DNA in living cells is generally processed via the generation and the protection of single-stranded DNA involving the binding of ssDNA-binding proteins (SSBs). The studies of SSB-binding mode transition and cooperativity are therefore critical to many cellular processes like DNA repair and replication. However, only a few atomic force microscopy (AFM) investigations of ssDNA nucleoprotein filaments have been conducted so far. The point is that adsorption of ssDN A–SSB complexes on mica, necessary for AFM imaging, is not an easy task. Here, we addressed this issue by using spermidine as a binding agent. This trivalent cation induces a stronger adsorption on mica than divalent cations, which are commonly used by AFM users but are ineffective in the adsorption of ssDNA–SSB complexes. At low spermidine concentration (<0.3 mM), we obtained AFM images of ssDNA–SSB complexes (E. coli SSB, gp32 and yRPA) on mica at both low and high ionic strengths. In addition, partially or fully saturated nucleoprotein filaments were studied at various monovalent salt concentrations thus allowing the observation of SSB-binding mode transition. In association with conventional biochemical techniques, this work should make it possible to study the dynamics of DNA processes involving DNA–SSB complexes as intermediates by AFM.

Saccharomyces cerevisiae Srs2 helicase was shown to displace Rad51 in vitro upon translocation on single-stranded DNA. This activity is sufficient to account for its antirecombination effect and for the elimination of otherwise dead-end recombination intermediates. Roles for the helicase activity are yet unknown. Because cells lacking Srs2 show increased incidence of mitotic crossovers, it was postulated that Srs2 promotes synthesis-dependent strand annealing (SDSA) by unwinding the elongating invading strand from the donor strand. We report here that synthetic DNA structures that mimic D loops are good substrates for the Srs2 helicase activity, that Srs2 translocates on RPA-coated ssDNA, and, furthermore, that the helicase activity is largely stimulated by the presence of Rad51 nucleoprotein filaments on double-stranded DNA. These properties strongly support the idea that Srs2 actively prevents crossovers by promoting SDSA.

Saccharomyces cerevisiae Srs2 helicase plays at least two distinct functions. One is to prevent recombinational repair through its recruitment by sumoylated Proliferating Cell Nuclear Antigen (PCNA), evidenced in postreplication-repair deficient cells, and a second one is to eliminate potentially lethal intermediates formed by recombination proteins. Both actions are believed to involve the capacity of Srs2 to displace Rad51 upon translocation on single-stranded DNA (ssDNA), though a role of its helicase activity may be important to remove some toxic recombination structures. Here, we described two new mutants, srs2R1 and srs2R3, that have lost the ability to hinder recombinational repair in postreplication-repair mutants, but are still able to remove toxic recombination structures. Although the mutants present very similar phenotypes, the mutated proteins are differently affected in their biochemical activities. Srs2R1 has lost its capacity to interact with sumoylated PCNA while the biochemical activities of Srs2R3 are attenuated (ATPase, helicase, DNA binding and ability to displace Rad51 from ssDNA). In addition, crossover (CO) frequencies are increased in both mutants. The different roles of Srs2, in relation to its eventual recruitment by sumoylated PCNA, are discussed.