The Srs2 anti-recombinase is a vital regulator of recombination activity in the cell. Although many aspects of its function have been described in vitro,
we undertook a cell biological approach to examine its intracellular organization and the consequences of disturbing its function in vivo. Using a fluorescently tagged Srs2, we visualized its localization to recombination foci and to replication forks in logarithmically growing cells (). The localization of Srs2 to recombination foci, surprisingly, does not require the presence of Rad51, the very protein it is known to dislodge from DNA and with which it interacts physically (Krejci et al., 2003
). Perhaps Srs2 senses the Rad51 filament indirectly through interactions with other members of the recombination machinery, through interactions with single-stranded DNA, or Srs2 is recruited specifically by modified forms of recombination proteins. In fact, we find that Siz1 is partially required for Srs2 recruitment to HR foci (), suggesting that sumoylation plays a role in this recruitment. However, in the absence of sumoylated Rad52 and Rad59, Srs2 recruitment is not impaired (unpublished data). Because the srs2
mutant lacking the SUMO-interaction domain does not show a measurable defect in localization to HR foci (srs2-ΔSIM
, Fig. S1 B), sumoylation of Srs2 itself may be one of the modifications important for its recruitment to HR foci. Possibly multiple proteins, both sumoylated and unsumoylated, may recruit Srs2 to HR foci. During S phase (cells with small- to mid-size buds), Srs2 appears as multiple small foci colocalizing with PCNA. Formation of these foci requires interaction with sumoylated PCNA because we observe no S phase Srs2 foci in mutants defective in PCNA sumoylation (pol30-RR
), or by mutating the Srs2 SIM domain (Fig. S1). These results support previous observations that Srs2 interacts preferentially with sumoylated PCNA and there is PCNASUMO
-dependent enrichment of Srs2 at replication forks (Papouli et al., 2005
). This cited report also showed that recombination proteins are increasingly recruited to replication forks in the absence of Srs2, and to a lesser extent, in the absence of PCNASUMO
. Likewise, we observe an increase in spontaneous HR foci in the pol30-RR
mutant, but find that Srs2 recruitment to HR foci is not impaired in this mutant (Figs. S1 and S2). This result suggests that when Srs2 is unable to be recruited to the replication fork directly, HR proteins can assemble there and can later recruit Srs2.
Although there is an increase in the number of spontaneous HR foci in the pol30-RR
mutant, there is an even larger accumulation of foci in the complete absence of Srs2. This accumulation is partially due to increased recruitment of HR proteins to replication forks (; Fig. S3 A; ). However, because there is no further induction of HR foci in srs2Δ
after HU addition, cells must retain some mechanism to inhibit HR at stalled replication forks even in the absence of Srs2 (). These observations suggest that there is still some restriction on recruitment of recombination proteins to forks other than Srs2, as long as replisome integrity is maintained. However, stalling replication forks in the absence of the Mec1 checkpoint kinase results in collapsed replication forks (Tercero and Diffley, 2001
), which then leads to strong induction of HR foci, both in the presence of Srs2 and even more so in its absence (). Together, these data imply that the recruitment of HR factors to intact replication forks is disfavored even in the absence of Srs2. Perhaps there are active mechanisms still in place under these conditions that restrict inappropriate HR. Alternatively, these differences may simply be due to the lower accumulation of ssDNA in the presence of stalled versus collapsed replication forks. In any case, these observations argue only a minor role for Srs2 in reversing HR intermediates at stalled forks.
Several lines of evidence suggest that Srs2 restricts inappropriate HR at multiple sites throughout the genome, and that unscheduled HR at the replication fork is probably not the only cause of the increased Rad51 and Rad54 focus levels in srs2Δ. First, colocalization data show that there are a number of Rad54 foci that are neither located at replication forks nor at sites of ssDNA in an srs2Δ (as determined by Rfa1 colocalization, ). Second, levels of Rad54 foci in the pol30 SUMO mutant (pol30-RR), which fails to recruit Srs2 specifically to replication forks, are not as high as in an srs2Δ (Fig. S3 A). Third, although srs2Δ rad54Δ double mutants are inviable, pol30-RR rad54Δ are viable (unpublished data), implying that the general role of Srs2 in recombination is the cause of lethality in rad54Δ. Thus, we suggest that Srs2 acts throughout the genome to restrict HR and that only a subset of its function is at replication forks.
An indication that HR foci form inappropriately in the absence of Srs2 comes from the observation that Rad51 and Rad54 foci can form in the absence of Rad52 if Srs2 is also absent. Furthermore, Rad51 and Rad54 colocalize to a similar extent in srs2Δ rad52Δ
double mutant as they do in wild-type cells, again indicating that these foci reflect similar structures (unpublished data). These foci are likely nonfunctional Rad51 filaments because srs2Δ rad52Δ
cells are as DNA damage sensitive as the rad52Δ
single mutant (), reflecting the crucial role for Rad52 in additional steps of HR. It is possible that some of these foci may be formed at structures that do not require HR, suggesting that in the absence of negative regulation by Srs2, Rad51 and Rad54 nucleate on sites that are not relevant to HR. Although it is not known whether Rad51 forms foci at dsDNA sites in vivo, Rad51 binds both single- and double-stranded DNA in vitro, with a slightly higher affinity for dsDNA (Shinohara et al., 1992
). It is of note that Srs2 dsDNA unwinding activity described by Dupaigne et al. (2008)
could act not only to displace Rad51 that is bound to dsDNA in legitimate HR structures, such as D-loops, but also to displace it from illegitimate dsDNA-Rad51 structures.We observe Rad51 foci at a low level in rad52Δ
cells but, unlike the foci formed in srs2Δ rad52Δ
double mutants, these aberrant foci are not inducible by gamma irradiation. Perhaps these foci form an aggregate or storage structure, as has been observed for RecA (Renzette et al., 2005
), or they are the result of binding to a DNA structure that is not formed by IR, such as dsDNA.
