Role for radA/sms in Recombination Intermediate Processing in Escherichia coli

RadA/Sms is a highly conserved eubacterial protein that sharessequence similarity with both RecA strand transferase and Lonprotease. We examined mutations in the radA/sms gene of Escherichiacoli for effects on conjugational recombination and sensitivityto DNA-damaging agents, including UV irradiation, methyl methanesulfonate(MMS), mitomycin C, phleomycin, hydrogen peroxide, and hydroxyurea(HU). Null mutants of radA were modestly sensitive to the DNA-methylatingagent MMS and to the DNA strand breakage agent phleomycin, withconjugational recombination decreased two- to threefold. Wecombined a radA mutation with other mutations in recombinationgenes, including recA, recB, recG, recJ, recQ, ruvA, and ruvC.A radA mutation was strongly synergistic with the recG Hollidayjunction helicase mutation, producing profound sensitivity toall DNA-damaging agents tested. Lesser synergy was noted betweena mutation in radA and recJ, recQ, ruvA, ruvC, and recA forsensitivity to various genotoxins. For survival after peroxideand HU exposure, a radA mutation surprisingly suppressed thesensitivity of recA and recB mutants, suggesting that RadA mayconvert some forms of damage into lethal intermediates in theabsence of these functions. Loss of radA enhanced the conjugationalrecombination deficiency conferred by mutations in Hollidayjunction-processing function genes, recG, ruvA, and ruvC. AradA recG ruv triple mutant had severe recombinational defects,to the low level exhibited by recA mutants. These results establisha role for RadA/Sms in recombination and recombinational repair,most likely involving the stabilization or processing of branchedDNA molecules or blocked replication forks because of its geneticredundancy with RecG and RuvABC.

INTRODUCTION

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Introduction

The radA/sms gene was initially identified in a screen for radiation-sensitivemutants of Escherichia coli (13). radA mutants showed a modestdecrease in survival after UV or X-irradiation exposure andin repair of DNA breaks (40). This phenotype is growth mediumdependent: in minimal medium, strains are less resistant andhave no radA-dependent component of survival. This may be relatedto the fact that in rich growth medium, E. coli cells have multiplesister chromosomes that can interact by recombination to effectrepair. The sms gene was defined as an open reading frame downstreamof, and coregulated with, serB, whose inactivation caused slightsensitivity to methyl methanesulfonate (MMS) (39). Later, smsand radA were identified as the same gene (46).

The RadA/Sms predicted protein sequence is a composite of threecharacteristic regions. It contains a putative zinc finger atits N terminus with a CXXC-X_n-CXXC motif. Its middle regionis related to the RecA strand exchange protein and the DnaBreplicative DNA helicase (24), containing both Walker A andWalker B boxes characteristic of ATPases. The highly conservedmotif among prokaryotic RadA orthologs, KNRFG, is found at theC-terminal edge of this RecA-related region. The C-terminal150 amino acids of RadA/Sms is related to Lon protease, an ATP-dependentserine protease that binds to DNA (51) and that regulates capsularpolysaccharide synthesis and the SOS response (16). The active-siteserine is present in E. coli RadA/Sms, but this residue is replacedby alanine in many of the eubacterial radA/sms orthologs. Orthologsof radA/sms carrying all three sequence motifs are ubiquitousamong the eubacteria and can be seen in the genomes of at least40 eubacterial genera to date. The radA gene of archaea, despiteits name, is not related to the radA/sms gene of eubacteria,except that both have similarity to recA of prokaryotes andRAD51 of eukaryotes. There is one bona fide eukaryotic radA/smsform in Arabidopsis thaliana, which may have entered the plantgenome via the chloroplast of eubacterial origin.

A role for radA/sms in recombination is suggested by the factsthat the repair pathways affected by radA are recA dependent(13) and that recombination mutants have concomitant defectsin repair of radiation-induced DNA lesions. However, radA/smsdoes not affect survival as severely as do other mutations thatresult in defects in double-strand-break repair, such as recA,recB, recC, or recN (40). As determined by conjugational recombination,radA mutants are not appreciably affected in the ability torecombine (13). However, conjugational recombination differssignificantly from repair recombination, and several mutationsresulting in strong defects in recombinational repair (suchas recJ, recN, recFOR, and ruvABC) have only minor effects onconjugational recombination frequencies (reviewed in reference27). A radA/sms ortholog mutant in Bacillus subtilis has beenreported to be defective in transformational inheritance (23).

