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

View ORCID ProfileAllison W. McClure, View ORCID ProfileJohn F.X. Diffley
doi: https://doi.org/10.1101/2021.04.09.439171
Allison W. McClure
1Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT
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John F.X. Diffley
1Chromosome Replication Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT
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  • For correspondence: john.diffley@crick.ac.uk
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Summary

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.

Competing Interest Statement

The authors have declared no competing interest.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted April 10, 2021.
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Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation
Allison W. McClure, John F.X. Diffley
bioRxiv 2021.04.09.439171; doi: https://doi.org/10.1101/2021.04.09.439171
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Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation
Allison W. McClure, John F.X. Diffley
bioRxiv 2021.04.09.439171; doi: https://doi.org/10.1101/2021.04.09.439171

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