Rad54 stabilizes Rad51 bound to ssDNA, but also removes Rad51 from dsDNA (Kiianitsa et al., 2002
; Solinger et al., 2002
; Mazin et al., 2003
), so it is conceivable that some of the increased Rad54 foci in srs2Δ
cells may indicate increased Rad54 dsDNA translocase activity in the absence of Srs2. However, the majority of detectable Rad54 foci are associated with Rad51 and ssDNA (96% of Rad54 foci colocalize with Rad51 in wild-type, and nearly 80% of Rad54 foci have detectable RPA association). Therefore, the majority of Rad54 foci are likely the result of binding to Rad51, most often on ssDNA.
The decreased requirement for Rad52 during Rad51 focus formation in srs2Δ suggests that Srs2 and Rad52 act antagonistically in the formation of Rad51 filaments. Indeed, addition of Srs2 to the in vitro Rad51-mediated strand exchange reaction inhibits formation of Rad51 filaments, whereas Rad52 protein allows their formation even in the presence of Srs2 (). Unlike Rad52, the Rad55/57 heterodimer does not strongly promote in vitro filament formation in the presence of Srs2, suggesting that Rad55/57 has a different or less potent mediator function than Rad52 in driving Rad51 filament formation. Indeed, Rad51 focus formation in the srs2Δ rad52Δ double mutant is greatly delayed, suggesting that additional impediments to Rad51 nucleation remain (). An obvious candidate is the ssDNA binding protein RPA, which competes for Rad51 binding sites. This competition is relieved by Rad52, which mediates the efficient replacement of RPA by Rad51. However, in an srs2Δ rad52Δ double mutant, Rad51 foci can eventually assemble, at the time when the foci in wild-type cells start to disappear. This observation suggests that in the absence of Srs2 activity, Rad51 filaments can be assembled even without Rad52. Despite the fact that Rad51 foci are able to form under these conditions, these foci do not represent recombination-proficient filaments.
Likewise, partially defective rad52
alleles can be suppressed by overexpression of Rad51 or deletion of Srs2 (Milne and Weaver, 1993
; Kaytor et al., 1995
; Schild, 1995
), suggesting that the role of Rad52 in filament formation may be separable from its other recombination functions. Furthermore, in Schizosaccharomyces pombe
, the HR defects and damage sensitivity of deletion of rad22
, its RAD52
homologue, is completely suppressed by deletion of an Srs2 orthologue, fbh1
(Osman et al., 2005
). All of these observations lead to the conclusion that defects in Rad52 mediator functions can be overcome by making Rad51 nucleation more favorable.
It is well accepted that Srs2 reverses toxic recombination intermediates, but our results show that expression of Srs2 also results in the disassembly of Rad51–Rad54 complexes from an appropriate site for HR, i.e., an I-SceI–induced DSB (). Because we are not directly visualizing Rad51, it remained possible that the decrease in Rad54 foci at the DSB was due to decreased recruitment of Rad54 to Rad51 foci caused by Srs2 expression. However, this is not the case because Rad54–Rad51 colocalization is similar both in the presence and absence of Srs2 (). Furthermore, because a large percentage of Rad51 foci colocalize with Rad54 foci, it is very likely that we are detecting the dismantling of Rad51 foci by proxy. In fact, even if only the subset of Rad51 foci that are represented by Rad54 foci were showing this dramatic threefold decrease at DSB sites (), it still supports the conclusion that Srs2 can dismantle HR complexes at appropriate sites.
As shown in , disruption of HR complexes “elsewhere” is nearly maximal at basal Srs2 expression levels, whereas the DSB-localized complexes require increased expression. We also find that Rad52 is preferentially enriched at the DSB-localized Rad54 foci. These data suggest that more Srs2 protein is required to shift the equilibrium to dismantle Rad54 foci at a DSB, due to the Rad52-mediated forward reaction. Although the nature of the foci outside of the I-SceI break site (i.e., “elsewhere”) is not precisely known, a portion of these foci are likely spontaneous lesions that are also appropriate sites for HR. However, because Srs2 is well established to reverse toxic HR complexes (Gangloff et al., 2000
), in all likelihood a high percentage of these foci are inappropriate.
How does the cell distinguish between appropriate and inappropriate sites for reformation of HR complexes? Given the central role for Rad52 in HR and the wide variety of interactions that it displays, we propose that Rad52 is involved in this decision-making process. Accordingly, Rad52 localizes most frequently to the marked DSB and these Rad52 foci are largely unaffected by expression of Srs2 (). These studies have led us to a new model to explain how Srs2 affects the regulation of recombination in vivo. Srs2 removes Rad51 from DNA indiscriminately, dissociating inappropriate Rad51 filaments, as well as those at appropriate sites. Rad52 guides Rad51 filaments to reform at appropriate locations, effectively channeling the HR machinery into bona fide substrates. In mammalian cells, BRCA2 may serve this same function, as orthologous mediator activities have been ascribed to it (San Filippo et al., 2006
; Shivji et al., 2006
) and BRCA2 is thought to target hRad51 to sites of damage (Venkitaraman, 2002
). We suggest that this mechanism serves as a recombination quality control point through the wanton destruction of recombination complexes followed by directed rebuilding of suitable recombination intermediates. In addition, Srs2 interactions with sumoylated proteins, e.g., PCNA at replication forks, may target its anti-recombinase activity to critical genomic locations, thus ensuring the presence of this quality control mechanism.