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.

Supplemental material for this article may be found at http://mcb.asm.org/.

We are grateful to Fred Alt for mice deficient for H2AX, ATM, and DNA-PKcs; Klaus Rajewsky for mice harboring immunoglobulin transgenes; Junjie Chen for mice deficient for 53BP1; and Ted Dawson for mice deficient for PARP1. We also thank Shaida Andrabi for advice on PARP1 genotyping and immunofluorescence, Juan Fu for technical assistance with B-cell purification, and Lillian Dasko-Vincent and Leslie Meslzer (in memoriam) at the Sidney Kimmel Comprehensive Cancer Center (SKCCC) Cell Imaging Core Facility for assistance with confocal microscopy and flow cytometry analysis.

We declare no conflict of interest.