Poly(ADP-Ribosyl) Glycohydrolase Prevents the Accumulation of Unusual Replication Structures during Unperturbed S Phase

Abstract

Poly(ADP-ribosyl)ation (PAR) has been implicated in various aspects of the cellular response to DNA damage and genome stability. Although 17 human poly(ADP-ribose) polymerase (PARP) genes have been identified, a single poly(ADP-ribosyl) glycohydrolase (PARG) mediates PAR degradation. Here we investigated the role of PARG in the replication of human chromosomes. We show that PARG depletion affects cell proliferation and DNA synthesis, leading to replication-coupled H2AX phosphorylation. Furthermore, PARG depletion or inhibition per se slows down individual replication forks similarly to mild chemotherapeutic treatment. Electron microscopic analysis of replication intermediates reveals marked accumulation of reversed forks and single-stranded DNA (ssDNA) gaps in unperturbed PARG-defective cells. Intriguingly, while we found no physical evidence for chromosomal breakage, PARG-defective cells displayed both ataxia-telangiectasia-mutated (ATM) and ataxia-Rad3-related (ATR) activation, as well as chromatin recruitment of standard double-strand-break-repair factors, such as 53BP1 and RAD51. Overall, these data prove PAR degradation to be essential to promote resumption of replication at endogenous and exogenous lesions, preventing idle recruitment of repair factors to remodeled replication forks. Furthermore, they suggest that fork remodeling and restarting are surprisingly frequent in unperturbed cells and provide a molecular rationale to explore PARG inhibition in cancer chemotherapy.C ellular responses are crucial for the adaptability and survival of a cell exposed to different types of endogenous and exogenous stress. The DNA damage response (DDR) consists of one such defense mechanism in response to different types of insults to the DNA. Poly(ADP)ribosylation of proteins is one of the quickest cellular responses to DNA damage and is brought about by proteins of the poly(ADP) ribose polymerase family (PARP), mostly PARP1 (1). Upon being recruited to sites of the DNA damage, NAD ϩ is used as a substrate by PARP to synthesize negatively charged poly(ADP-ribosyl)ation (PAR) polymers onto itself and also its target proteins (1). Through this posttranslational modification, PARP targets a variety of nuclear proteins to facilitate the recruitment of DNA repair factors to sites of damage (2, 3). Accordingly, PARP-1 or PARP-2-deficient mice and mouse embryonic fibroblasts show chromosomal aberrations and various DNA repair defects (4-6).Inhibition of PARP has become a promising therapeutic approach for the treatment of certain types of cancer (7). It was shown that PARP inhibitors could selectively kill homologous recombination (HR)-deficient cancer cells (8, 9). The reason behind the sensitivity of HR-deficient cells to PARP inhibition is thought to be the accumulation of single-stranded DNA (ssDNA) breaks in the absence of PAR synthesis, leading to replication fork collapse and double-stranded breaks (DSBs), which would then require HR factors for repair (8,10). Recently, PARP activity has also been reported to play a ro...

The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability

“…Their increase is not simply due to more capture of EdU-labeled DNA since other histones remain relatively unchanged. PARG also travels with undamaged forks, which is consistent with the observation that it is needed to prevent the accumulation of abnormal replication structures in an unperturbed S phase (Mortusewicz et al, 2011; Ray Chaudhuri et al, 2015). Thus, a net increase in PAR at stalled forks may be mediated by the decrease in PARG activity.…”

(ADP-ribosyl)hydrolases: structure, function, and biology

“…Importantly, PARG depletion leads to hypersensitivity to genotoxic and replication stress and, consequently, it was proposed as a novel target for modern chemotherapeutic approaches (James et al 2016;Pillay et al 2019). In addition to its functions in DNA repair, PARG activity seems to be involved in the progression of replication forks and recovery from persistent replication stress (Illuzzi et al 2014;Ray Chaudhuri et al 2015). These observations are in agreement with the interaction of PARG with the replication helicase PCNA and its localization to replication foci during S-phase ( Fig.…”

Posttranscriptional Regulation of PARG mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors

“…This is the first study to show that DNA damage triggers activation of PARG, which is directly related to the ability of HuR to rapidly stabilize specific mRNA (i.e., PARG) (17, 18, 20, 32). The link between PARG activation and DNA repair is emerging (48–50). Our data indicate that in response to (or during) DNA damage, an HuR-dependent increase in PARG expression and activity (i.e.…”