We examined here the role of radA in DNA damage survival andconjugational recombination. In addition, we constructed doublemutants with mutations in both radA and other known recombinationfunction genes to reveal properties of radA that may be redundantto those of other genes. Sensitivity to various types of DNA-damagingagents, such as UV irradiation, MMS, hydrogen peroxide, hydroxyurea(HU), and phleomycin, were assayed. The radA/sms gene does indeedplay a role in recombination and DNA repair. Its effects onrepair were revealed most strongly in mutants deficient in postsynapticDNA processing, especially those lacking the RecG branch migrationhelicase, suggesting that RadA may also play a role in thistype of branched-structure processing.

MATERIALS AND METHODS

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Materials and Methods

Strains and plasmids. Strains were grown at 37°C as previously described on Luria-Bertani(LB) medium, consisting of 1% Bacto Tryptone, 0.5% yeast extract,0.5% sodium chloride, and, for plates, 1.5% agar (50). For transductionsand preparation of P1 phage lysates, cultures were grown inLCG medium, consisting of LB medium supplemented with 1% glucose,2 mM calcium chloride, and, for plates, 1% agar. LCG top agarcontained 0.7% agar. The following antibiotics at the indicatedconcentrations were used: ampicillin and streptomycin, 100 µg/mleach; kanamycin, 30 to 60 µg/ml; tetracycline and chloramphenicol,15 µg/ml each. Isogenic strains in an AB1157 backgroundwere constructed by P1 transduction (37) and are listed in Table1.

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TABLE 1. Escherichia coli K-12 strains

Construction of radA mutant alleles. The radA gene was amplified by PCR using Taq polymerase (Promega,Inc.) with the primers 5' CTGAATTCAG AAGTAATTGCTCGCCCG and 5'ATCCGGCACG GTCGGCTGCTGCGACAT from chromosomal DNA derived fromwild-type E. coli K-12 strain MG1655. The PCR product was digestedwith EcoRI and BstBI and ligated into vector pBS SK- (Stratagene,Inc.) that had been cut with EcoRI and ClaI, producing plasmidpSTL307. The radA1::kan allele was constructed by insertionof a BamHI fragment of mini-Tn10-kan (48) into the unique BglIIsite of radA, producing plasmid pSTL310. This plasmid was digestedwith HincII and BglI restriction endonucleases and transformedby electroporation (14) into strain STL941 (31), with selectionfor kanamycin resistance (Km^r) and screening for ampicillinsensitivity (Ap^s). A P1 transducing lysate from the resultingdisruptant, STL1542, was used to convert AB1157 to Km^r, yieldingSTL1815. The location of the kan insertion within radA was confirmedby its linkage in transductional crosses with CAG18429 (zjh-606::Tn10),CAG18430 (zji-202::Tn10), and CAG18442 (thr-34::Tn10) (43).Because STL1815 developed an auxotrophy for cysteine in an uncharacterizedgene, all experiments employed a Km^r transductant of STL1815into AB1157, strain STL5280, which does not carry this additionalauxotrophy. The radA3 ${Delta}$ ::FRT allele with a precise deletion ofthe entire radA coding region was constructed by the methodof Datsenko and Wanner (12) with PCR primers 5' CCGCCATCCTGCGGGCGGCACAGCATTAACGAGGTACACCTGTAGGCTGGAGCTGCTTCGand 5' TCAGGTAATCAAATGACGACATATCTCCCTCCGTATATCTCATATGAATATCCTCCTTAGto amplify the kan gene of plasmid pKD4 flanked by FRT site-specificrecombination sites and radA-specific sequences. The resultantPCR fragment carried a 40-bp homologous region where the allelewas substituted by recombination into the chromosome of strainBW26308, producing STL5821. From this strain, the allele wastransduced into AB1157 by using P1, with selection for Km^r (producingstrain STL6036); subsequent deletion of the kan gene at theflanking FRT sites was accomplished by transformation with FLPrecombinase-encoding plasmid pCP20, selecting for Ap^r at 30°C.After streaking STL6036 onto LB medium at 42°C to cure theplasmid, deletion strain STL6430 was produced. Genetic crosseswith CAG18442 (42) confirmed the appropriate genetic location,and Southern blotting and PCRs confirmed deletion of the radAcoding sequence.

