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Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation.

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journal contribution
posted on 27.08.2021, 11:38 by Allison W Mcclure, John FX Diffley
The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

Funding

Crick (Grant ID: 10066, Grant title: Diffley FC001066) Wellcome Trust (Grant ID: 106252/Z/14/Z, Grant title: WT 106252/Z/14/Z) Wellcome Trust (Grant ID: 219527/Z/19/Z, Grant title: WT 219527/Z/19/Z) European Research Council (Grant ID: 669424 - CHROMOREP, Grant title: ERC 669424 - CHROMOREP)

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