Interactions of human mismatch repair proteins MutSα and MutLα with proteins of the ATR-Chk1 pathway

Yiyong Liu, Yanan Fang, Hongbing Shao, Laura Lindsey-Boltz, Aziz Sancar, Paul Modrich

Research output: Contribution to journalArticle

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Abstract

At clinically relevant doses, chemotherapeutic SN1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSα and MutLα. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the 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, Claspin, and Chk1 and interaction of MutLα with TopBP1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1, and Chk1 and of MutLα with TopBP1. MutSα-Claspin and MutLα-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα- and MutSα-dependent fashion after N-methyl-N′-nitro-N- nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.

Original languageEnglish (US)
Pages (from-to)5974-5982
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number8
DOIs
StatePublished - Feb 19 2010
Externally publishedYes

Fingerprint

DNA Mismatch Repair
Replication Protein A
Repair
Chromatin
Methylnitronitrosoguanidine
Proteins
Chromatin Immunoprecipitation
Chemical activation
Proliferating Cell Nuclear Antigen
Post Translational Protein Processing
Immunoprecipitation
Assays
DNA
Cells
Pharmaceutical Preparations
MutL Proteins
Experiments

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Interactions of human mismatch repair proteins MutSα and MutLα with proteins of the ATR-Chk1 pathway. / Liu, Yiyong; Fang, Yanan; Shao, Hongbing; Lindsey-Boltz, Laura; Sancar, Aziz; Modrich, Paul.

In: Journal of Biological Chemistry, Vol. 285, No. 8, 19.02.2010, p. 5974-5982.

Research output: Contribution to journalArticle

Liu, Yiyong ; Fang, Yanan ; Shao, Hongbing ; Lindsey-Boltz, Laura ; Sancar, Aziz ; Modrich, Paul. / Interactions of human mismatch repair proteins MutSα and MutLα with proteins of the ATR-Chk1 pathway. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 8. pp. 5974-5982.
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abstract = "At clinically relevant doses, chemotherapeutic SN1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSα and MutLα. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the 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, Claspin, and Chk1 and interaction of MutLα with TopBP1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1, and Chk1 and of MutLα with TopBP1. MutSα-Claspin and MutLα-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα- and MutSα-dependent fashion after N-methyl-N′-nitro-N- nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.",
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