DNA damage survival assays. For UV survival determinations, cells were grown in LB liquidmedium to exponential stage (optical density at 600 nm [OD_600],0.4 to 0.6), serially diluted in 56/2 buffer (50), and platedon LB agar plates. The plates were immediately irradiated withvarious doses of UV (254 nm) and incubated at 37°C in thedark overnight. The total viable cells in serially diluted unirradiatedcells were determined. Resistance to MMS was determined forat least eight independent cultures in 2 ml of LB medium afterovernight growth, and resistance to mitomycin C (MMC), phleomycin,and HU was determined for at least eight independent culturesin 2 ml of LB medium after growth to exponential phase (OD₆₀₀,0.4 to 0.6). To score for survival of cells to these DNA-damagingagents, the cultures were serially diluted in 56/2 buffer andplated directly onto LB plates containing 0.1% MMS, 1 µgof MMC/ml, 0.5 µg of phleomycin/ml, or 10 mM HU. Chemicalswere purchased from Sigma, Inc. Plates containing MMS were usedwithin 48 h. Hydrogen peroxide sensitivity was measured withexponentially growing liquid cultures, with hydrogen peroxideadded to cultures to a final concentration of 5 mM. After incubationof shaking cultures at 37°C for 20 min, 50 µg of catalaseper ml was added directly to the cultures to inactivate theperoxide. The cultures were serially diluted in 56/2 bufferand plated directly onto LB plates. Total viable cells weredetermined by serial dilution with 56/2 buffer followed by platingon LB medium. Cell counts were determined on all platesafterovernight growth at 37°C.

Assay for conjugational recombination. All strains were assayed for conjugational inheritance in parallelwith the radA⁺ control strain. Matings were performed usinga 10:1 recipient-to-donor ratio, with recipient cells grownto an OD₆₀₀ of 0.4 and donor cells grown to an OD₆₀₀ of 0.3.The matings were allowed to proceed for 30 min at 37°C,and then the cultures were shaken vigorously and serially dilutedin 56/2 buffer. STL6804 was used as the Hfr donor in recombinationproficiency tests. (The radA gene was mutated in the Hfr donorto prevent complementation of radA in the zygote due to earlytransfer of radA⁺ in the cross.) The Leu⁺ Ser⁺ Sm^r recombinantswere selected on streptomycin-minimal medium plates lackingleucine after serial dilution and plating. As a control forthe efficiency of conjugal transfer, the strains were also crossedwith STL7130, a similar Hfr donor lysogenic for lambda at attB.Production of zygotically induced infective centers was assayedas previously described (30). Although the data are not reported,none of the strains listed below (see Table 3) had any defectsin conjugational transfer of lambda. Viable counts were determinedby plating serially diluted unmated cultures on LB plates. Allplates were counted after 1 or 2 days of growth at 37°C.

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TABLE 3. Conjugational inheritance in radA mutants^a

RESULTS

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Results

A modest effect of single radA mutations on DNA survival and recombination. The original allele of radA is radA100, which encodes a mutationin one of the conserved cysteine residues in the putative zincfinger of the RadA protein (46). We generated two additionalmutant alleles: radA1::kan, a simple insertion, and rad3 ${Delta}$ ::FRT,a complete and precise deletion of the entire open reading frame.We tested the survival of these three single mutants to a groupof DNA-damaging agents to which recA and other recombinationmutants of E. coli K-12 are sensitive.

