Localization of recombination proteins and Srs2 reveals anti-recombinase function in vivo

Abstract

Homologous recombination (HR), although an important DNA repair mechanism, is dangerous to the cell if improperly regulated. The Srs2 “anti-recombinase” restricts HR by disassembling the Rad51 nucleoprotein filament, an intermediate preceding the exchange of homologous DNA strands. Here, we cytologically characterize Srs2 function in vivo and describe a novel mechanism for regulating the initiation of HR. We find that Srs2 is recruited separately to replication and repair centers and identify the genetic requirements for recruitment. In the absence of Srs2 activity, Rad51 foci accumulate, and surprisingly, can form in the absence of Rad52 mediation. However, these Rad51 foci do not represent repair-proficient filaments, as determined by recombination assays. Antagonistic roles for Rad52 and Srs2 in Rad51 filament formation are also observed in vitro. Furthermore, we provide evidence that Srs2 removes Rad51 indiscriminately from DNA, while the Rad52 protein coordinates appropriate filament reformation. This constant breakdown and rebuilding of filaments may act as a stringent quality control mechanism during HR.

Remodeling of the Rad51 DNA Strand-Exchange Protein by the Srs2 Helicase

“…Consistent with this suggestion, Rothstein and colleagues (Burgess et al 2009) recently demonstrated that during mitosis the in vivo depletion of Srs2 permits Rad54, thus possibly Rad51, loading onto chromatin, even in the absence of Rad52, indicating that Srs2 depletion reduces the requirement for Rad52. Moreover, the deletion of the SRS2 gene in mitosis suppresses DNA damage defect of the rad55 or rad57 mutants (Liu et al 2011).…”

“…This might be simply due to delayed repair events. Moreover, overexpression of Srs2 decreases the Rad54-GFP that binds to DSBs (Burgess et al 2009), which indirectly supports this idea that Srs2 disrupts Rad51 on the mitotic DSB site, given that Rad54 binds to Rad51 ensembles (Colavito et al 2009). For this study, we combined cytological characterization of chromosome spreads and genetic overexpression of Srs2 during meiosis to demonstrate that Srs2 could disrupt Rad51 filaments on chromosomes in vivo.…”

“…Cytological studies of an srs2 deletion strain indicated that ionizing radiation (IR)-induced Rad54-GFP foci increase in the srs2 mutant relative to wild-type cells (Burgess et al 2009), suggesting a postassembly role for Srs2 in regulating Rad51 filaments. However, other scenarios can explain the persistence of these Rad54-GFP foci.…”

Unloading of homologous recombination factors is required for restoring double‐stranded DNA at damage repair loci

“…At the C‐terminus, Srs2 contains a variety of regulatory motifs, which are modulated through post‐translational modifications and/or required for the interactions of Srs2 with other proteins, including PCNA, and these are important for its role at replication forks (Papouli et al , 2005; Pfander et al , 2005; Burgess et al , 2009) and regulation of the D‐loop extension (Burkovics et al , 2013). However, most of the C‐terminus was not required for the role of Srs2 in DSB repair via de novo telomere addition, BIR and SSA (Fig 8A–D).…”

“…The Srs2 helicase inhibits HR machinery by disassembling Rad51 filament and reducing DNA extension, as demonstrated in vitro (Burkovics et al , 2013; Krejci et al , 2003; Veaute et al , 2003). This function is believed to be important for repression of excessive recombination, particularly at replication forks where Srs2 is recruited and regulated through its C‐terminal domain (Papouli et al , 2005; Pfander et al , 2005; Burgess et al , 2009). Loss of Srs2 results in a paradoxical phenotype.…”