Differential Requirement for H2AX and 53BP1 in Organismal Development and Genome Maintenance in the Absence of Poly(ADP)ribosyl Polymerase 1

Abstract

Combined deficiencies of poly(ADP)ribosyl polymerase 1 (PARP1) and ataxia telangiectasia mutated (ATM) result in synthetic lethality and, in the mouse, early embryonic death. Here, we investigated the genetic requirements for this lethality via analysis of mice deficient for PARP1 and either of two ATM-regulated DNA damage response (DDR) factors: histone H2AX and 53BP1. We found that, like ATM, H2AX is essential for viability in a PARP1-deficient background. In contrast, deficiency for 53BP1 modestly exacerbates phenotypes of growth retardation, genomic instability, and organismal radiosensitivity observed in PARP1-deficient mice. To gain mechanistic insights into these different phenotypes, we examined roles for 53BP1 in the repair of replication-associated double-strand breaks (DSBs) in several cellular contexts. We show that 53BP1 is required for DNA-PKcs-dependent repair of hydroxyurea (HU)-induced DSBs but dispensable for RPA/ RAD51-dependent DSB repair in the same setting. Moreover, repair of mitomycin C (MMC)-induced DSBs and sister chromatid exchanges (SCEs), two RAD51-dependent processes, are 53BP1 independent. Overall, our findings define 53BP1 as a main facilitator of nonhomologous end joining (NHEJ) during the S phase of the cell cycle, beyond highly specialized lymphocyte rearrangements. These findings have important implications for our understanding of the mechanisms whereby ATM-regulated DDR prevents human aging and cancer.DNA double-strand breaks (DSBs) arise constantly in mammalian cells from endogenous and exogenous sources (35). Defective DSB repair leading to cellular senescence or apoptosis, or aberrant repair to form chromosomal rearrangements, has been linked to aging and cancer in humans (22, 31). To prevent these deleterious outcomes, mammalian cells have evolved the DNA damage response (DDR), a network of factors that sense and signal DSBs to promote their repair (29). ataxia telangiectasia mutated (ATM) is a phophoinositide 3-kinase (PI3K)-like kinase that regulates the functions of hundreds of substrates during DDR, including histone H2AX and 53BP1 (41). Ultimately, the DDR restores DNA strand continuity via either of two main DSB repair pathways: homologous recombination (HR), an error-free pathway that operates only during the S/G 2 phases (70), or nonhomologous end joining (NHEJ), a versatile but error-prone pathway that operates throughout the cell cycle (37, 44).Poly(ADP)ribosyl polymerase 1 (PARP1) regulates, among other processes, transcription, cell death, and DNA repair (57). In the last context, PARP1 senses single-strand breaks (SSBs) and promotes their repair via base excision repair (BER) (15). Although PARP1 is not a component of the BER pathway per se, PARP1-deficient cells accumulate SSBs, which become substrates for HR-mediated repair upon replication (10, 21). ATM and other DDR factors may play important roles in DSB recognition and recruitment of the HR machinery in this setting (42). In addition, PARP1 also catalyzes PAR formation at de novo-generated DNA DSBs (28...

Ataxia Telangiectasia Mutated (ATM) Is Dispensable for Endonuclease I-SceI-induced Homologous Recombination in Mouse Embryonic Stem Cells

“…To maintain genome stability, this ATM-dependent alternative repair pathway along with ATMindependent HR may be required to properly repair DSBs, at least those arising in euchromatin. As PARP inhibition selectively kills cells defective for HR (16), it is consistent that H2AX Ϫ/Ϫ cells, which are defective for HR, are sensitive to PARP inhibitors, and combined PARP1 and H2AX deficiency causes embryonic lethality (45). In contrast, as loss of ATM does not impair HR, HR defects do not appear to be a contributing factor to the molecular mechanism of synthetic lethality imposed by combined loss of ATM and PARP1/2.…”

“…Inhibitor Olaparib-ATM or H2AX deficiency in combination with PARP1 or PARP2 deficiency causes embryonic lethality (17,18,45). It is unclear whether ATM/H2AX deficiency contributes to this synthetic lethality through the same molecular mechanism.…”

RNF168 ubiquitylates 53BP1 and controls its response to DNA double-strand breaks

“…53BP1 also plays important roles in NHEJ (13,28,29). Although recently 53BP1 has been reported to be important for the choice between the NHEJ and homologous recombination (HR) repair pathways (30)(31)(32), previous studies demonstrated that 53BP1 is not required for HR (33) and IRIF for Rad51, a recombinase integral for HR (34,35). We therefore examined the effect of Rnf168 deficiency on DSB repair using reporter plasmids for NHEJ and HR (36)(37)(38).…”

Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells

“…In a recent study, disabling NHEJ was shown to reverse the DNA-repair defects and chromosomal instability of FANCD2 mutants exposed to platinum cross-linking agents (11). Moreover, ablation of 53BP1, a molecule recently demonstrated to facilitate NHEJmediated DSB repair (38) in addition to its other roles (39), also rescued the genotoxicity of DNA-damaging agents in a BRCA1 background (34,40). These earlier studies provide support for a model in which unrestricted NHEJ could induce genomic instability and eventual lethality in HR-deficient cells.…”