Unlike the results of a previous report (13), we were unableto demonstrate any consistent defect in survival after UV lightexposure for the radA1 single mutant (Fig. 1 and 2), althoughslight sensitivity was evident in one of the six experiments(Fig. 1A). This is not an allele effect, since we were alsounable to demonstrate UV sensitivity for the other radA alleles,radA100 and radA3 ${Delta}$ (B. Levinson, C. E. Beam, and S. T. Lovett,unpublished results). As reported earlier (39), these mutantsdid show sensitivity to the methylating agent MMS (Table 2),although their decrease in survival was much less than the morethan four orders of magnitude of killing seen for the recA mutantof E. coli at the same MMS concentration. None of the mutantswas sensitive to oxidative damage in the presence of hydrogenperoxide, and all showed a weak sensitivity to the cross-linkingagent MMC. The presumed null alleles radA1 and radA3, but notthe radA100 point mutation, conferred modest sensitivity tophleomycin, a compound related to the antitumor agent bleomycin,that induces strand breaks in DNA (4, 44). Conversely, HU, aninhibitor of deoxynucleotide synthesis, was an effective killerof the radA100 point mutant but had much less effect on thepresumed null mutants carrying radA1 or radA3. The last resultssuggest that the radA100 putative zinc finger mutation has specificand limited effects on RadA function in vivo. The radA singlemutants had modest, if any, defects in conjugational inheritance(Table 3), results in agreement with a previously publishedreport (13).

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FIG. 1. Synergy of radA with mutations affecting recombinational UV repair. Shown are UV survival curves for AB1157-derived strains both singly and doubly deficient for radA, recA, recJ, recQ, and recB. (A) AB1157, rec⁺ ( ${blacksquare}$ ); STL5280, radA1::kan (•); JC10287, recA ${Delta}$ ( ${blacklozenge}$ ); STL4799, recA ${Delta}$ radA1::kan ( ${lozenge}$ ); (B) AB1157, rec⁺ ( ${blacksquare}$ ); STL5280, radA1::kan (•); STL1548, recQ1802::Tn3 ( ${blacktriangleup}$ ); JC12123, recJ284::Tn10 ( ${blacklozenge}$ ); STL5048, recQ1802::Tn3 radA1::kan ( ${Delta}$ ); STL5042, recJ284::Tn10 radA1::kan ( ${lozenge}$ ); (C) AB1157, rec⁺ ( ${blacksquare}$ ); STL5280, radA1::kan (•); N2101, recB268::Tn10 ( ${blacktriangleup}$ ); STL5480, recB268::Tn10 radA1::kan ( ${Delta}$ ). Error bars indicate standard errors of the determinations.

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FIG. 2. RadA is synergistic with Holliday junction-processing genes in UV repair. UV survival curves are shown for E. coli strains both singly and multiply deficient for radA, recG, ruvA, and ruvC. All strains assayed for UV survival were derived from the AB1157 background. (A) AB1157, rec⁺ ( ${blacksquare}$ ); STL5280, radA1::kan (•); N2096, ruvA ${Delta}$ 63 ( ${blacktriangleup}$ ); CS140, ruvC53 ( ${blacklozenge}$ ); STL5046, ruvC53 radA1::kan ( ${lozenge}$ ); STL5037, ruvA ${Delta}$ 63 radA1::kan ( ${Delta}$ ); (B) AB1157, rec⁺ ( ${blacksquare}$ ); STL5280, radA1::kan (•); N4452, recG ${Delta}$ 265::cat ( ${blacktriangleup}$ ); STL6588, recG ${Delta}$ 265::cat radA1::kan ( ${Delta}$ ); (C) AB1157, rec⁺ ( ${blacksquare}$ ); STL5280, radA1::kan (•); STL6586, recG ${Delta}$ 265::cat ruvC53 ( ${lozenge}$ ); STL6640, recG ${Delta}$ 265::cat radA1::kan ruvC53 ( ${blacklozenge}$ ); STL6571, recG ${Delta}$ 265::cat ruvA ${Delta}$ 63( ${Delta}$ ); STL6592, recG ${Delta}$ 265::cat radA1::kan ruvA ${Delta}$ 63 ( ${blacktriangleup}$ ). Error bars indicate standard errors of the determinations.

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TABLE 2. Relative survival of radA and rec mutant strains to DNA-damaging agents^a

Effect of radA when combined with other recombination mutations. Recombination and recombinational repair are mediated in E.coli by several genetically distinct pathways (7, 21, 27). Inaddition, some processing events in recombination are redundant;for this reason, mutational effects are sometimes not manifestunless other functions are also mutated. We therefore placedthe radA1 insertion allele in combination with mutations ingenes encoding several known recombination functions (reviewedin reference 22), including recA (DNA strand exchange protein),recB (component of DNA nuclease/helicase complex), recJ (single-strandDNA exonuclease), recQ (DNA helicase), recG (branch migrationhelicase), ruvA (component of branch migration helicase), andruvC (Holliday junction endonuclease).

