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  • DNA replication checkpoint kinases in both yeast and human c


    DNA replication checkpoint kinases in both yeast and human M 1145 sale upregulate dNTP levels under replication stress to mediate their essential function at replication forks (Yeeles et al., 2013). It is possible that upregulation of dNTP levels under replication stress helps fork restart once replication stress is terminated. We suggest that elevated levels of dNTPs also help prevent the generation of long stretches of ssDNA at origins that have already fired. ssDNA is a known inducer of replication catastrophe and genome instability in both yeast and human cells. Unwinding of dsDNA by the MCM helicase through uncoupling of MCM and DNA polymerase activity is essential for the activation of the replication checkpoint during S phase (Byun et al., 2005, Tercero et al., 2003). Our observations of the excessive ssDNA and MCM localization in rad53-1 mutant cells suggest that activated Rad53 is required to prevent excessive DNA unwinding by the MCM helicase under replication stress. Therefore, we propose that a feedback mechanism exists between dsDNA unwinding by the MCM helicase and DNA replication checkpoint pathway. Under replication stress, Pol ε, while having a greater contact with the nascent leading-strand DNA at active forks, backtracks to bind dsDNA at HU-stalled forks (Yu et al., 2017). The change in association mode of Pol ε binding to dsDNA at HU-stalled forks was also detected in rad53-1 mutant cells (data M 1145 sale not shown). Therefore, we suggest that Pol ε disengages from the active DNA synthesis mode and moves passively with active CMG helicase in rad53-1 mutant cells under replication stress. The MCM helicase also moves ahead of the site of actual DNA synthesis in cells lacking Mrc1 (Katou et al., 2003). While Mrc1 functions upstream of Rad53 under replication stress, Rad53 and Mec1 also phosphorylate Mrc1 (Alcasabas et al., 2001). Moreover, Mrc1 promotes leading-strand DNA synthesis in vitro (Yeeles et al., 2017). Therefore, it is tempting to speculate that Rad53 mediates its essential function at DNA replication forks through Mrc1. However, unlike Rad53, Mrc1 is not essential. Therefore, Rad53 likely also regulates other replisome components to perform its essential function. Pol ε stimulates the CMG helicase activity (Kang et al., 2012), so we speculate that Rad53 likely regulates the Pol ε-CMG interaction under replication stress, which in turn couples leading and lagging-strand DNA synthesis.
    Author Contributions Z.Z., H.G., and C.Y. conceived the project. C.Y. performed almost all experiments. H.G. performed all bioinformatics and statistical analysis. H.G. and J.H. performed some of the experiments. S.D. performed the in vitro DNA replication experiments under the guidance of D.R.; S.S. and A.C. measured dNTP levels. H.G., C.Y., and Z.Z. wrote the original draft. D.R. edited the manuscript extensively and all authors read the paper.
    Acknowledgments We thank Drs. Bruce Stillman, Xiaolan Zhao, Rodney Rothstein, Alberto Ciccia, Lorraine Symington, Jean Gautier, and Erik Johansson for discussions and comments on this manuscript. We thank Drs. Symington, Diffley, Wittenberg, Kunkel, and Kolodner for yeast strains and plasmids and Dr. Brill for antibodies against yeast RPA. This study is supported by NIH grants R35GM118015 (Z.Z.) and R01GM107239 (D.R.) and by the Swedish Cancer Society and the Swedish Research Council (A.C.).
    Introduction DNA double-strand breaks (DSBs) are among the most severe types of genomic damage threatening cellular viability. They occur physiologically during processes that generate antibody diversity in immune cells or genetic variability in germ cells and can arise following exposure to exogenous agents such as ionizing radiation (IR) or chemotherapeutic drugs. Understanding how cells respond to DSBs lies at the heart of evaluating the efficacy of radio- and chemotherapy as well as assessing risks from low-dose IR exposure (Jackson and Bartek, 2009).