N-terminus of hMLH1 confers interaction of hMutLa and hMutLb with hMutSa

Mismatch repair is a highly conserved system that ensures replication ®delity by repairing mispairs after DNA synthesis. In humans, the two protein heterodimers hMutSa (hMSH2-hMSH6) and hMutLa (hMLH1-hPMS2) constitute the centre of the repair reaction. After recognising a DNA replication error, hMutSa recruits hMutLa, which then is thought to transduce the repair signal to the excision machinery. We have expressed an ATPase mutant of hMutLa as well as its individual subunits hMLH1 and hPMS2 and fragments of hMLH1, followed by examination of their interaction properties with hMutSa using a novel interaction assay. We show that, although the interaction requires ATP, hMutLa does not need to hydrolyse this nucleotide to join hMutSa on DNA, suggesting that ATP hydrolysis by hMutLa happens downstream of complex formation. The analysis of the individual subunits of hMutLa demonstrated that the hMutSa±hMutLa 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, hMutLb (hMLH1±hPMS1). We show that hMutLb interacts as ef®ciently with hMutSa as hMutLa, and that it predominantly binds to hMutSa via hMLH1 as well.