Chl1 helicase controls replication fork progression by regulating dNTP pools.

Amandine Batté,Sushma Sharma,Haico Van Attikum,Sophie C Van Der Horst,Mireille Tittel-Elmer,Andrei Chabes,Su Ming Sun,Jolanda Van Leeuwen

doi:10.26508/lsa.202101153

Abstract

Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.

Highlights

Faithful duplication of the genome by DNA replication in the S phase of the cell cycle is crucial for the maintenance of genomic stability
Analysis of these interactions revealed that CHL1 is a major hub in the cohesin network (Fig 1A), showing interactions with genes involved in sister chromatid cohesion (SCC) (RTS1, VIK1, DCC1, RAD61, and CHL4), chromosome segregation (BUB1, BUB3, CTF3, and MAD3) and DNA replication (POL30 [PCNA], and POL2 [DNA polymerase ε])
Systematic mapping of synthetic genetic interactions in yeast revealed an interaction between CHL1 and MRC1 (Sun et al, 2020), a known gene involved in the replication stress response, suggesting a role for CHL1 in this response

Summary

Introduction

Faithful duplication of the genome by DNA replication in the S phase of the cell cycle is crucial for the maintenance of genomic stability. This process can be compromised when replication forks encounter obstacles such as DNA lesions, secondary DNA structures, natural pause sites, or covalent protein-DNA crosslinks, that can lead to replication fork stalling (Zeman & Cimprich, 2014). The DNA replication checkpoint is a major pathway of this surveillance mechanism mediated by the highly conserved Mec1/ATR and Rad53/Chk kinases. The sensor kinase Mec1/ATR detects the accumulation of replication protein A (RPA)-coated ssDNA at stalled forks and promotes the phosphorylation of the effector kinase Rad53/Chk. In Saccharomyces cerevisiae, activated Rad maintains replication fork integrity and controls inhibition of late origin firing, up-regulation of dNTP pools and activation of DNA damage repair genes (Huang et al, 1998; Lopes et al, 2001; Sogo et al, 2002; Zhao & Rothstein, 2002; Zegerman & Diffley, 2010)

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