N-terminus of hMLH1 confers interaction of hMutLalpha and hMutLbeta with hMutSalpha.
Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSalpha (hMSH2-hMSH6) and hMutLalpha (hMLH1-hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSalpha recruits hMutLalpha, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLalpha as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSalpha using a novel interaction assay. We show that, although the interaction requires ATP, hMutLalpha does not need to hydrolyse this nucleotide to join hMutSalpha on DNA, suggesting that ATP hydrolysis by hMutLalpha happens downstream of complex formation. The analysis of the individual subunits of hMutLalpha demonstrated that the hMutSalpha-hMutLalpha interaction is predominantly conferred by hMLH1. Further experiments revealed that only the N-terminus of hMLH1 confers this interaction. In contrast, only the C-terminus stabilised and co-immunoprecipitated hPMS2 when both proteins were co-expressed in 293T cells, indicating that dimerisation and stabilisation are mediated by the C-terminal part of hMLH1. We also examined another human homologue of bacterial MutL, hMutLbeta (hMLH1-hPMS1). We show that hMutLbeta interacts as efficiently with hMutSalpha as hMutLalpha, and that it predominantly binds to hMutSalpha via hMLH1 as well.
Plotz
Guido
G
2nd Department of Medicine, University of the Saarland, Kirrberger Strasse, D-66421 Homburg/Saar, Germany.
Raedle
Jochen
J
Brieger
Angela
A
Trojan
Jörg
J
Zeuzem
Stefan
S
D048868:Adaptor Proteins, Signal Transducing; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002352:Carrier Proteins; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D017384:Sequence Deletion
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; C501486:MLH1 protein, human; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C088996:PMS1 protein, human; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C094934:PMS2 protein, human; C497172:MSH2 protein, human; D000070976:Mismatch Repair Endonuclease PMS2; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D045643:DNA Repair Enzymes