Cell Cycle-Specific Functions and Regulation of the DNA Nuclease SNM1B

[摘要] DNA replication is a fundamental cellular process that ensures the accurate duplication and transmission of genetic information.The replication machinery is frequently challenged by endogenous and exogenous sources of DNA lesions or barriers, which can interfere with replication fork progression.If stalled replication forks are not properly stabilized and restarted, they can collapse, resulting in the generation of DNA double-strand breaks (DSBs).Mis-repaired breaks lead to increased genome instability in the form of chromosomal deletions, amplifications, and aberrant rearrangements.These detrimental alterations characterize many human diseases and can contribute to malignant transformation and the progression of cancer.Therefore, the cell has evolved intricate mechanisms to promote the efficient resolution of replication stress.The repair of stalled or collapsed replication forks requires precise nucleolytic processing of DNA intermediate structures.However, the molecular details of these processing events are not well defined.We describe a critical role for the DNA nuclease, SNM1B, in the resolution of replication stress and uncover potential mechanisms for its regulation.We found that SNM1B is dispensable for the detection of stalled forks, as it localizes independently to sites of stress after early-response proteins.DNA fiber analyses and immunofluorescence assays also revealed that SNM1B is important for efficient replication restart and for preventing the accumulation of single-stranded DNA intermediates and DSBs at stalled forks.Using a chromosomally integrated substrate that induces site-specific replication fork arrest in mammalian cells, we showed that SNM1B is required for the recombination-mediated repair of DSBs after fork collapse.Together, these findings highlight the importance of SNM1B in accurately processing DNA intermediates and in preserving genomic integrity during DNA replication. DNA repair pathways are tightly controlled and coordinated within the cell cycle to safeguard genetic information.The regulation of nucleases is especially important as inappropriate DNA resection can lead to fragile DNA structures or mis-repaired substrates.Little is known about the regulation of the SNM1B nuclease; thus, we sought to identify the mechanisms underlying the control of its functions and stability within the cell cycle.We found that SNM1B protein levels are stabilized during S-phase in response to agents that induce replication stress and that SNM1B is phosphorylated on its C-terminus in G2-phase and mitosis.Investigations using non-phosphorylatable mutants demonstrated that SNM1B phosphorylation is dispensable for its localization to stalled forks, but may promote protein stability by preventing polyubiquitination during mitosis.Overall, these studies define a novel role for SNM1B in resolving stalled replication forks and provide insight into the regulatory mechanisms necessary for the prevention of replication-associated DNA damage.

[发布机构] University of Michigan

[效力级别] DNA Replication Stress

[关键词] DNA Repair;DNA Replication Stress;SNM1B Nuclease;Molecular;Cellular and Developmental Biology;Science;Cellular & Molecular Biology