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Interactions of human mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR‐Chk1 pathway
Author(s) -
Liu Yiyong,
Fang Yanan,
Shao Hongbing,
Lindseyboltz Laura,
Sancar Aziz,
Modrich Paul
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.492.10
Subject(s) - chromatin , dna mismatch repair , chromatin immunoprecipitation , dna damage , dna repair , immunoprecipitation , proliferating cell nuclear antigen , microbiology and biotechnology , chemistry , dna , biology , biochemistry , gene , gene expression , promoter
At clinically relevant doses, chemotherapeutic S N 1 DNA methylating agents induce an ATR‐mediated checkpoint response in human cells that is dependent on functional mismatch repair activities MutSα and MutLα. To better understand the mechanisms linking mismatch repair to ATR‐Chk1 checkpoint activation, we have systematically examined interactions of human MutSα and MutLα with proteins of the ATR‐Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co‐immunoprecipitation, we have detected interaction of MutSα with ATR, TopBP1, BRCA1, Claspin and Chk1, and interaction of MutLα with TopBP1, BRCA1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9 or RPA in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1 and Chk1, and MutLα with TopBP1. Use of a modified chromatin immunoprecipitation assay showed that PCNA, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα‐ and MutSα‐dependent fashion after treatment with N ‐methyl‐ N ′‐nitro‐ N ‐nitrosoguanidine. While our failure to observe chromatin enrichment of RPA, Claspin, Rad17‐RFC, and Rad9‐Rad1‐Hus1 could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation. This study was financially supported by the National Institute of General Medical Sciences and the Howard Hughes Medical Institute.