Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing.

In many eubacteria, coexpression of recX with recA is essential for attenuation of the deleterious effects of recA overexpression; however, the molecular mechanism has remained enigmatic. Here T he past several years have seen a considerable progress in our understanding of the central role of Escherichia coli RecA protein in homologous recombination, DNA repair, restoration of stalled replication forks, induction of SOS response, and mutagenesis (1, 2). The much-studied homologous recombination process in vitro is the three-strand exchange reaction between circular single-and linear double-stranded DNA (1-3). In this model, the reaction proceeds in three sequential phases: (i) The presynaptic polymerization of RecA protein on singlestranded DNA; (ii) synapsis, the homologous alignment of nucleoprotein filament with linear double-stranded DNA; and (iii) unidirectional strand exchange (1-3). The mechanistic aspects of homologous recombination promoted by the prototype E. coli RecA protein may arguably be the best understood, but understanding its counterparts from other organisms will be essential to establish the generality of the phenomenon. To this end, we have described the biochemical characterization and x-ray structure of M. tuberculosis RecA (4-6).In some eubacteria, recX is located on the same coding strand downstream of recA (7). In Streptomyces lividans, Mycobacterium smegmatis, Mycobacterium tuberculosis, Pseudomonas aeruginosa, or Thiobacillus ferrooxidans, the ORFs of recA and recX overlap and the two genes are cotranscribed (7-12). It is known that overexpression of recA in recX mutants of S. lividans, M. smegmatis, or P. aeruginosa, but not mutant RecA, lead to induction of deleterious effects (8, 10, 13). However, the molecular mechanisms by which recX attenuates the deleterious effects induced by recA overexpression has remained unknown. Using M. tuberculosis as a model, we explored the mechanism by which RecA is regulated by RecX. Here, we show that RecX interacts directly with RecA in vitro and in vivo resulting in suppression of ATPase and strand exchange, processes that are central to homologous recombination. The negative regulation of RecA by RecX implies that RecX might act as an antirecombinase to quell inappropriate recombinational repair during normal DNA metabolism. (14) and their concentrations determined as described (15). Negatively supercoiled (form I) and circular single-stranded M13 DNA (ssDNA) was prepared as described (16). The concentrations are expressed in moles of nucleotide residues. Materials and Methods Purification of RecX. E. coli BL21(DE3)[pLysS] strain harboringM. tuberculosis recX gene on plasmid pET15b was cultured in 1 liter of LB medium containing 50 g͞ml ampicillin and 34 g͞ml chloramphenicol at 37°C. At mid-exponential phase (A 600 ϭ 0.4), recX expression was induced by adding isopropyl ␤-Dthiogalactoside (IPTG) to a final concentration of 0.5 mM and incubated for 4 h. All subsequent steps were performed at 4°C unless indicated otherwise. Cell paste (8 g) was...

Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination. Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants. Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2. ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard. Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation. In its helicase action, Srs2 prefers substrates with a 3 ss overhang, and ϳ10 bases of 3 overhanging DNA is needed for efficient targeting of Srs2 to the substrate. Even though a 3 overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein. The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the singlestrand DNA-binding factor replication protein A.DNA helicases are ubiquitous among prokaryotes and eukaryotes. These enzymes utilize the free energy derived from the hydrolysis of a nucleoside triphosphate to disrupt the hydrogen bonds that bind the complementary strands of duplex DNA. DNA helicases play an essential role in virtually every aspect of nucleic acid metabolism, including transcription, replication, recombination, and repair (reviewed in 1-3).We focus on the mechanism of homologous recombination in eukaryotes and are interested in the biology of DNA helicases that influence recombination events. One such helicase is encoded by the Saccharomyces cerevisiae SRS2 1 (Suppressor of RAD Six-screen mutant 2) gene. Srs2 protein belongs to the SF1 helicase family and contains regions of similarity to the bacterial UvrD, Rep, and PcrA helicases (4). A mutant form of SRS2 was initially identified as a suppressor of the radiationsensitivity of rad6 and rad18 mutants that are defective in post-replicative DNA repair (4, 5). Subsequent studies revealed that suppression of rad6 and rad18 mutations by the srs2 mutation is due to heightened recombination mediated by the RAD52 epistasis group of genes (6). Consistent with this observation, srs2 mutants show a hyper-recombination phenotype (7,8). Together, these genetic observations indicate that SRS2 negatively regulates RAD52-mediated homologous recombination. It has been suggested that by antagonizing the recombination machinery, Srs2 ensures the channelling of DNA lesions that arise during DNA replication into the Rad6/Rad18-mediated repair reactions (4). Hence, in rad6 and rad18 mutants, repair of the replication-associated DNA lesions by recombination is inefficient unless SRS2 is inactivat...

Single-stranded DNA-binding proteins (SSB) play an important role in most aspects of DNA metabolism including DNA replication, repair, and recombination. We report here the identification and characterization of SSB proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis. Sequence comparison of M. smegmatis SSB revealed that it is homologous to M. tuberculosis SSB, except for a small spacer connecting the larger amino-terminal domain with the extreme carboxyl-terminal tail. The purified SSB proteins of mycobacteria bound single-stranded DNA with high affinity, and the association and dissociation constants were similar to that of the prototype SSB. The proteolytic signatures of free and bound forms of SSB proteins disclosed that DNA binding was associated with structural changes at the carboxyl-terminal domain. Significantly, SSB proteins from mycobacteria displayed high affinity for cognate RecA, whereas Escherichia coli SSB did not under comparable experimental conditions. Accordingly, SSB and RecA were coimmunoprecipitated from cell lysates, further supporting an interaction between these proteins in vivo. The carboxyl-terminal domain of M. smegmatis SSB, which is not essential for interaction with ssDNA, is the site of binding of its cognate RecA. These studies provide the first evidence for stable association of eubacterial SSB proteins with their cognate RecA, suggesting that these two proteins might function together during DNA repair and/or recombination.

Single-stranded DNA-binding proteins play an important role in homologous pairing and strand exchange promoted by the class of RecA-like proteins. It is presumed that SSB facilitates binding of RecA on to ssDNA by melting secondary structure, but direct physical evidence for the disruption of secondary structure by either SSB or RecA is still lacking. Using a series of oligonucleotides with increasing amounts of secondary structure, we show that stem loop structures impede contiguous binding of RecA and affect the rate of ATP hydrolysis. The electrophoretic mobility shift of a ternary complex of SSB-DNA-RecA and a binary complex of SSB-DNA are similar; however, the mechanism remains obscure. Binding of RecA on to stem loop is rapid in the presence of SSB or ATPgammaS and renders the complex resistant to cleavage by HaeIII, to higher amounts of competitor DNA or low temperature. The elongation of RecA filament in a 5' to 3' direction is halted at the proximal end of the stem. Consequently, RecA nucleates at the loop and cooperative binding propagates the RecA filament down the stem causing its disruption. The pattern of modification of thymine residues in the loop region indicates that RecA monomer is the minimum binding unit. Together, these results suggest that SSB plays a novel role in ensuring the directionality of RecA polymerization across stem loop in ssDNA. These observations have fundamental implications on the role of SSB in multiple aspects of cellular DNA metabolism.