Nuclear poly(A)-binding protein 1 is an ATM target and essential for DNA double-strand break repair.

Michal Gavish-Izakson,Yosef Shiloh,Rosario Prados-Carvajal,Pablo Huertas,Reuven Agami,Tamar Geiger,Yael Ziv,Georgina D Barnabas,Ran Elkon,Bhagya Bhavana Velpula,Alejandro Pineiro Ugalde

doi:10.1093/nar/gkx1240

Abstract

The DNA damage response (DDR) is an extensive signaling network that is robustly mobilized by DNA double-strand breaks (DSBs). The primary transducer of the DSB response is the protein kinase, ataxia-telangiectasia, mutated (ATM). Here, we establish nuclear poly(A)-binding protein 1 (PABPN1) as a novel target of ATM and a crucial player in the DSB response. PABPN1 usually functions in regulation of RNA processing and stability. We establish that PABPN1 is recruited to the DDR as a critical regulator of DSB repair. A portion of PABPN1 relocalizes to DSB sites and is phosphorylated on Ser95 in an ATM-dependent manner. PABPN1 depletion sensitizes cells to DSB-inducing agents and prolongs the DSB-induced G2/M cell-cycle arrest, and DSB repair is hampered by PABPN1 depletion or elimination of its phosphorylation site. PABPN1 is required for optimal DSB repair via both nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR), and specifically is essential for efficient DNA-end resection, an initial, key step in HRR. Using mass spectrometry analysis, we capture DNA damage-induced interactions of phospho-PABPN1, including well-established DDR players as well as other RNA metabolizing proteins. Our results uncover a novel ATM-dependent axis in the rapidly growing interface between RNA metabolism and the DDR.

Highlights

The double-strand break (DSB) is a severe DNA lesion when generated by internal or external DNA damaging agents
Halazonetis, anti-53BP1 polyclonal antibody was obtained from Novus Biologicals, LLC (Littleton CO, USA), anti-pS139-H2AX monoclonal antibody was obtained from Merck Millipore (Darmstadt, Germany), anti-poly(A)-binding protein 1 (PABPN1) and anti-BRCA1 antibodies were obtained from Abcam (Cambridge, MA, USA), phospho-antibodies against phosphorylated Ser 95 of PABPN1 and phosphorylated KAP-1 were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA), anti-ATM obtained from Sigma–Aldrich
Following 30 min of treatment with the DSB-inducing chemical, neocarzinostatin (NCS), we observed in cellular extracts a signal that was markedly reduced upon PABPN1 depletion using RNAi (Figure 1A)

Summary

Introduction

The double-strand break (DSB) is a severe DNA lesion when generated by internal or external DNA damaging agents. DSBs vigorously trigger the DNA damage response (DDR), an elaborate signaling network that reaches out to all cellular compartments and mobilizes numerous cellular processes [3,4,5]. This network is based on a core of dedicated DDR players and vast, temporary recruitment of additional proteins from other physiological circuits. Additional, minor resection-dependent pathways are single-strand annealing (SSA) and alternative end-joining (Alt-EJ) reviewed in [7,8]. Of these pathways, only HRR is error-free.

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Results

Conclusion