| Abstract: |
Failure to reactivate stalled or collapsed DNA replication forks is a potential source of genomic instability. Homologous recombination (HR) is a major mechanism for repairing the DNA damage resulting from replication arrest. The single-strand DNA (ssDNA)-binding protein, replication protein A (RPA), plays a major role in multiple processes of DNA metabolism. However, the role of RPA2 hyperphosphorylation, which occurs in response to DNA damage, had been unclear. Here, we show that hyperphosphorylated RPA2 associates with ssDNA and recombinase protein Rad51 in response to replication arrest by hydroxyurea (HU) treatment. In addition, RPA2 hyperphosphorylation is critical for Rad51 recruitment and HR-mediated repair following HU. However, RPA2 hyperphosphorylation is not essential for both ionizing radiation (IR)-induced Rad51 foci formation and I-Sce-I endonuclease-stimulated HR. Moreover, we show that expression of a phosphorylation-deficient mutant of RPA2 leads to increased chromosomal aberrations following HU treatment but not after exposure to IR. Finally, we demonstrate that loss of RPA2 hyperphosphorylation results in a loss of viability when cells are confronted with replication stress whereas cells expressing hyperphosphorylation-defective RPA2 or wild-type RPA2 have a similar sensitivity to IR. Thus, our data suggest that RPA2 hyperphosphorylation plays a critical role in maintenance of genomic stability and cell survival after a DNA replication block via promotion of HR. © The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org. |
| Keywords: |
controlled study; protein phosphorylation; unclassified drug; hydroxyurea; dna replication; metabolism; cell viability; dna damage; homologous recombination; cell survival; in situ hybridization, fluorescence; dna repair; immunofluorescence; cell line, tumor; phosphorylation; wild type; radiation exposure; chromosome aberration; blotting, western; fluorescence in situ hybridization; genetic recombination; genetic transfection; recombination, genetic; genomic instability; tumor cell line; western blotting; plasmid; ionizing radiation; genetic stability; replication factor a; chromosome aberrations; single stranded dna; cell strain mcf 7; replication protein a; rad51 protein; mutant; cell stress; rad51 recombinase; comet assay; replication factor a 2; rad51 protein, human; rpa2 protein, human
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