Highlights d Relocation of collapsed forks to NPCs depends on sumoylation by Mms21/Nse2 d Targets of sumoylation important for relocation are RPA, Rad52, Rad59, and Smc5 d Resection mediated by Mre11, Exo1, and Sgs1 is required for relocation to the NPC d RPA sumoylation prevents Rad51 binding to collapsed forks before NPC anchoring
Background: Dpb11 is required for the initiation of DNA replication. The replication fork helicase is composed of Cdc45, Mcm2-7, and GINS. Results: Dpb11 recruits Cdc45 to Mcm2-7, and Dpb11 blocks GINS interaction with Mcm2-7. Dpb11 also binds to ssDNA, and this interaction releases Dpb11 from Mcm2-7. Conclusion: Dpb11 helps control assembly of the replication fork helicase. Significance: A mechanism for Dpb11 function is described.
The DNA damage response relies on protein modifications to elicit physiological changes required for coping with genotoxic conditions. Besides canonical DNA damage checkpointmediated phosphorylation, DNA damage-induced sumoylation has recently been shown to promote genotoxin survival. Crosstalk between these two pathways exists in both yeast and human cells. In particular, sumoylation is required for optimal checkpoint function, but the underlying mechanisms are not wellunderstood. To address this question, we examined the sumoylation of the first responder to DNA lesions, the ssDNA-binding protein complex replication protein A (RPA) in budding yeast (Saccharomyces cerevisiae). We delineated the sumoylation sites of the RPA large subunit, Rfa1 on the basis of previous and new mapping data. Findings using a sumoylation-defective Rfa1 mutant suggested that Rfa1 sumoylation acts in parallel with the 9-1-1 checkpoint complex to enhance the DNA damage checkpoint response. Mechanistically, sumoylated Rfa1 fostered an interaction with a checkpoint adaptor protein, Sgs1, and contributed to checkpoint kinase activation. Our results suggest that SUMO-based modulation of a DNA damage sensor positively influences the checkpoint response.
The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin in srs2 mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions.
The homologous recombination (HR) machinery plays multiple roles in genome maintenance. Best studied in the context of DNA double-stranded break (DSB) repair, recombination enzymes can cleave, pair, and unwind DNA molecules, and collaborate with regulatory proteins to execute multiple DNA processing steps before generating specific repair products. HR proteins also help to cope with problems arising from DNA replication, modulating impaired replication forks or filling DNA gaps. Given these important roles, it is not surprising that each HR step is subject to complex regulation to adjust repair efficiency and outcomes as well as to limit toxic intermediates. Recent studies have revealed intricate regulation of all steps of HR by the protein modifier SUMO, which has been increasingly recognized for its broad influence in nuclear functions. This review aims to connect established roles of SUMO with its newly identified effects on recombinational repair and stimulate further thought on many unanswered questions.Homologous recombination is critical for several aspects of life, ranging from DNA repair and genome duplication to gamete production. Our understanding of HR pathways has benefited from a combination of assay systems. In cells, the generation of a defined DSB allows quantitative assessment of the status of the broken DNA molecules and the repair proteins at a temporal resolution, as well as determination of the genetic requirement for each step of repair (Haber 2016). Extensive biochemical analyses and more recently single molecule experiments have further defined the activities of HR enzymes and elucidated how they can collaborate in multiple HR steps. Several recent reviews have discussed these findings in detail (Symington et al. 2014;Heyer 2015;Ranjha et al. 2018); thus, we give only a brief overview here for each HR step to provide the context of SUMO-based regulation. As the HR machinery and its sumoylation are best examined in budding yeast, we use this system as an index for summarizing SUMO-based control. We also discuss additional regulation in mammalian cells and highlight their similarities and differences with those found in yeast. It is noteworthy that SUMO plays important roles in modulating protein recruitment to damaged chromatin and in other DNA break repair pathways. As these topics have been well covered in other reviews (Schwertman et al. 2016; Garvin and Morris 2017), they are not addressed here in order to maintain the focus on the regulation of core HR machinery.
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