Interactions of human mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway.
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.
Liu
Yiyong
Y
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Fang
Yanan
Y
Shao
Hongbing
H
Lindsey-Boltz
Laura
L
Sancar
Aziz
A
Modrich
Paul
P
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D053843:DNA Mismatch Repair / Q000187:drug effects / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D000070956:MutL Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C500929:MutLalpha protein, human; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; C113575:Rad17 protein, human; D051716:Replication Protein A; C110513:TOPBP1 protein, human; D008769:Methylnitronitrosoguanidine; C069930:rad9 protein; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D000070956:MutL Proteins; D045643:DNA Repair Enzymes