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

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Abstract

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.

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Allison McClure

The Francis Crick Institute, London, United Kingdom

Competing interests
The authors declare that no competing interests exist.
John Diffley

The Francis Crick Institute, London, United Kingdom

For correspondence
john.diffley@crick.ac.uk

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-5184-7680

Funding

Wellcome Trust (106252/Z/14/Z)

John Diffley

Wellcome Trust (219527/Z/19/Z)

John Diffley

European Commission (669424-CHROMOREP)

John Diffley

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.