Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein a complex formation.
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.
Kim
Kyung
K
Department of Biochemistry, New York University School of Medicine, 550 First Ave., MSB-383, New York, NY 10016, USA.
Dimitrova
Diana D
DD
Carta
Kristine M
KM
Saxena
Anjana
A
Daras
Mariza
M
Borowiec
James A
JA
D064007:Ataxia Telangiectasia Mutated Proteins; D002166:Camptothecin / Q000633:toxicity; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D018797:Cell Cycle Proteins / Q000502:physiology; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004249:DNA Damage; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009154:Mutation / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011839:Radiation, Ionizing; D051716:Replication Protein A; D013539:Simian virus 40 / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000502:physiology; D000097796:Nucleolin
D064007:Ataxia Telangiectasia Mutated Proteins; D002166:Camptothecin; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D016601:RNA-Binding Proteins; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D000097796:Nucleolin; C493956:CDKN1A protein, human; C497167:RPA1 protein, human; C576324:ATM protein, human; C576597:ATR protein, human
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