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.

Edited by Patrick SungThe assembly of the replication fork helicase during S phase is key to the initiation of DNA replication in eukaryotic cells. One step in this assembly in budding yeast is the association of Cdc45 with the Mcm2-7 heterohexameric ATPase, and a second step is the assembly of the tetrameric GINS (GG-Ichi-Nii-San) complex with Mcm2-7. Dbf4-dependent kinase (DDK) and S-phase cyclin-dependent kinase (S-CDK) are two S phase-specific kinases that phosphorylate replication proteins during S phase, and Dpb11, Sld2, Sld3, Pol ⑀, and Mcm10 are factors that are also required for replication initiation. However, the exact roles of these initiation factors in assembly of the replication fork helicase remain unclear. Key to the initiation of DNA replication is the assembly and activation of the replication fork helicase, the 11-subunit assembly that provides single-stranded DNA templates for the replicative polymerases (1). The replication fork helicase is called Cdc45, GINS (CMG) 3 and is comprised of the Mcm2-7 heterohexameric ATPase and the essential helicase stimulatory factors Cdc45 and GINS (2-5). Cdc45 is a single protein, whereas GINS is a four-subunit complex comprised of Sld5, Psf1, Psf2, and Psf3 (5, 6).The Mcm2-7 complex is a single hexamer when it is free in solution, but when loaded onto DNA in late M phase and G 1 by Orc, Cdc6, and Cdt1 in the presence of ATP, Mcm2-7 forms a double hexamer, with the N termini of the Mcm2-7 proteins forming interhexameric contacts (7,8). In S phase, several changes occur that lead to assembly and activation of the replication fork helicase. Mcm2-7 assembles with Cdc45 and GINS to form the CMG helicase, single-stranded DNA is extruded from the central channel of Mcm2-7 (i.e. origin melting), and the Mcm2-7 double hexamers separate to initiate bidirectional unwinding (8 -11).Five essential DNA replication initiation proteins are also required for replication initiation, and these proteins are Sld2, Sld3, Dpb11, Pol ⑀,. In addition, two S phase-specific kinases, S-CDK and DDK, are required for replication initiation (18 -20). S-CDK phosphorylates Sld2 and Sld3, and phosphorylation of Sld2 and Sld3 results in the formation of a ternary complex with Dpb11 (21,22). In addition, DDK phosphorylates Mcm2, Mcm4, and Mcm6, and DDK phosphorylation of Mcm2-7 is required for cell growth (23)(24)(25).It was shown previously, using an in vitro reconstitution assay, that DDK phosphorylation of Mcm4 and Mcm6 stimulates Sld3 binding to Mcm2-7 with subsequent recruitment of Cdc45 to . GINS recruitment to Mcm2-7 requires all of the initiation factors aside from Mcm10 (27), and GINS binds directly to Dpb11 (28).We report here that Dpb11 stimulates DDK phosphorylation of Mcm4 alone, soluble Mcm2-7, or Mcm2-7 that is loaded onto origin dsDNA. The human homolog of Dpb11, TopBP1, stimulates human DDK phosphorylation of human Mcm4, suggesting that the reaction is conserved from yeast to human. We also find that the DDK phosphomimetic mutant of Mcm4 binds substantially more tightly t...

Replication Protein A (RPA) is a heterotrimeric complex that binds to single-stranded DNA (ssDNA) and recruits over three dozen RPA-interacting proteins to coordinate multiple aspects of DNA metabolism including DNA replication, repair, and recombination. Rtt105 is a molecular chaperone that regulates nuclear localization of RPA. Here, we show that Rtt105 binds to multiple DNA binding and protein-interaction domains of RPA and configurationally staples the complex. In the absence of ssDNA, Rtt105 inhibits RPA binding to Rad52, thus preventing spurious binding to RPA-interacting proteins. When ssDNA is available, Rtt105 promotes formation of high-density RPA nucleoprotein filaments and dissociates during this process. Free Rtt105 further stabilizes the RPA-ssDNA filaments by inhibiting the facilitated exchange activity of RPA. Collectively, our data suggest that Rtt105 sequesters free RPA in the nucleus to prevent untimely binding to RPA-interacting proteins, while stabilizing RPA-ssDNA filaments at DNA lesion sites.

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