DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation

Rémy Bétous,Siem Van Der Laan,Marie-Jeanne Pillaire,Petr Grúz,Caixia Guo,Domenico Maiorano,Emma Ohl-Séguy,Naoko Niimi,Errol Friedberg,Laura Pierini,Bénédicte Recolin,Christophe Cazaux,Takehiko Nohmi,Jean-Sébastien Hoffmann

doi:10.1038/emboj.2013.148

Abstract

Formation of primed single-stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR-mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA-mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y-family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9-1-1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9-1-1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.

Highlights

Common impediments to replication fork progression include exogenous and endogenous DNA damage, natural structured DNA, or tightly associated DNA–protein complexes that disturb the progression of the replicative DNA polymerases a, d and e
Significant RNAimediated depletion of Pol k was achieved without significantly altering the expression of other replicative or Y-family TLS DNA polymerases (Supplementary Figure S1C)
DinB/ Pol k-like polymerases are found in all domains of life and are among the most highly conserved of all the TLS DNA polymerases (Waters et al, 2009)

Summary

Introduction

Common impediments to replication fork progression include exogenous and endogenous DNA damage, natural structured DNA, or tightly associated DNA–protein complexes that disturb the progression of the replicative DNA polymerases a, d and e. Cells have evolved several mechanisms to deal with the constant challenge of replication stress These include systems to detect and repair damaged DNA, and replication checkpoints that sense stalled replication forks in S phase to direct appropriate cellular responses (Nyberg et al, 2002; Ciccia and Elledge, 2010). The ATR signalling pathway is crucial in regulating the replication stress response to a large array of insults such as DNA damaging agents and chemicals that cause replication arrest (Melo and Toczyski, 2002; Cimprich and Cortez, 2008). The regulatory protein Claspin has been shown to be required for ATR-dependent phosphorylation of Chk

Methods

Results

Conclusion