With respect to UV survival, the radA mutation exacerbated theUV sensitivity of several mutants, including the recA, recJ,recQ (Fig. 1A and B), recG, ruvA, and possibly ruvC mutants(Fig. 2A and B). The effect was especially great with recG:although neither single mutant showed pronounced survival defects,the double mutant was severely affected. The recG radA ruv triplemutants were as sensitive (Fig. 2C), if not more so, than therecA mutants (Fig. 1A) that are defective in all pathways ofhomologous recombination and in induction of the SOS response.No effect on the recB mutants was afforded by addition of theradA mutation (Fig. 1C). The RecG, RuvA, and RuvC proteins playa role in the processing of branched recombinational intermediates,such as Holliday junctions, and therefore act at a late stepin recombination pathways (42, 49). The RecQ and RecJ proteinsin concert may reveal single-strand DNA to initiate recombination(reviewed in reference 21). There is some evidence that theseproteins also play a role in processing of replication forksstalled by UV lesions (9). The RecJ exonuclease also has a postsynapticrole in stabilization of joint molecules, both in vitro andin vivo (8, 15)

In other DNA damage survival assays (Table 2), genetic syntheticeffects were again strongest between radA and recG, where radAwas found to sensitize recG mutants to all agents tested 5-to 500-fold. The sensitivity of the double mutants approachedor exceeded the sensitivity of recA mutants that are deficientin homologous recombination and induction of the SOS response.For MMC, phleomycin, and hydrogen peroxide, although both recGand radA single mutants were only modestly sensitive, the recGradA double mutant showed profound defects in survival. Thisgenetic effect is consistent with the idea that recG and radA/smsshare some redundant role in DNA damage tolerance or repair.

Mutants in the Holliday junction helicase and resolvase, RuvAand RuvC, were very sensitive to all agents tested. The additionof a mutation in radA exacerbated DNA damage sensitivity ofruvA or ruvC mutants to all agents except the replication inhibitorHU. Strains with mutations in recJ (encoding exonuclease) andrecQ (encoding helicase), like those with mutations in radA,were only modestly sensitive to the array of DNA-damaging agents,with the strongest killing effects demonstrated by either MMSor phleomycin. A mutation in radA was found to sensitize recJand recQ mutants to the killing effect of MMS and recQ and recBmutants to that of phleomycin.

An unexpected but consistent result was the suppression of sensitivityof recA and recB mutants to hydrogen peroxide and to HU by mutationsin radA. This contrasts to the slightly enhancing effect ofa radA mutation on the extreme sensitivity to UV light (Fig.1), MMS, MMC, and phleomycin (Table 2) conferred by recA. Thesuppressive result seems to suggest that functional RadA mayconvert some kinds of DNA damage, such as that after oxidationor replication inhibition, into a form that is lethal in theabsence of RecABCD-dependent repair.

Several different double and triple mutant combinations weretested for effects on conjugational recombination (Table 3).Although conjugational recombination was reduced by approximately4 orders of magnitude by a mutation in recA, mutations in radA,recG, ruvA, or ruvC reduced recombination no more than about10-fold. A mutation in radA substantially reduced residual recombinationof recG or ruv mutants, and the radA recG ruv triple mutantsachieved recombination deficiency comparable to that of recAmutants. Again, this result is consistent with the idea thatRadA plays a role in recombination that is redundant to thatof the Holliday junction-processing proteins RecG and RuvABC.

DISCUSSION

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Discussion

The ubiquity of the radA/sms gene in bacterial genomes suggeststhat it has played an important role in the promotion of cellgrowth or survival. We show here that the radA/sms gene is requiredfor efficient repair of certain forms of DNA damage and is requiredfor genetic recombination in a step that is apparently redundantto that provided by the Holliday junction-processing proteinsRecG and RuvABC. For promotion of survival after DNA damage,in particular, RadA/Sms has genetic effects that are often highlyredundant to those of the RecG helicase, such that only theloss of both functions results in severe sensitivity to genotoxinsand radiation.

