title: Functional analysis of human MLH1 variants using yeast and in vitro mismatch repair assays.
abstract: The functional characterization of nonsynonymous single nucleotide polymorphisms in human mismatch repair (MMR) genes has been critical to evaluate their pathogenicity for hereditary nonpolyposis colorectal cancer. We previously established an assay for detecting loss-of-function mutations in the MLH1 gene using a dominant mutator effect of human MLH1 expressed in Saccharomyces cerevisiae. The purpose of this study is to extend the functional analyses of nonsynonymous single nucleotide polymorphisms in the MLH1 gene both in quality and in quantity, and integrate the results to evaluate the variants for pathogenic significance. The 101 MLH1 variants, which covered most of the reported MLH1 nonsynonymous single nucleotide polymorphisms and consisted of one 3-bp deletion, 1 nonsense and 99 missense variants, were examined for the dominant mutator effect by three yeast assays and for the ability of the variant to repair a heteroduplex DNA with mismatch bases by in vitro MMR assay. There was diversity in the dominant mutator effects and the in vitro MMR activities among the variants. The majority of functionally inactive variants were located around the putative ATP-binding pocket of the NH(2)-terminal domain or the whole region of the COOH-terminal domain. Integrated functional evaluations contribute to a better prediction of the cancer risk in individuals or families carrying MLH1 variants and provide insights into the function-structure relationships in MLH1.
authors.affiliation: Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University Hospital, Tohoku University, Sendai, Japan.
authors.forename: Masanobu
authors.initials: M
authors.lastname: Takahashi
authors.forename: Hideki
authors.initials: H
authors.lastname: Shimodaira
authors.forename: Corinne
authors.initials: C
authors.lastname: Andreutti-Zaugg
authors.forename: Richard
authors.initials: R
authors.lastname: Iggo
authors.forename: Richard D
authors.initials: RD
authors.lastname: Kolodner
authors.forename: Chikashi
authors.initials: C
authors.lastname: Ishioka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D045325:HCT116 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C501486:MLH1 protein, human; D009687:Nuclear Proteins; D020033:Protein Isoforms; D000070957:MutL Protein Homolog 1