Novel Checkpoint Response to Genotoxic Stress Mediated by Nucleolin-Replication Protein A Complex Formation

Abstract

Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-RPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.

ACKNOWLEDGMENTS

We thank John Hirsch for assistance with FACS analysis, Eric Rubin (UMDNJ) for providing the GST-nucleolin expression vectors, Cristina Cardoso for the pENeGFP RPA34 plasmid, and Trever Bivona and Mark Philips (NYU School of Medicine) for kindly providing the H-Ras and Ras-binding-domain constructs and other reagents and for their invaluable advice on FRET. We also thank Angus Wilson for insightful comments on our studies and Vitaly Vassin for helpful discussions.

This study was supported by NIH grant AI29963, DOD Breast Cancer Research Program DAMD17-03-1-0299, Philip Morris grant 15-B0001-42171, the NYU Cancer Institute, and the Rita J. and Stanley Kaplan Comprehensive Cancer Center (NCI P30CA16087). Purchase of the confocal microscope was funded by the Shared Instrumentation Grant Program of the NIH (S10 RR017970).