Mutations in the MutSα interaction interface of MLH1 can abolish DNA mismatch repair

Abstract

MutLα, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSα and assembles and controls further repair enzymes. We tested if the interaction of MutLα with DNA-bound MutSα is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLα for MutSα is located on the edge of an extensive β-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein–protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL–MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLα–MutSα interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.

Expression Defect Size among Unclassified MLH1 Variants Determines Pathogenicity in Lynch Syndrome Diagnosis

“…Missense variants were generated via site-directed mutagenesis (QuikChange II Kit, Stratagene) and confirmed by direct sequencing. HEK293T cells were transiently transfected with 5 mg of vector DNA and 20 mL of polyethyleneimine (1 mg/mL, linear, 25 kDa, Polysciences) and extracted as described previously (24,25). The extracts were analyzed via SDS-PAGE and immunoblotting (using anti-MLH1, G168-728, BD Biosciences, and anti-PMS2, E-19, and anti-b-actin, C2, from Santa Cruz Biotechnologies).…”

“…This RNA had either been prepared fresh or came from samples that had been stored at À80 C for less than one month and not thawed more than twice. Reverse transcription was conducted for 10 minutes at 25 C, followed by 50 minutes at 50 C, with M-MLV reverse transcriptase (50U, RNase H Minus point mutant, Promega) and 250 ng of random primers (Promega) according to the manufacturers' recommendations in a total volume of 25 mL. The cDNA samples were stored at À20 C. Primer and probe sequences were designed using FileBuilder software and produced by Applied Biosystems.…”

A conserved MutS homolog connector domain interface interacts with MutL homologs

“…Recently, mutations in the N-terminal domain of Mlh1 were shown to eliminate Msh2-Msh6 binding, although it is unclear whether the mutations affect a region directly involved in complex assembly (27). In addition, the mispair binding domain of Msh6 and the mispair that is being recognized are unlikely to be parts of the interface as revealed by the genetics of an msh6 allele encoding the mispair binding domain of Msh3 (28).…”

A new mutation in Muir-Torre syndrome associated with familiar transmission of different gastrointestinal adenocarcinomas

“…This mutation leading to an Arg>Pro change, has not been published yet in english medical literature, and in the international databases (11). The heterodimer united from the hMLH1 and hPMS2 called MutLα has an important role to correct the errors generated during DNA replication (12). During this repair process MutLα has an ATP-dependent connection with the MutSα which identifies the DNA mismatches (12).…”

“…The heterodimer united from the hMLH1 and hPMS2 called MutLα has an important role to correct the errors generated during DNA replication (12). During this repair process MutLα has an ATP-dependent connection with the MutSα which identifies the DNA mismatches (12). Arg265 is situated in the MutS homologs interaction domain, which is an evolutionally conservative region of hMLH1.…”