Nucleic Acids Research, 2003, Vol. 31, No. 12 3217-3226
© 2003 Oxford University Press
N-terminus of hMLH1 confers interaction of hMutL
and hMutLß with hMutS
2nd Department of Medicine, University of the Saarland, Kirrberger Straße, D-66421 Homburg/Saar, Germany and 1 2nd Department of Medicine, University of Frankfurt, Theodor Stern-Kai 7, D-60590 Frankfurt, Germany
*To whom correspondence should be addressed. Tel: +49 6841 16 23201; Fax: +49 6841 16 23267; Email: zeuzem{at}uniklinik-saarland.de
Mismatch repair is a highly conserved system that ensures replication fidelity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutS
(hMSH2-hMSH6) and hMutL (hMLH1-hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutS recruits hMutL, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutL as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutS using a novel interaction assay. We show that, although the interaction requires ATP, hMutL does not need to hydrolyse this nucleotide to join hMutS on DNA, suggesting that ATP hydrolysis by hMutL happens downstream of complex formation. The analysis of the individual subunits of hMutL demonstrated that the hMutS–hMutL 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, hMutLß (hMLH1–hPMS1). We show that hMutLß interacts as efficiently with hMutS as hMutL, and that it predominantly binds to hMutS via hMLH1 as well.
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