The maintenance of genome stability is critical for cell survival and for the proper development of an organism. It requires a network of genes that must be coordinated during various DNA metabolic processes. In the budding yeast Saccharomyces cerevisiae
, the SLX5
) and SLX8
genes are among the guardians of genomic stability. Originally identified as genes required for the viability of cells lacking Sgs1 (the homolog of human BLM and WRN), both SLX5
were subsequently shown to be required for the viability or fitness of many other strains with mutations that affect genomic integrity (29
). Particularly, these genes exhibit extensive interactions with genes involved in replication or replication fork stability, such as RAD27
, and DBF2
, suggesting a role for these genes in replication and/or repair (31
). Consistent with this view, the deletion of SLX5
leads to a 150- to 200-fold increase in gross chromosomal rearrangement and a 4-fold increase in spontaneous mutation rates (48
). These findings point to the importance of Slx5 and Slx8 in the maintenance of genome stability.
Based on biochemical and genetic evidence, Slx5 and Slx8 proteins function as a complex (29
). Several recent studies suggest that the Slx5-Slx8 complex participates in the sumoylation pathway, which entails the addition of a small ubiquitin-like modifier (SUMO) to the target proteins. Sumoylation requires the sequential action of E1, E2, and E3 enzymes; while only a single E1 and a single E2 exist in previously studied organisms, multiple E3s have been found and are thought to confer substrate specificities (19
). It was shown previously that mutations in Slx5 or Slx8, as well as mutations in several proteins involved in the sumoylation pathway, can restore transcription in a mot1
transcriptional regulator mutant (45
). The same study showed that slx5
Δ and slx8
Δ mutants are synthetic lethal or sick with mutations in SUMO (SMT3
), SUMO E1 (AOS1/UBA2
), SUMO E2 (UBC9
), and two SUMO E3s (SIZ1
). These genetic data are consistent with findings in earlier reports that Slx5 interacts with SUMO in two-hybrid assays (13
). All of these results support a role for the Slx5-Slx8 complex in sumoylation. However, it is not clear if such a role is specific to the transcriptional functions of the complex or if it is related to the functions of the complex in genome stability.
Sumoylation and the reverse process, desumoylation, are tightly linked with genomic stability. The mutation of the enzymes or the regulators of the sumoylation and desumoylation pathway (called the SUMO pathway for simplicity) can lead to sensitivity to DNA-damaging agents, recombination defects, and the disruption of chromosomal structures (reviewed in references 19
). In higher eukaryotes, defects in the SUMO pathway can lead to cancer and developmental abnormalities (reviewed in reference 36
). Recent studies, including several using proteomic and genomic approaches, revealed a dozen SUMO substrates, such as the replicative clamp (proliferating cell nuclear antigen [PCNA]) and the central recombination protein Rad52, involved in various DNA metabolism processes (35
). Further examination of the effects of sumoylation has revealed several mechanisms by which the SUMO pathway can regulate genome stability. Among these mechanisms, two entail the regulation of homologous recombination during replication. In one, the Siz1-dependent sumoylation of PCNA recruits the antirecombinase Srs2, which can disassemble Rad51 filaments from single-stranded DNA (ssDNA) and thus disfavors homologous recombination (reviewed in reference 36
). In the other, the SUMO E3 Mms21 mediates sumoylation to counteract the accumulation of Rad51-dependent recombinogenic structures at damaged replication forks (2
While both the aforementioned regulatory mechanisms target Rad51-dependent recombination processes, it is unclear if any regulation is imposed on Rad51-independent recombination processes, such as single-strand annealing (SSA) and a subset of break-induced replication (BIR) pathways. SSA refers to the annealing of two 3′ ssDNA tails containing complementary sequences; this reaction is catalyzed by the strand annealing activity of Rad52 and can be facilitated by Rad59 when the repeat length is short (reviewed in references 11
). During BIR, ssDNA from a break site anneals with a homologous sequence and a DNA replication fork is established. Although BIR is efficiently mediated by Rad51, it may also occur in the absence of Rad51. Both SSA and BIR are useful for the repair of damaged chromosomes or the restarting of replication forks, yet they can be mutagenic by generating deletions, amplifications, and translocations (reviewed in references 11
). Therefore, it is conceivable that both SSA and BIR require regulation to minimize their deleterious effects, and such regulation would be critical for genomic stability.
In this report, we show that the absence of the Slx5-Slx8 complex leads to an increase of both Rad51-dependent and -independent recombination. While such increases may be explained by a higher incidence of DNA lesions during replication, our results suggest that the Slx5-Slx8 complex confers an additional negative regulation of Rad51-independent recombination and modulates the sumoylation of several enzymes in this pathway. Our results also suggest that the regulation of Rad51-independent recombination by the Slx5-Slx8 complex is particularly important for the stability of repetitive sequences and for the maintenance of extrachromosomal DNA.