Functional analysis of HNPCC-related missense mutations in MSH2.
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.
Lützen
Anne
A
Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark.
de Wind
Niels
N
Georgijevic
Dubravka
D
Nielsen
Finn Cilius
FC
Rasmussen
Lene Juel
LJ
D000818:Animals; D001483:Base Sequence; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics* / Q000378:metabolism; D053843:DNA Mismatch Repair / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics / Q000378:metabolism; D051718:MutS Homolog 2 Protein / Q000737:chemistry / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D010641:Phenotype; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D004273:DNA, Neoplasm; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C497172:MSH2 protein, human; C497173:Msh2 protein, mouse; D051719:MutS DNA Mismatch-Binding Protein; D051718:MutS Homolog 2 Protein; C497175:MutS protein, E coli