dmged site involves ssocition of UvrA dimer with UvrB followed by the loding rection itself. Both ssocition of the two subunits (Orren & Sncr, 1989) nd the loding of UvrB (Vn Houten et l., 1988) re ATP dependent. We hve found (dt not shown) tht in UvrA-100 nd UvrA-001 the ssocition of UvrA with UvrB is gretly reduced. Thus, it ppers tht the ATPse ctivities of both UvrA nd UvrB re essentil for loding but the two ATPses function t different steps of the overll rection. n conclusion, we believe UvrA to hve two domins defined by the A1-B2 nd A3-B3 segments defined by Doolittle et l. (1986). Ech domin hs its own zinc finger, nd ech domin is presumed to fold independently nd to hve n ATP binding/hydrolysis ctivity s well s DNA binding ctivity conferred by the zinc fingers. This premise is directly ddressed in the following pper (Myles & Sncr, 1991). REFERENCES A-Shwi, M. K., Prsonge, D., & Senior, A. E. (1988) J. Biol. Chem. 263, Brdford, M. M. (1976) Anl. Biochem. 72, Chothi, C. (1975) Nture (London) 254, Colowick, S. P., & Womck, F. C. (1969) J. Biol. Chem. 244, Dombroski, A. J., Brennn, C. A., Sper, P., & Pltt, T. (1988) J. Biol. Chem. 263, Dombroski, A. J., LDine, J. R., Cross, R. L., & Pltt, T. (1988b) J. Biol. Chem. 263, Doolittle, R. R., Johnson, M. S., Husin,., Vn Houten, B., Thoms, D. C., & Sncr, A. (1986) Nture (London) 323, Duncn, T. M., Prsonge, D., & Senior, A. E. (1986) FEBS Lett. 208, 1-6. Foote, S. J., Thompson, J. K., Cowmn, A. F., & Kemp, D. J. (1989) Cell 57, Fry, D. C., Kuby, S. A., & Mildvn, A. S. (1985) Biochemistry 24, Fry, D. C., Kuby, S. A., & Mildvn, A. S. (1986) Proc. Ntl. Acd. Sci. U.S.A. 83, Fry, D. C., Byler, D. M., Susi, H., Brown, E. M., Kuby, S. A., & Mildvn, A. S. ( 988) Biochemistry 27, Gorbleny, A. E., & Koonin, E. V. (1990) J. Mol. Biol. 213, Gros, P., Rymond, M., Bell, J., & Housmn, D. (1988) Mol. Cell. Biol. 8, Grossmn, L., & Yeung, A. T. (1990) Photochem. Photobiol. 51, Higgins, C. F., Hiles,. D., Slmond, G. P. C., Gill, D. R., Downie, J. A., Evns,. J., Hollnd,. B., Gry, L., Buckel, S. D., Bell, A. W., & Hermodson, M. A. (1986) Nture (London) 323, Hummel, J. P., & Dreyer, W. J. (1962) Biochim. Biophys. Act 63, Husin,., Vn Houten, B., Thoms, D. C., & Sncr, A. (1986) J. Biol. Chem. 261, Kunkel, T. A., Roberts, J. D., & Zkour, R. A. (1987) Methods Enzymol. 154, Lemmli, U. K. (1970) Nture (London) 227, Linewever, H., & Burk, D. (1934) J. Am. Chem. SOC. 56, Myles, G. M., & Sncr, A. (1991) Biochemistry (second of three ppers in this issue). Nvrtnm, S., Myles, G. M., Strnge, R. W., & Sncr, A. (1989) J. Biol. Chem. 264, Oh, E. Y., Clssen, L., Thiglingm, S., Mzure, S., & Grossmn, L. (1989) Nucleic Acids Res. 17, Orren, D. K., & Sncr, A. (1988) UCLA Symp. Mol. Cell. Biol. 83, Orren, D. K., & Sncr, A. (1989) Proc. Ntl. Acd. Sci. U.S.A. 86, Orren, D. K., & Sncr, A. (1990) J. Biol. Chem. 265, Pi, E. F., Schsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, Prsonge, D., Wile-Mounts, S., & Senior, A. E. (1987) J. Biol. Chem. 263, Prsonge, D., A-Shwi, M. K., & Senior, A. E. (1988) J. Biol. Chem. 262, Ro, R., Pgn, J., & Senior, A. E. (1988) J. Biol. Chem. 263, Sncr, A., & Rupp, W. D. (1983) Cell 33, Sncr, A., & Sncr, G. B. (1988) Annu. Rev. Biochem. 57, Snger, F., Nicklen, S., & Coulson, A. R. (1977) Proc. Ntl. Acd. Sci. U.S.A. 74, Sctchrd, G. (1949) Ann. N.Y. Acd. Sci. 51, Scott, J. F., Eisenberg, S., Bertsch, L. L., & Kornberg, A. (1977) Proc. Ntl. Acd. Sci. U.S.A. 74, Seeberg, E., & Steinum, A. L. (1982) Proc. Ntl. Acd. Sci. U.S.A. 79, Seeley, T. W., & Grossmn, L. (1989) Proc. Ntl. Acd. Sci. U.S.A. 86, Seeley, T. W., & Grossmn, L. (1990) J. Biol. Chem. 265, Sung, P., Higgins, D., Prksh, L., & Prksh, S. (1988) EMBO J. 7, Tbor, S., & Richrdson, D. D. (1987) Proc. Ntl. Acd. Sci. U.S.A. 84, Thoms, D. C., Levy, M., & Sncr, A. (1985) J. Biol. Chem. 260, Vn Houten, B., (1990) Microbiol. Reu. 54, Vn Houten, B., Gmper, H., Sncr, A., & Herst, J. E. (1987) J. Biol. Chem. 262,

