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Phosphorylation of RPA2 Is Required For TopBP1 Recruitment and ATR Activation
Author(s) -
Liu Shengqin,
Glanzer Jason,
Oakley Greg
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.875.2
Subject(s) - phosphorylation , dna damage , replication protein a , microbiology and biotechnology , chromatin , biology , chemistry , dna , dna binding protein , biochemistry , transcription factor , gene
Replication protein A (RPA) is a heterotrimer, single strand DNA binding protein involved in multiple aspects of DNA metabolism. The N‐terminus of the RPA2 subunit is phosphorylated at multiple sites during cell cycle progression and in response to DNA damage. To understand the role of RPA2 phosphorylation, we generated stable cell lines expressing wild‐type and mutant forms of RPA with down regulation of endogenous RPA2 by shRNA. The inability to phosphorylate Ser4 and Ser8 resulted in a loss of phosphorylation of Ser12 and Thr21, but not Ser33 in response to etoposide treatment. Cells expressing mutant RPA2 showed a defective G2/M checkpoint and failed to halt mitotic entry after etoposide treatment. Decreased phosphorylation of Chk1 and Mre11, suggested ATR activation in response to etoposide was compromised in cells expressing mutant RPA2. TopBP1, a key activator of ATR, is recruited to DNA lesions by Rad9. Rad9 also interacts with RPA through binding with RPA1. Defective phosphorylation of the mutant RPA increased interaction with Rad9 and inhibited phosphorylation of Rad9. The decrease in the phosphorylation of Rad9 correlated with a loss of TopBP1 recruitment to chromatin in response to DNA damage. Taken together, these results suggest that phosphorylation of RPA may regulate RPA function by altering its interaction with other key proteins in the DNA damage response pathway.