PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress.

Giuditta Illuzzi,Elise Fouquerel,Kristina Rehmet,Jean-Christophe Amé,Françoise Dantzer,Heinz-Peter Nasheuer,Valérie Schreiber,Aurélia Noll

doi:10.1093/nar/gku505

Abstract

Poly(ADP-ribosyl)ation is involved in numerous bio-logical processes including DNA repair, transcription and cell death. Cellular levels of poly(ADP-ribose) (PAR) are regulated by PAR polymerases (PARPs) and the degrading enzyme PAR glycohydrolase (PARG), controlling the cell fate decision between life and death in response to DNA damage. Replication stress is a source of DNA damage, leading to transient stalling of replication forks or to their collapse followed by the generation of double-strand breaks (DSB). The involvement of PARP-1 in replicative stress response has been described, whereas the consequences of a deregulated PAR catabolism are not yet well established. Here, we show that PARG-deprived cells showed an enhanced sensitivity to the replication inhibitor hydroxyurea. PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A association with collapsed forks resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation.

Highlights

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification of proteins mediated by Poly(ADP-ribose) polymerases (PARPs)
Some cells could not resume cell cycle progression after the prolonged HU treatment. These results suggest that PAR glycohydrolase (PARG) is dispensable for the restart of stalled replication forks but required for efficient recovery from replicative stress conditions known to trigger replication fork collapse
Homologous recombination is impaired in high PAR HUtreated shPARG cells To evaluate the consequence of PARG deficiency and defective RPA2 loading onto chromatin on the homologous recombination (HR)-mediated recovery from prolonged replication fork stalling, we examined the recruitment of RAD51 onto chromatin and formation of RAD51 foci following an HU treatment of 24 h and after different release time points

Summary

Introduction

Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification of proteins mediated by Poly(ADP-ribose) polymerases (PARPs). PARylation is a reversible modification, PAR catabolism is mediated mainly by poly(ADP-ribose) glycohydrolase (PARG), encoded by a single gene but present as multiple isoforms localized in different cellular compartments [3,4]. In cell-based models, the depletion of all PARG isoforms using either siRNA or shRNA strategies does not necessarily affect cell viability in unstressed conditions. Upon genotoxic insults, these PARG-deficient cells revealed increased cell death and impaired repair of single- and double-strand breaks (SSB and DSB, respectively) and of oxidized bases [6,7,8], thereby highlighting the key functions of PARG, like PARP-1, in DNA damage response

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