11 3834 Biochemistry 1991, 30, Vn Houten, B., Gmper, H., Herst, J. E., & Sncr, A. (1988) J. Biol. Chem. 263, Yohd, M., Oht, S., Hisbori, T., & Kgw, Y. (1988) Biochim. Biophys. Act 933, Wlker, J. E., Srste, M., Runswick, M. J., & Gy, N. J. Zoller, M. J., & Smith, M. (1983) Method Enzymol. 100, ( 1982) EMBO J., soltion nd Chrcteriztion of Functionl Domins of UvrAt Gry M. Mylest nd Aziz Sncr* Deprtment of Biochemistry nd Biophysics, University of North Crolin School of Medicine, Chpel Hill, North Crolin Received November 8, 1990; Revised Mnuscript Received Jnury 15, 1991 ABSTRACT: The sequence of Escherichi coli UvrA protein suggests tht it my fold into two functionl domins ech possessing DNA binding nd ATPse ctivities. We hve tken two pproches to physiclly isolte polypeptides corresponding to the two puttive domins. First, 180 bse pir DNA segment encoding multiple collgense recognition sequences ws inserted into UvrA's puttive interdomin hinge region. This UvrA derivtive ws purified nd digested with collgense, nd the resulting 70-kD N-terminl nd 35-kD C-terminl frgments were purified. Both frgments possessed nonspecific DNA binding ctivity, but only the N-terminl domin retined its nucleotide binding cpcity s evidenced by mesurements of ATP hydrolysis nd by ATP photo-cross-linking. Together, the two frgments filed to substitute for UvrA in reconstituting (A)BC excinuclese nd, therefore, were presumed to be unble to lod UvrB onto dmged DNA. Second, the DNA segments encoding the two domins were fused to gene. The UvrA N-terminl domin-@-glctosidse fusion protein ws overproduced nd purified. This fusion protein hd ATPse ctivity, thus confirming tht the mino-terminl domin does possess n intrinsic ATPse ctivity independent of ny interction with the crboxy terminus. Our results show tht UvrA hs two functionl domins nd tht the specificity for binding to dmged DNA is provided by the proper three-dimensionl orienttion of one zinc finger motif reltive to the other nd is not n intrinsic property of n individul zinc finger domin. U v r A, which is one of the three subunits of Escherichi coli (A)BC excinuclese (Sncr & Sncr, 1988; Selby & Sncr, 1990), is n ATPse nd DNA binding protein with higher ffinity for dmged thn for undmged DNA (Seeberg & Steinum, 1982). Anlysis of the mino cid sequence of UvrA (Husin et l., 1986) hs reveled 3 ATPse A nd B segments interspersed with 3 zinc finger motifs ech of mino cids (Doolittle et l., 1986). t ppers tht the second zinc finger motif is degenerte nd, therefore, hs lost its zinc chelting cpcity; thus, UvrA contins only 2 mol of Zn2+ per monomer pprently chelted by the first nd third motifs (Nvrtnm et l., 1989). Similrly, of the three pirs of ATPse A nd B segments, B1 nd A2 pper to be severely truncted (Doolittle et l., 1986). ndeed, mutgenesis of the "invrint" Lys residue in the A2 segment nd the invrint Asp residue in the B segment hd no effect on the protein's in vivo ctivity. This hs led to the proposl tht the two ATPse ctivities of UvrA were mde up of A1-B2 nd A3- B3 segments, respectively (Myles et l., 1991), nd tht ech of these ATPse units ws ssocited with zinc finger, Al-B2 with Znl nd A3-B3 with Zn3, to constitute two domins ech with its own DNA binding nd ATPse ctivities. 'This work ws supported by Grnts GM32833 from the Ntionl nstitutes of Helth nd PCM from the Ntionl Science Foundtion nd in prt by Grnt CTR1872 from The Council for Tobcco Reserch nc. * Correspondence should be ddressed to this uthor. *Present ddress: Fred Hutchinson Cncer Reserch Center, 1124 Columbi St.. Settle, WA n this pper, we describe the use of genetic engineering methods to isolte frgments of UvrA contining A-Zn 1-B2 (70 kd) nd A3-Zn3-B3 (35 kd) sequences. Our results show tht ech of these frgments constitutes domin with nonspecific DNA binding ctivity but tht only the Al- Znl-B2 domin possesses ATPse ctivity when not physiclly linked to the crboxy-terminl A3-Zn3-B3 domin. MATERALS AND METHODS Mterils. The wild-type UvrA, UvrB, nd UvrC proteins were purified s described previously (Thoms et l., 1985). Collgense from Achromobcter iophgus ws purchsed from Boehringer Mnnheim Biochemicls; it ws suspended t 0.12 mg/ml in TEN 7.4 nd stored t 4 OC. Nco ws obtined from Promeg; T4 kinse, bcteril lkline phosphtse, BmH, nd Su3A were from Bethesd Reserch Lbortories; nd T4 ligse nd T4 DNA polymerse were from Bio-Rd. [2,fk3H]ATP(30 Ci/mmol), [LY-~~PATP (3000 Ci/mmol), nd [Y-~~PATP (7000 Ci/mmol) were from CN Biomedicls, nc.; [3H]thymidine (82.4 Ci/mmol) ws from New Englnd Nucler. Single-strnd DNA-cellulose nd heprin-grose ffinity resins were from Sigm, AcA-34 gel filtrtion resin ws from BM Biotechnics, nd DEAE-Bio-Gel ws from Bio-Rd. Silver-stining regents were purchsed from Bio-Rd, PTG nd ONPG were from Boehringer Mnnheim Biochemicls, nd the TLC pltes (Polygrm ce1300 PE) were from Brinkmnn nstruments /9 1 / $02.50/ Americn Chemicl Society