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Molecular and Cellular Biology, March 2005, p. 2463-2474, Vol. 25, No. 6
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.6.2463-2474.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Kyung Kim,1 Diana D. Dimitrova,1 Kristine M. Carta,1 Anjana Saxena,1 Mariza Daras,1 and James A. Borowiec1*

Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York1

Received 19 May 2004/ Returned for modification 14 June 2004/ Accepted 13 December 2004

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.

* Corresponding author. Mailing address: Department of Biochemistry, New York University School of Medicine, 550 First Ave., MSB-383, New York, NY 10016. Phone: (212) 263-8453. Fax: (212) 263-8166. E-mail: james.borowiec{at}med.nyu.edu.

Molecular and Cellular Biology, March 2005, p. 2463-2474, Vol. 25, No. 6
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.6.2463-2474.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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