Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

Geylani Can,Anastasia Christine Kauerhof,Dominik Macak,Philip Zegerman

doi:10.1016/j.molcel.2018.11.025

Geylani Can, Anastasia Christine Kauerhof + Show 2 more

Open Access

https://doi.org/10.1016/j.molcel.2018.11.025

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Journal: Molecular Cell	Publication Date: Dec 27, 2018
Citations: 31	License type: cc-by

Affiliation: Wellcome/Cancer Research UK Gurdon Institute

Abstract

SummaryAcross eukaryotes, disruption of DNA replication causes an S phase checkpoint response, which regulates multiple processes, including inhibition of replication initiation and fork stabilization. How these events are coordinated remains poorly understood. Here, we show that the replicative helicase component Cdc45 targets the checkpoint kinase Rad53 to distinct replication complexes in the budding yeast Saccharomyces cerevisiae. Rad53 binds to forkhead-associated (FHA) interaction motifs in an unstructured loop region of Cdc45, which is phosphorylated by Rad53 itself, and this interaction is necessary for the inhibition of origin firing through Sld3. Cdc45 also recruits Rad53 to stalled replication forks, which we demonstrate is important for the response to replication stress. Finally, we show that a Cdc45 mutation found in patients with Meier-Gorlin syndrome disrupts the functional interaction with Rad53 in yeast. Together, we present a single mechanism by which a checkpoint kinase targets replication initiation and elongation complexes, which may be relevant to human disease.

Highlights

Eukaryotic DNA replication is tightly regulated to ensure that the genome is replicated in its entirety in every cell cycle
The first step in replication involves the formation of the pre-replicative complex at origins in G1 phase, a process called ‘‘licensing’’ (Remus and Diffley, 2009)
Replication initiation at licensed origins can only occur in S phase due to the activation of the S-phase cyclin-dependent kinase (S-Cyclin-dependent kinase (CDK)) and Dbf4-dependent (DDK) kinases (Labib, 2010)

Summary

Introduction

Eukaryotic DNA replication is tightly regulated to ensure that the genome is replicated in its entirety in every cell cycle. Sld binding to Mcm facilitates the recruitment of Cdc, which is a critical component of the active form of the replicative helicase, called the CMG (Cdc45Mcm-GINS) complex (Bell and Labib, 2016). Cyclin-dependent kinase (CDK), on the other hand, phosphorylates Sld and an additional initiation factor, Sld, which via phospho-interactions with Dpb results in the recruitment of GINS and the leadingstrand polymerase (Pol ε) to origins (Tanaka and Araki, 2013). DDK and CDK are required both for the ordered assembly of the active CMG complex and for the recruitment of additional proteins to form the multi-subunit replication machinery, called the replisome (Tanaka and Araki, 2013)

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