The Slx5-Slx8 Complex Affects Sumoylation of DNA Repair Proteins and Negatively Regulates Recombination

Abstract

Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized to replication centers. In addition, like SUMO E2 mutants, slx8⌬ mutants exhibited clonal lethality, which was due to the overamplification of 2m, an extrachromosomal plasmid. Interestingly, in both SUMO E2 and slx8⌬ mutants, clonal lethality was rescued by deleting genes required for Rad51-independent recombination but not those involved in Rad51-dependent events. These results suggest that sumoylation negatively regulates Rad51-independent recombination, and indeed, the Slx5-Slx8 complex affected the sumoylation of several enzymes involved in early steps of Rad51-independent recombination. We propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins.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 (or HEX3) 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 and SLX8 were subsequently shown to be required for the viability or fitness of many other strains with mutations that affect genomic integrity (29,31,40). Particularly, these genes exhibit extensive interactions with genes involved in replication or replication fork stability, such as RAD27, POL32, ELG1, and DBF2, suggesting a role for these genes in replication and/or repair (31). Consistent with this view, the deletion of SLX5 or SLX8 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 (29,47,48). 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 ...

Nuclear organization in genome stability: SUMO connections

“…Moreover, SUMO is implicated in the maintenance of rDNA stability by suppressing illegitimate recombination among the yeast rDNA repeats [22]. Consistent with this widespread effect on genomic integrity, it was found that many other proteins involved in DNA repair are sumoylated, notably PCNA, Rad52, Rfa1, Rfa2, Sgs1 helicase, as well as its human homologues, BLM and WRN [23][24][25][26][27][28]. Many of these proteins are found either transiently or stably associated with PML-NBs in mammalian cells in response to DNA damage (reviewed in [20]).…”

“…Mutations in genes involved in the SUMO pathway, such as Ubc9, Mms21, Ulp1, Slx5 and Slx8, render cells sensitive to DNA damage, and many proteins involved in the maintenance of genome stability have been shown to become sumoylated during the damage response [23][24][25][26][27][28]. Specifically, it has been shown that the SUMO pathway plays an important role in homologous recombination and the resumption of stalled/collapsed replication forks [27,[36][37][38].…”

“…Moreover, it contributes to the maintenance of telomeric repeats by recombination in telomerasedeficient cells [21]. Based on the structural homology Resolution of recombination intermediate [27] Mms21 SUMO E3 ligase Sister chromatid exchange [37] Resolution of recombination intermediate [27] ALT maintenance of telomeres [21] Siz1 SUMO E3 ligase PCNA sumoylation (recruit Srs2) [44,45] Ulp1 SUMO protease Suppression of recombination during replication [56] Slx5/Slx8 STUbL Maintenance of replication fork stability [25,53] Alternative lengthening of telomeres [59] www.cell-research.com | Cell Research…”

“…Slx5 and Slx8 form a heterodimeric Ub ligase that specifically recognizes sumoylated substrate through SUMO interaction motifs (SIMs). Both Slx5 and Slx8 harbor such SIMs, while Slx8 bears a RING finger domain with Ub ligase activity [25,[46][47][48][49][50][51]. The Slx5 protein physically interacts with components of the proteasome [52], and deletion of either SLX5 or SLX8 leads to the accumulation of sumoylated proteins [47,51].…”

Opening the DNA repair toolbox: Localization of DNA double strand breaks to the nuclear periphery

“…55 This is associated with an increase in the number and duration of Rad52 foci during S-phase, suggesting that Slx5/8 may act to suppress damage that occurs during replication and to channel that damage into faster repair pathways. The relevant substrates for Slx5/8 are not currently known, though it has been suggested that they may target the degradation of proteins involved in Rad51-independent recombination pathways.…”