Recombinational repair processes can, in various ways, alleviateblocks to DNA replication imposed by genotoxic assaults andrestore chromosome integrity (for reviews, see references 10,20, 21, 36). Lesions in DNA, such as those produced by UV lightor by the cross-linking agent MMC, can block DNA polymerasesduring replication, leading to the accumulation of single-strandDNA gaps or double-strand breaks. Other agents can directlyor indirectly via processing enzymes cause the accumulationof single-strand nicks or double-strand breaks. Recombinationcan restore broken chromosomes or broken replication forks ina manner dependent on the double-strand-break-processing helicase/nucleaseRecBCD and the strand invasion protein RecA. In contrast, single-strandDNA gaps are filled by a recombinational mechanism involvingRecA and the RecFOR proteins (45).

Recombinational reactions involve the formation of branchedintermediate structures that are processed by the RecG helicaseand by the RuvAB helicase in concert with the Holliday junctionendonuclease RuvC. RecG and the Ruv proteins have redundanteffects on genetic recombination and DNA repair (25), such thatloss of either function causes a modest reduction whereas theloss of both has a severe effect. We show here that RadA/Smsjoins this redundant team of processing enzymes; loss of allthree functions produces a strain with severe conjugationalrecombination defects that is comparable to recA strand transferasemutants of E. coli. Since conjugational recombination occursprimarily via the RecBCD-dependent pathway (7, 27), RadA/Smsis implicated in recombination initiated from double-strandends (Fig. 3).

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FIG. 3. Double-strand-break (DSB)-mediated recombination. A broken fork can be repaired by recombinational reactions. (Likewise, ends of conjugative DNA or transducing fragments can be integrated via this mechanism.) Double-strand ends are resected by RecBCD nuclease; RecBCD also assists in loading of RecA onto single-strand DNA. The RecA-single-strand DNA filament promotes strand invasion into a homologous duplex molecule (the sister chromosome), forming a D-loop intermediate. Branch migration helicases can extend the region of pairing to form a Holliday junction (HJ), which can be resolved by cleavage mediated by Holliday junction endonucleases such as RuvC. Ligation of strand scissions restores an intact recombinant chromosome. RadA may participate in recombination by stabilizing any of these joint intermediates or by mediating branch migration or cleavage.

In addition to a role in genetic recombination, the Hollidayjunction-processing proteins of E. coli may contribute to theprocess of replication fork regression (Fig. 4). When a forkis stalled, perhaps by lesions or tightly bound proteins, thenascent strands can anneal to one another, producing a four-waybranched or "chicken foot" structure. This junction can be cleavedby Holliday junction endonuclease RuvC in vivo (40). Fork regressionserves two important purposes in DNA repair (18): (i) it reconvertsparental template DNA in the fork region into a double strandso that excision repair can remove replication-blocking lesions,and (ii) it allows a template switch reaction so that one nascentstrand can provide a template for the other, overcoming blocksto replication on the leading strand parental template. As bothRuvAB and RecG have been implicated in the regression reaction(34, 35, 41) along with RecJ/RecQ (9), it is possible that RadAplays a similar and partially redundant role in the processingof regressed forks that accounts for its effects on DNA damagesurvival. One explanation for the suppressive effect that radAmutations have on the sensitivity of recA and recB mutants toperoxide and HU is that RadA converts damaged or stalled forksinto double-strand breaks, which recA and recB mutants are unableto process. In the absence of RadA, these lesions can be channeledinto other repair pathways. Such an effect may also explainsimilar suppressive effects of various Bacillus subtilis recombinationmutants with mutation in radA/sms (5).

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FIG. 4. Fork regression and repair. Lesions can stall DNA polymerase. Fork regression catalyzed by RecG or RuvAB helicase activity can move the lesion into double-strand DNA, where it can be repaired. 5' to 3' degradation of the lagging strand by RecJ/RecQ may facilitate repair when the lagging strand has moved ahead of the leading strand. The regressed fork forms a Holliday junction, which can be cleaved by RuvC. Double-strand break repair can then restore integrity of the fork. Alternatively, RecBCD degradation of the double-strand arm or reversed branch migration can restore a fork structure.

