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.
Highlights
In response to DNA replication stress such as low nucleotide levels or DNA damage, a cascade of events is orchestrated by the DNA replication checkpoint to ensure genome protection
DNA replication stress is detected by proteins that activate the apical protein kinase Mec1 in Saccharomyces cerevisiae (ATR in humans) (Pardo et al, 2017; Saldivar et al, 2017)
Rad53 inhibition of origin firing in vitro via Dbf4 and Sld3 To understand in molecular detail how Rad53 regulates DNA replication, we have exploited the reconstitution of DNA replication with purified budding yeast proteins
Summary
In response to DNA replication stress such as low nucleotide levels or DNA damage, a cascade of events is orchestrated by the DNA replication checkpoint to ensure genome protection. Active Rad coordinates a broad response to promote cell survival by regulating damage-dependent transcription, cell cycle, deoxyribonucleotide triphosphate (dNTP) levels, and replication origin firing (Bastos de Oliveira et al, 2012; Krishnan et al, 2004; Paulovich and Hartwell, 1995; Santocanale and Diffley, 1998; Travesa et al, 2012; Zegerman and Diffley, 2010; Zhao et al, 1998). Rad inhibits origin firing through multiple, redundant phosphorylation events of two essential firing factors, Sld and Dbf; non-phosphorylatable mutants of Sld and Dbf, when combined, show the same fast progression through S phase as rad mutants (Zegerman and Diffley, 2010). The Rad targets involved in regulating replication fork stability are currently unclear, but several studies have implicated the Mec1-Rad checkpoint in replication fork slow-down in response to replication stress suggesting there may be a link between replication fork rate and stability (Bacal et al, 2018; Kumar and Huberman, 2009; Mutreja et al, 2018; Seiler et al, 2007)
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