Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage.
RPA (replication protein A) is an essential factor for DNA DSB (double-strand break) repair and cell cycle checkpoint activation. The 32 kDa subunit of RPA undergoes hyperphosphorylation in response to cellular genotoxic insults. However, the potential involvement of hyperphosphorylated RPA in DSB repair and checkpoint activation remains unclear. Using co-immunoprecipitation assays, we showed that cellular interaction of RPA with two DSB repair factors, Rad51 and Rad52, was predominantly mediated by the hyperphosphorylated species of RPA in cells after UV and camptothecin treatment. Moreover, Rad51 and Rad52 displayed higher affinity for the hyperphosphorylated RPA than native RPA in an in vitro binding assay. Checkpoint kinase ATR (ataxia telangiectasia mutated and Rad3-related) also interacted more efficiently with the hyperphosphorylated RPA than with native RPA following DNA damage. Consistently, immunofluorescence microscopy demonstrated that the hyperphosphorylated RPA was able to co-localize with Rad52 and ATR to form significant nuclear foci in cells. Our results suggest that hyperphosphorylated RPA is preferentially localized to DSB repair and the DNA damage checkpoint complexes in response to DNA damage.
Wu
Xiaoming
X
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA.
Yang
Zhengguan
Z
Liu
Yiyong
Y
Zou
Yue
Y
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000235:genetics* / Q000528:radiation effects; D004260:DNA Repair; D005786:Gene Expression Regulation; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D051135:Rad51 Recombinase / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism*; D014466:Ultraviolet Rays
D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; D051135:Rad51 Recombinase