RadA has an unusual motif structure, with a putative Zn fingerN-terminal region, a region similar to RecA, including WalkerA and Walker B boxes conserved in ATPases, and a C-terminaldomain similar to that of Lon protease. Despite its similarityto RecA, we have no evidence that RadA can replace the RecAprotein; on the contrary, most RadA-dependent genetic functionsrequire RecA as well. In this way, RadA resembles the familyof Rad51 (and RecA-related) paralogs in eukaryotes that functionin concert with the Rad51 strand exchange protein (which playsa role comparable to RecA in prokaryotes) (33). In Saccharomycescerevisiae, two of the paralogs, Rad55 and Rad57, act as "mediator"proteins to load Rad51 onto presynaptic filaments in vitro (47).The Dmc1 protein, also homologous to Rad51, is a meiosis-specificfactor that works in concert with Rad51 to promote meiotic recombination(3) and has weak strand exchange properties itself (17, 19,38). In sequence comparisons, RadA and the eukaryotic Rad51-relatedproteins show the most extensive similarity to Dmc1. Sequencealignment of the vertebrate Dmc1 protein extends through theputative Zn finger region of the RadA protein, with two of thefour cysteines conserved and the other two replaced by lysinein the Dmc1 sequence (Fig. 5); two flanking invariant glycinesare also shared between RadA and Dmc1.

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FIG. 5. Alignment of E. coli RadA/Sms with human Dmc1 showing similarity through the putative Zn finger region. Arrows indicate the conserved cysteines of the Zn finger.

Zn fingers can act as motifs promoting DNA binding or proteininteractions (2). The function of RadA's putative Zn fingeris important in repair, since the original radA100 mutationresults in a cysteine-to-tyrosine change at one of the invariantresidues (46). In our DNA damage survival assays, the radA100mutant was as sensitive as two null mutants in radA to the alkylatingagent MMS. However, in assays of survival after exposure toother agents, the radA100 mutant behaved differently than thenull mutants. Unlike the null mutants, the radA100 mutant wasnot sensitive to the DNA breakage antibiotic phleomycin. Moreover,the radA100 mutant showed extreme sensitivity to the replicationinhibitor HU, whereas neither null mutant was appreciably sensitive,suggesting that RadA processing in the absence of the Zn fingeris worse for the HU-exposed cell than the absence of RadA altogether.These results must mean that Zn finger is essential for onlyone of multiple functions of the RadA protein, which is notunexpected given its predicted multiple-domain structure.

The role of the Lon protease domain of RadA is unknown. Althoughmany radA/sms open reading frames are annotated in some genomedatabases as encoding "probable" or "predicted" ATP-dependentproteases, no protease activity has ever been experimentallydemonstrated for RadA/Sms. The active-site serine of the comparableLon domain is converted to alanine in many of the RadA bacterialforms (although E. coli RadA/Sms does retain the active-siteserine), which in our opinion makes its improbable that a Lon-likeprotease activity contributes to its biological function. Assaysdetecting Lon protease have failed to demonstrate protease activityof purified RadA protein (A. Long, S. T. Lovett, and L. Hedstom,unpublished results). However, this domain is conserved amongthe RadA/Sms family, and truncations of this domain fail tocomplement radA (D. Resnicow and S. T. Lovett, unpublished results).This suggests that the Lon protease domain of RadA/Sms playsan important function, perhaps by protein or nucleic acid interactionssimilar to those of the Lon protease.

ACKNOWLEDGMENTS

This work was supported by U.S. Public Health Service grantsT32 GM07122 (to C.E.B. and C.J.S.) and RO1 GM51753 from theNational Institute of General Medical Sciences.

We thank M. Berlyn of the E. coli genetic stock center and A.J. Clark, R. Kolodner, R. G. Lloyd, B. Michel, N. Sargentini,and B. Wanner for providing strains used in this study. We acknowledgeD. Resnicow and B. Levinson for their work on the complementationanalysis and cloning of radA mutants.

FOOTNOTES

* Corresponding author. Mailing address: Rosenstiel Basic Medical Sciences Research Center MS029, Brandeis University, Waltham, MA 02454-9110. Phone: (781) 736-2497. Fax: (781) 736-2405. E-mail: lovett{at}brandeis.edu.

Present address: Chemical Abstracts Services, Columbus, OH 43202-1505.

REFERENCES

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