Functional analysis of HNPCC-related missense mutations in MSH2

TY - JOUR

T1 - Functional analysis of HNPCC-related missense mutations in MSH2

AU - Lützen, Anne

AU - de Wind, Niels

AU - Georgijevic, Dubravka

AU - Nielsen, Finn Cilius

AU - Rasmussen, Lene Juel

PY - 2008/10/14

Y1 - 2008/10/14

N2 - Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.

AB - Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.

KW - Animals

KW - Base Sequence

KW - Colorectal Neoplasms, Hereditary Nonpolyposis

KW - DNA Mismatch Repair

KW - DNA, Neoplasm

KW - Escherichia coli

KW - Escherichia coli Proteins

KW - Humans

KW - Mice

KW - Models, Molecular

KW - MutS DNA Mismatch-Binding Protein

KW - MutS Homolog 2 Protein

KW - Mutagenesis, Site-Directed

KW - Mutation, Missense

KW - NIH 3T3 Cells

KW - Phenotype

KW - Protein Structure, Tertiary

KW - Recombinant Proteins

KW - Two-Hybrid System Techniques

U2 - 10.1016/j.mrfmmm.2008.08.015

DO - 10.1016/j.mrfmmm.2008.08.015

M3 - Journal article

C2 - 18822302

SN - 0027-5107

VL - 645

SP - 44

EP - 55

JO - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis

JF - Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis

IS - 1-2

ER -