DNA helicase Srs2 disrupts the Rad51 presynaptic filament

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.

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…Thus, the 3 -end of the leading strand is elongated beyond the lesion on the lagging strand as template. Srs2 could discourage D-loop formation acting at a presynaptic stage (Krejci et al, 2003;Veaute et al, 2003) or a postsynaptic stage by disrupting the D-loop to relocate the formerly arrested, now elongated, strand back onto its original template. This is similar to its function in promoting SDSA in double-strand break repair ( Figure 5E; Ira et al, 2003;Dupaigne et al, 2008).…”

“…Monoubiquitinated PCNA can be polyubiquitinated by the Rad5-Ubc13-Mms2 complex (Hofmann and Pickart, 1999) to allow error-free mechanisms (Torres-Ramos et al, 2002). Alternatively, K164 of PCNA can be SUMOylated to regulate recombination at the fork (Pfander et al, 2005;Moldovan et al, 2006) via a direct interaction with Srs2 (Pfander et al, 2005), a 3 -5 DNA helicase (Rong and Klein, 1993), thought to exert a negative control on HR (Krejci et al, 2003;Veaute et al, 2003).…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…Genetic data indicate that Srs2 negatively regulates recombination [70,71] possibly by reversal of intermediate recombination structures [72][73][74][75]. Indeed, the DNA strand exchange mediated by Rad51 is inhibited by Srs2 through disruption of the Rad51-ssDNA filaments [76,77], and it turns out that sumoylated PCNA has increased affinity for Srs2 [78,79] and represses the Rad52-dependent recombination pathway [80]. These observations collectively support the hypothesis that Srs2 serves as a molecular switch between homologous recombination and DDT [6], and further confirm that the sensor for this switch is the state of PCNA modification.…”

Multiple Genetic Pathways Involving the Caenorhabditis elegans Bloom's Syndrome Genes him-6, rad-51, and top-3 Are Needed To Maintain Genome Stability in the Germ Line

“…The phenotype of sgs1 mutants includes a slight reduction in growth rate, a 10-fold increase in chromosome missegregation, an elevated mitotic recombination frequency, a high sensitivity to methyl methanesulfonate and hydroxyurea (HU) (15,66), and premature aging as a consequence of an increased number of extrachromosomal ribosomal DNA circles in the cell (17,51,60,61). Sgs1p acts redundantly with the budding yeast helicase Srs2p, which has been shown to inhibit loading of Rad51p onto single-stranded DNA (ssDNA) in vitro (33,56). In addition, the sgs1 mutants display some meiotic defects such as delayed meiotic prophase and defective chromosome synapsis (16,45).…”