SUMO-Chain-Regulated Proteasomal Degradation Timing Exemplified in DNA Replication Initiation.

Similar to ubiquitin, SUMO forms chains, but the identity of SUMO-chain-modified factors and the purpose of this modification remain largely unknown. Here, we identify the budding yeast SUMO protease Ulp2, able to disassemble SUMO chains, as a DDK interactor enriched at replication origins that promotes DNA replication initiation. Replication-engaged DDK is SUMOylated on chromatin, becoming a degradation-prone substrate when Ulp2 no longer protects it against SUMO chain assembly. Specifically, SUMO chains channel DDK for SUMO-targeted ubiquitin ligase Slx5/Slx8-mediated and Cdc48 segregase-assisted proteasomal degradation. Importantly, the SUMOylation-defective ddk-KR mutant rescues inefficient replication onset and MCM activation in cells lacking Ulp2, suggesting that SUMO chains time DDK degradation. Using two unbiased proteomic approaches, we further identify subunits of the MCM helicase and other factors as SUMO-chain-modified degradation-prone substrates of Ulp2 and Slx5/Slx8. We thus propose SUMO-chain/Ulp2-protease-regulated proteasomal degradation as a mechanism that times the availability of functionally engaged SUMO-modified protein pools during replication and beyond.

DNA bending facilitates the error-free DNA damage tolerance pathway and upholds genome integrity.

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.

DNA damage checkpoint and recombinational repair differentially affect the replication stress tolerance of Smc6 mutants.

DNA damage checkpoint and recombinational repair are both important for cell survival of replication stress. Because these two processes influence each other, isolation of their respective contributions is challenging. Research in budding yeast shows that removal of the DNA helicase Mph1 improves survival of cells with defective Smc5/6 complex under replication stress. mph1 is known to reduce the levels of recombination intermediates in smc6 mutants. Here, we show that mph1 also hyperactivates the Mec1 checkpoint. We dissect the effects of recombination regulation and checkpoint hyperactivation by altering the checkpoint circuitry to enhance checkpoint signaling without reducing recombination intermediate levels. We show that these approaches, similar to mph1, lead to better survival of smc6 cells upon transient replication stress, likely by ameliorating replication and chromosomal segregation defects. Unlike mph1, however, they do not suppress smc6 sensitivity to chronic stress. Conversely, reducing the checkpoint response does not impair survival of smc6 mph1 mutants under chronic stress. These results suggest a two-phase model in which smc6 mutant survival upon transient replication stress can be improved by enhancing Mec1 checkpoint signaling, whereas smc6 sensitivity to chronic stress can be overcome by reducing recombination intermediates.

The Smc5-Smc6 complex regulates recombination at centromeric regions and affects kinetochore protein sumoylation during normal growth.

The Smc5-Smc6 complex in Saccharomyces cerevisiae is both essential for growth and important for coping with genotoxic stress. While it facilitates damage tolerance throughout the genome under genotoxin treatment, its function during unperturbed growth is mainly documented for repetitive DNA sequence maintenance. Here we provide physical and genetic evidence showing that the Smc5-Smc6 complex regulates recombination at non-repetitive loci such as centromeres in the absence of DNA damaging agents. Mutating Smc6 results in the accumulation of recombination intermediates at centromeres and other unique sequences as assayed by 2D gel analysis. In addition, smc6 mutant cells exhibit increased levels of Rad52 foci that co-localize with centromere markers. A rad52 mutation that decreases centromeric, but not overall, levels of Rad52 foci in smc6 mutants suppresses the nocodazole sensitivity of these cells, suggesting that the Smc6-mediated regulation of recombination at centromeric regions impacts centromere-related functions. In addition to influencing recombination, the SUMO ligase subunit of the Smc5-Smc6 complex promotes the sumoylation of two kinetochore proteins and affects mitotic spindles. These results suggest that the Smc5-Smc6 complex regulates both recombination and kinetochore sumoylation to facilitate chromosomal maintenance during growth.

The Saccharomyces cerevisiae Esc2 and Smc5-6 proteins promote sister chromatid junction-mediated intra-S repair.

Recombination is important for DNA repair, but it can also contribute to genome rearrangements. RecQ helicases, including yeast Sgs1 and human BLM, safeguard genome integrity through their functions in DNA recombination. Sgs1 prevents the accumulation of Rad51-dependent sister chromatid junctions at damaged replication forks, and its functionality seems to be regulated by Ubc9- and Mms21-dependent sumoylation. We show that mutations in Smc5-6 and Esc2 also lead to an accumulation of recombinogenic structures at damaged replication forks. Because Smc5-6 is sumoylated in an Mms21-dependent manner, this finding suggests that Smc5-6 may be a crucial target of Mms21 implicated in this process. Our data reveal that Smc5-6 and Esc2 are required to tolerate DNA damage and that their functionality is critical in genotoxic conditions in the absence of Sgs1. As reported previously for Sgs1 and Smc5-6, we find that Esc2 physically interacts with Ubc9 and SUMO. This interaction is correlated with the ability of Esc2 to promote DNA damage tolerance. Collectively, these data suggest that Esc2 and Smc5-6 act in concert with Sgs1 to prevent the accumulation of recombinogenic structures at damaged replication forks, likely by integrating sumoylation activities to regulate the repair pathways in response to damaged DNA.