No abstract

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.

Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G 2 /M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded(ss)DNAdependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1⌬ or rad54⌬ mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.DNA helicases perform important functions in various chromosomal transactions, including replication, repair, recombination, and transcription (1, 2). These proteins utilize the chemical energy from the hydrolysis of a nucleoside triphosphate to dissociate DNA structures and nucleoprotein complexes. Interestingly, mutations in several DNA helicases are involved in the pathogenesis of human diseases. For instance, mutations in the XPB and XPD helicases, which constitute subunits of the transcription factor TFIIH that has a dual role in nucleotide excision repair, lead to the cancer prone syndrome xeroderma pigmentosum (3). Furthermore, mutations in the BLM, WRN, and RecQ4 proteins, members of the RecQ helicase family, cause the cancer-prone Bloom, Werner, and RothmundThomson syndromes, respectively (4, 5).We are interested in the biology of various DNA helicases that influence homologous recombination and DNA repair processes. One such helicase is encoded by the Saccharomyces cerevisiae SRS2 gene, altered forms of which were first described as either suppressors of the DNA damage sensitivity of rad6 and rad18 mutants (6) or as hyperrecombination mutants (7). Detailed genetic analyses have shown that a major function of SRS2 is to attenuate homologous recombination activity to allow for the channeling of certain DNA lesions into the RAD6/ RAD18-mediated postreplication repair pathway (8, 9). Accordingly, srs2 mutants are sensitive to DNA damaging agents and show a hyperrecombination phenotype. Genetic deletion of the RAD51 or RAD52, key members of the RAD52 epis...

We have determined the nucleotide sequence of the dihydrofolate reductase-thymidylate synthetase (DHFR-TS) gene of the protozoan parasite Leishmania major (dihydrofolate reductase, EC 1.5.1.3 and thymidylate synthase, EC 2.1.1.45). The DHFR-TS protein is encoded by a single 1560-base-pair open reading frame within genomic DNA, in contrast to vertebrate DHFRs or mouse and phage T4 TSs, which contain intervening sequences. Comparisons of the DHFR-TS sequence with DHFR and TS sequences of other organisms indicate that (i) the order of enzymatic activities within the bifunctional polypeptide chain is DHFR followed by TS, (ii) the Leishmania bifunctional DHFR-TS evolved independently and not through a phage T4-related intermediate, and (iii) the rate ofevolution of both the DHFR and TS domains has not detectably changed despite the acquisition of new functional properties by the bifunctional enzyme. The Leishmania gene is 86% G+C in the third codon position, in contrast to genes of the parasite Plasmodium falciparum, which exhibit an opposite bias toward A+T. The DHFR-TS locus is encoded within a region of DNA amplified in methotrexate-resistant lines, as previously proposed.