PARP1-dependent Kinetics of Recruitment of MRE11 and NBS1 Proteins to Multiple DNA Damage Sites

Guy G. Poirier,Amélie Rodrigue,Jean-François Haince,Ugo Déry,Darin McDonald,Michael J. Hendzel,Jean-Yves Masson

doi:10.1074/jbc.m706734200

Guy G. Poirier, Amélie Rodrigue

Open Access

https://doi.org/10.1074/jbc.m706734200

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Abstract

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Highlights

Chromatin-associated proteins rapidly become poly(ADP-ribosyl)ated in the vicinity of DNA lesions [5]
Laser-induced microirradiation produces a variety of DNA lesions, including SSBs, and base damage, but it is best known for its ability to produce double strand breaks (DSBs)
Poly(ADP-ribose) polymerase 1 (PARP1) Is Required for Rapid Recruitment of MRE11 and NBS1 at DSB Sites—Because the selective recruitment of a specialized DNA damage sensor might be required to enhance the efficiency of different DNA repair pathways, we extended our analysis to the recruitment kinetics of the MRN complex

Summary

Introduction

Chromatin-associated proteins rapidly become poly(ADP-ribosyl)ated in the vicinity of DNA lesions [5]. Dynamics of PARP1 Recruitment to DNA Damage Lesions— We carried out live cell imaging analysis using MEFs derived from the PARP1 mutant mice and complemented with a functional GFP-tagged PARP1 protein (Fig. 1a).

Results

Conclusion