Functional Analysis of Human MLH1 Variants Using Yeast and In vitro Mismatch Repair Assays Masanobu Takahashi, 1 Hideki Shimodaira, 1 Corinne Andreutti-Zaugg, 2 Richard Iggo, 2 Richard D. Kolodner, 3 and Chikashi Ishioka 1 1 Department of Clinical Oncology, Institute of Development, Aging and Cancer, and Tohoku University Hospital, Tohoku University, Sendai, Japan; 2 Oncogene Group, Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland; and 3 Ludwig Institute for Cancer Research, University of California at San Diego, La Jolla, California 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 nonsynon- ymous 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 con- sisted 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 . [Cancer Res 2007;67(10):4595–604] Introduction Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common familial cancer syndromes caused by mainly germ-line mutations in DNA mismatch repair (MMR) genes, such as MLH1 , MSH2 , MSH6 , and PMS2 . MMR contributes to genome integrity by correcting replication errors, particularly mismatch base pairs or slippages in simple repeat sequences. The MMR system is well conserved from Escherichia coli to mammals, and the E. coli MMR system, where MutS, MutL, and MutH complexes function, has been well analyzed. In mammalian cells, hetero- dimers of MutS homologues (MSH2-MSH6 and MSH2-MSH3) recognize replication errors, and the heterodimer of the MutL homologue (MLH1-PMS2) interacts with MutS homologues and recruits further repair proteins. In the two MMR genes, MLH1 and MSH2 , which account for the majority of HNPCC kindred mutations, 31.6% of the MLH1 mutations and 19.4% of the MSH2 mutations are missense (nonsynonymous single nucleotide polymorphisms) according to the InSiGHT database. 4 Pathogenic significances of nonsynon- ymous single nucleotide polymorphisms are not easily evaluated without a functional assay. Generally, this makes genetic diagnosis more difficult, especially when phenotype-genotype segregation analysis is limited because of an ethical issue, the small number of family members, or other reasons. Therefore, functional analysis has been needed to interpret the pathogenicity of MLH1 variants in genetic diagnoses of HNPCC. Several groups, including ours, have attempted to resolve this issue by analyzing the functional significance of MLH1 variants by various methods (1–8). We found a dominant mutator effect (DME) of wild-type MLH1 on interference in the yeast MMR system and evaluated the pathogenicity of 20 MLH1 missense variants using this effect for a yeast-based functional assay (1). One of the other strategies was focusing on the well-defined functions of MLH1, ATPase activity in the NH 2 terminus, and the binding abilities with PMS2 in the COOH terminus (2, 4, 7). Another strategy was based on the assessment of total MMR activity such as in vitro MMR assay, monitoring the MMR rate of cell extracts for heteroduplex DNA-containing mismatch bases (4, 5), or yeast system measuring the replication error rate resulting from the expressed yeast-human hybrid proteins or equivalent yeast variants (3). An alternative approach was to analyze the effects of an MLH1 variant for the expression of MLH1 mRNA and protein and the proliferation rate of cells when an MLH1 variant was introduced into the cells (6). Recently, MLH1 variants were characterized for the multiple functional properties of wild-type MLH1, including protein expression, subcellular localization, MLH1-PMS2 interaction, and MMR efficiency (8). However, it still seems to be important to establish the database on the functional effects of as many variants as possible by various methods and integrate the accumulated data for better understanding of MLH1 variants. Beyond functional analyses, crystal structure analyses of E. coli MutL were used to predict the structural alterations of MLH1 variants (9, 10). Besides the NH 2 terminus, the crystal structure of the COOH terminus of E. coli MutL was identified recently (11). Consideration of the protein structure permits more comprehensive analysis of MLH1 and is thought to provide useful information for interpreting the pathogenesis of MLH1 variants in genetic diagnoses. In this study, we examined 101 MLH1 variants in yeast assays and an in vitro MMR assay, to compare these two assays and to develop the functional database for a large number of variations. Requests for reprints: Chikashi Ishioka, Department of Clinical Oncology, Institute of Development, Aging and Cancer, and Tohoku University Hospital, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22- 717-8547; Fax: 81-22-717-8548; E-mail: chikashi@idac.tohoku.ac.jp. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3509 4 http://www.insight-group.org/ www.aacrjournals.org 4595 Cancer Res 2007; 67: (10). 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