Open AccessNovel Checkpoint Response to Genotoxic Stress Mediated by Nucleolin-Replication Protein A Complex Formation
Author(s) -
Kyung Kim,
Diana D. Dimitrova,
Kristine M. Carta,
Anjana Saxena,
Mariza Daras,
James A. Borowiec
Publication year - 2005
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.25.6.2463-2474.2005
Subject(s) - nucleolin , biology , replication protein a , dna replication , dna damage , microbiology and biotechnology , replication factor c , nucleoplasm , eukaryotic dna replication , dna , nucleolus , dna binding protein , genetics , transcription factor , gene , nucleus
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.