Role of MutSalpha in the recognition of iododeoxyuridine in DNA.
We have previously demonstrated that both the MLH1 and MSH2 status impact the DNA levels of the halogenated thymidine (dThd) analogues iododeoxyuridine (IdUrd) and bromodeoxyuridine (BrdUrd), and thereby radiosensitization induced by these analogues, indirectly implicating both mismatch repair (MMR) proteins in the removal of these bases from DNA. More recent data from our group demonstrate that base excision repair (BER) also impacts IdUrd-DNA levels, supporting a role for the BER pathway in IdUrd removal as well. In this study, we have examined more direct interactions between the MSH2 protein and the processing of IdUrd incorporated in DNA. Our data demonstrate that the MutSalpha (MSH2/MSH6) complex binds specifically to DNA containing an IdUrd-G mismatch, using both purified human MutSalpha as well as nuclear extracts from Msh2-proficient and-deficient mouse cell lines. MutSalpha binding to a IdUrd-G is better recognized than a G-T mismatch in the same sequence context. In addition, MSH2 protein can be found colocalized with IdUrd-DNA using confocal microscopy in G(1) synchronized cells after treatment with IdUrd. Consistent with our recent publication, coadministration of IdUrd and a chemical inhibitor of BER, methoxyamine (MX), also increases the extent of MSH2 nuclear colocalization with IdUrd. Furthermore, we show that the extent of MSH2 colocalization with IdUrd in G(1)-synchronized human tumor cells varies with MLH1 status, suggesting a role for the MLH1 protein in stabilizing the interaction between the MSH2 protein and DNA containing IdUrd. These data, both in vitro and in vivo, suggest direct involvement of MSH2 in processing IdUrd in DNA.
D048868:Adaptor Proteins, Signal Transducing; D020137:Base Pair Mismatch; D002352:Carrier Proteins; D002467:Cell Nucleus / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016193:G1 Phase / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006898:Hydroxylamines / Q000494:pharmacology; D007065:Idoxuridine / Q000378:metabolism* / Q000494:pharmacology; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C094443:G-T mismatch-binding protein; D006898:Hydroxylamines; C501486:MLH1 protein, human; C501485:Mlh1 protein, mouse; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D004247:DNA; C005214:methoxyamine; C497172:MSH2 protein, human; D000070957:MutL Protein Homolog 1; D051718:MutS Homolog 2 Protein; D007065:Idoxuridine