The role of RPA2 phosphorylation in homologous recombination in response to replication arrest.
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.
Department of Radiation Oncology, Washington University School of Medicine, St. Louis, MO 63108, USA.
Wei
W
Shi
Zhihui
Z
Feng
Jiuqin
J
Zhang
Ignacio
I
Gonzalez-Suarez
Robert P
RP
Vanderwaal
Xiaohua
X
Wu
Simon N
SN
Powell
Joseph L
JL
Roti Roti
Susana
S
Gonzalo
Junran
J
Zhang
D015153:Blotting, Western; D045744:Cell Line, Tumor; D002869:Chromosome Aberrations; D020552:Comet Assay; D004249:DNA Damage; D042822:Genomic Instability; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D017404:In Situ Hybridization, Fluorescence; D010766:Phosphorylation; D051135:Rad51 Recombinase / Q000378:metabolism; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000378:metabolism*
D051716:Replication Protein A; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; C516794:RPA2 protein, human; D006918:Hydroxyurea