HSSB1 phosphorylation is dynamically regulated by DNA-PK and PPP-family protein phosphatases

Nicholas W Ashton,Nicolas Paquet,Sally L Shirran,Emma Bolderson,Ruvini Kariawasam,Christine Touma,Azadeh Fallahbaghery,Roland Gamsjaeger,Liza Cubeddu,Catherine Botting,Pamela M Pollock,Kenneth J O’Byrne,Derek J Richard

doi:10.1016/j.dnarep.2017.03.006

Abstract

The maintenance of genomic stability is essential for cellular viability and the prevention of diseases such as cancer. Human single-stranded DNA-binding protein 1 (hSSB1) is a protein with roles in the stabilisation and restart of stalled DNA replication forks, as well as in the repair of oxidative DNA lesions and double-strand DNA breaks. In the latter process, phosphorylation of threonine 117 by the ATM kinase is required for hSSB1 stability and efficient DNA repair. The regulation of hSSB1 in other DNA repair pathways has however remained unclear. Here we report that hSSB1 is also directly phosphorylated by DNA-PK at serine residue 134. While this modification is largely suppressed in undamaged cells by PPP-family protein phosphatases, S134 phosphorylation is enhanced following the disruption of replication forks and promotes cellular survival. Together, these data thereby represent a novel mechanism for hSSB1 regulation following the inhibition of replication.

Highlights

DNA replication constitutes one of the most important processes within the cell and allows for the accurate duplication of genomic information prior to cell division
Cells depleted of endogenous DNA-PKcs or expressing an enzymatically deficient kinase mutant are unable to efficiently restart stalled replication forks and show reduced clonogenic survival when treated with hydroxyurea [7]
In this article we demonstrate that Human single-stranded DNA-binding protein 1 (hSSB1) S134 phosphorylation is required for clonogenic survival of cells treated with the replication stress compounds hydroxyurea, aphidicolin and camptothecin, as well as establish that phosphorylation is primarily a result of DNA-PK activity

Summary

Introduction

DNA replication constitutes one of the most important processes within the cell and allows for the accurate duplication of genomic information prior to cell division While this process occurs rapidly in unperturbed cells, numerous endogenous and exogenous sources may either slow or stall replication fork progression [1]. Such disruption results in the exposure of single-stranded DNA (ssDNA) that must be stabilised to prevent replication fork collapse and double-stranded DNA break formation [2]. In addition to ATR, numerous additional regulatory enzymes are activated by replication stress, including the related PI3K-like kinases, ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK) [6,7,8]. Activation of these enzymes results in the coordinated regulation of numerous DNA repair proteins and is exemplified by the ATM, ATR and DNA-PKmediated phosphorylation of the RPA 32 kDa subunit (RPA32) [8,9,10]

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Results

Conclusion