Interactions of human mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway.

Published

Journal Article

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 MutSalpha and MutLalpha. 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 MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSalpha with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLalpha with TopBP1 and Claspin. We were unable to detect interaction of MutSalpha or MutLalpha with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSalpha with ATR, TopBP1, and Chk1 and of MutLalpha with TopBP1. MutSalpha-Claspin and MutLalpha-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 MutLalpha- and MutSalpha-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.

Full Text

Duke Authors

Cited Authors

  • Liu, Y; Fang, Y; Shao, H; Lindsey-Boltz, L; Sancar, A; Modrich, P

Published Date

  • February 2010

Published In

Volume / Issue

  • 285 / 8

Start / End Page

  • 5974 - 5982

PubMed ID

  • 20029092

Pubmed Central ID

  • 20029092

Electronic International Standard Serial Number (EISSN)

  • 1083-351X

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.m109.076109

Language

  • eng