PCNA-MutSalpha-mediated binding of MutLalpha to replicative DNA with mismatched bases to induce apoptosis in human cells.
Modified bases, such as O6-methylguanines, are produced in cells exposed to alkylating agents and cause apoptosis. In human cells treated with N-methyl-N-nitrosourea, we detected a protein complex composed of MutSalpha, MutLalpha and PCNA on damaged DNA by immunoprecipitation method using chromatin extracts, in which protein-protein interactions were stabilized by chemical crosslinking. Time course experiments revealed that MutSalpha, consisting of MSH2 and MSH6 proteins, and PCNA bind to DNA to form an initial complex, and MutLalpha, composed of MLH1 and PMS2, binds to the complex when the DNA is damaged. This sequential mode of binding was further confirmed by the findings that the association of PCNA-MutSalpha complex on chromatin was observed even in the cells that lack MLH1, whereas in the absence of MSH2 no association of MutLalpha with the chromatin was achieved. Moreover, reduction in the PCNA content by small-interfering RNA or inhibition of DNA replication by aphidicolin, an inhibitor of DNA polymerase, significantly reduced the levels of the PCNA-MutSalpha-MutLalpha complex and also suppressed an increase in the caspase-3 activity, a hallmark for the induction of apoptosis. These observations imply that the induction of apoptosis is coupled with the progression of DNA replication through the action of PCNA.
Hidaka
Masumi
M
Department of Molecular Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan. hidaka@beri.or.jp
Takagi
Yasumitsu
Y
Takano
Tomoko Y
TY
Sekiguchi
Mutsuo
M
D017209:Apoptosis; D020137:Base Pair Mismatch; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D045643:DNA Repair Enzymes; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008770:Methylnitrosourea; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism
D002843:Chromatin; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; C500929:MutLalpha protein, human; D009363:Neoplasm Proteins; D018809:Proliferating Cell Nuclear Antigen; D011518:Proto-Oncogene Proteins; D006147:Guanine; D008770:Methylnitrosourea; D004247:DNA; C008449:O-(6)-methylguanine; C497172:MSH2 protein, human; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D045643:DNA Repair Enzymes