HR is essential for genome maintenance and must be tightly controlled to avoid of loss of heterozygosity, chromosome translocations and gene deletions (1
). Recent studies have documented several examples of HR proteins and associated factors being post-translationally modified by the SUMO protein. In particular, defects in SUMOylation of Rad52, Rad59 and RPA result in genomic context-dependent changes in HR and chromosomal rearrangements (31
). Also, during S-phase, Srs2 (a helicase that blocks recombinational repair by disrupting Rad51 nucleoprotein filaments) is recruited to replication forks via interaction with SUMOylated PCNA (proliferating cell nuclear antigen), thereby preventing assembly of Rad51 nucleoprotein filaments on ssDNA associated with the replication forks (32
In this study we have strived to determine the effects of SUMOylation on the Rad52 protein. Using in vitro biochemical assays, we have found that Rad52 is SUMOylated in a manner that is stimulated by ssDNA and dependent on the carboxyl-terminal DNA binding domain. The stimulation is specific for ssDNA, as dsDNA has no effect on the level of SUMOylation. In addition, the CD analysis revealed that binding of ssDNA by Rad52 is accompanied by a conformational change. By replacing each of the SUMOylation-targeted lysines with arginine, we observed that increasing SUMOylation in the presence of ssDNA is directed to residue K253. In addition, we have found that RPA-bound, but not Rad51-bound, ssDNA promotes Rad52 SUMOylation, suggesting that Rad52 SUMOylation is favoured prior to the formation of Rad51 nucleoprotein filament during HR in cells. Significantly, while Rad52 SUMOylation has no effect on its oligomerization and interaction with RPA and Rad51, a small percentage (<10%) of SUMOylated Rad52 markedly decreases its affinity to ssDNA and dsDNA, and causes a reduction of its DNA annealing activity.
Collectively, our biochemical results suggest that upon binding to resected ssDNA tails coated with RPA, Rad52 undergoes conformational change that can promote its efficient SUMOylation. Because SUMOylation does not affect Rad52 interaction with RPA and Rad51 or its oligomerization, this modification does not appear to function by altering protein–protein interactions. Instead, our results suggest that SUMOylation attenuates Rad52 strand annealing activity and prompts its disassociation from DNA. This can provide a mechanism either for favouring appropriate pathways over others or for dynamic exchange of Rad52 on DNA. Consistently with previous reports (15
), we find that SUMOylation of Rad52 in vivo
suppresses rDNA recombination and favours single-strand annealing over gene conversion in direct repeat recombination at the LEU2
locus. In addition, we show that both the N-terminal (K43,44) and central (K253) SUMOylation sites contribute to these effects. Further, we show in this study that Rad52 SUMOylation reduces meiotic spore viability and appears to favour BIR events. While the mechanism for these effects remains to be determined, it may reflect a requirement of Rad52 dynamics in these pathways or a role of SUMO in facilitating events unique to these pathways.
Finally, it is noteworthy that the effects of SUMO on Rad52 activity are somewhat reminiscent of that on thymine-DNA glycosylase, SUMOylation of which induce its dissociation from the abasic site (33
). However, different from thymine-DNA glycosylase, Rad52 functions in concert with several additional mediator and recombination proteins, such that its regulation is more complex and defects in any single form of regulation can be buffered by other mechanisms. Our in vivo
results suggest that this is likely the case, as the lack of Rad52 SUMOylation does not dramatically affect several mitotic or meiotic recombination processes examined here. However, the observed alteration of recombination pathway usage as well as the changes of duration times of recombination foci in rad52
SUMOylation defective mutants indicate the modification of Rad52 can indeed influence recombination pathway choice or efficiency. An intriguing possibility is that SUMOylation, in conjunction with other Rad52 functions such as antagonizing Srs2 (34
), helps to fine-tune the efficiency of recombinational repair. In addition, as other recombination proteins and Rad52-interacting factors are also subject to SUMOylation, the presence and modification of these factors may collectively provide a quality control mechanism to direct HR pathway choice depending on substrate types and the chromosomal environment (36
). This work brings initial characterization of the role of SUMOylation during HR; additional studies will need to further our understanding of the underlying molecular mechanism.