Identification of MLH2/hPMS1 dominant mutations that prevent DNA mismatch repair function

AbstractInactivating mutations affecting key mismatch repair (MMR) components lead to microsatellite instability (MSI) and cancer. However, a number of patients with MSI-tumors do not present alterations in classical MMR genes. Here we discovered that specific missense mutations in the MutL homolog MLH2, which is dispensable for MMR, confer a dominant mutator phenotype in S. cerevisiae. MLH2 mutations elevated frameshift mutation rates, and caused accumulation of long-lasting nuclear MMR foci. Both aspects of this phenotype were suppressed by mutations predicted to prevent the binding of Mlh2 to DNA. Genetic analysis revealed that mlh2 dominant mutations interfere with both Exonuclease 1 (Exo1)-dependent and Exo1-independent MMR. Lastly, we demonstrate that a homolog mutation in human hPMS1 results in a dominant mutator phenotype. Our data support a model in which yeast Mlh1-Mlh2 or hMLH1-hPMS1 mutant complexes act as roadblocks on DNA preventing MMR, unraveling a novel mechanism that can account for MSI in human cancer.

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224 Specific Nucleotides That Flank Coding Microsatellites of Activin Receptor 2 (ACVR2) Determine Exonic Selectivity and Control Frameshift Mutation Rates in Defective DNA Mismatch Repair (MMR)

Gastroenterology ◽

10.1016/s0016-5085(10)60194-1 ◽

2010 ◽

Vol 138 (5) ◽

pp. S-42-S-43

Author(s):

Heekyung Chung ◽

Jenny Lai ◽

Joy Holmstrom ◽

Dennis J. Young ◽

John M. Carethers

Keyword(s):

Mismatch Repair ◽

Frameshift Mutation ◽

Dna Mismatch Repair ◽

Mutation Rates ◽

Activin Receptor ◽

Dna Mismatch ◽

And Control

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Both microsatellite length and sequence context determine frameshift mutation rates in defective DNA mismatch repair

Human Molecular Genetics ◽

10.1093/hmg/ddq151 ◽

2010 ◽

Vol 19 (13) ◽

pp. 2638-2647 ◽

Cited By ~ 13

Author(s):

Heekyung Chung ◽

Claudia G. Lopez ◽

Joy Holmstrom ◽

Dennis J. Young ◽

Jenny F. Lai ◽

...

Keyword(s):

Mismatch Repair ◽

Frameshift Mutation ◽

Dna Mismatch Repair ◽

Mutation Rates ◽

Sequence Context ◽

Dna Mismatch

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Mutator Phenotypes Conferred by MLH1Overexpression and by Heterozygosity for mlh1Mutations

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.3177 ◽

1999 ◽

Vol 19 (4) ◽

pp. 3177-3183 ◽

Cited By ~ 119

Author(s):

Polina V. Shcherbakova ◽

Thomas A. Kunkel

Keyword(s):

Mismatch Repair ◽

Germ Line ◽

Genome Instability ◽

Human Cancer ◽

Missense Mutations ◽

Wild Type ◽

Mutator Phenotype ◽

Yeast Strains ◽

Germ Line Mutations ◽

Mutator Effect

ABSTRACT Loss of DNA mismatch repair due to mutation or diminished expression of the MLH1 gene is associated with genome instability and cancer. In this study, we used a yeast model system to examine three circumstances relevant to modulation of MLH1function. First, overexpression of wild-type MLH1 was found to cause a strong elevation of mutation rates at three different loci, similar to the mutator effect of MLH1 gene inactivation. Second, haploid yeast strains with any of six mlh1 missense mutations that mimic germ line mutations found in human cancer patients displayed a strong mutator phenotype consistent with loss of mismatch repair function. Five of these mutations affect amino acids that are homologous to residues suggested by recent crystal structure and biochemical analysis of Escherichia coli MutL to participate in ATP binding and hydrolysis. Finally, using a highly sensitive reporter gene, we detected a mutator phenotype of diploid yeast strains that are heterozygous for mlh1 mutations. Evidence suggesting that this mutator effect results not from reduced mismatch repair in the MLH1/mlh1 cells but rather from loss of the wild-type MLH1 allele in a fraction of cells is presented. Exposure to bleomycin or to UV irradiation strongly enhanced mutagenesis in the heterozygous strain but had little effect on the mutation rate in the wild-type strain. This damage-induced hypermutability may be relevant to cancer in humans with germ line mutations in only one MLH1 allele.

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Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

Journal of Bacteriology ◽

10.1128/jb.01435-09 ◽

2010 ◽

Vol 192 (13) ◽

pp. 3452-3463 ◽

Cited By ~ 17

Author(s):

Nicole M. Dupes ◽

Brian W. Walsh ◽

Andrew D. Klocko ◽

Justin S. Lenhart ◽

Heather L. Peterson ◽

...

Keyword(s):

Bacillus Subtilis ◽

Dna Replication ◽

Mismatch Repair ◽

Mutation Frequency ◽

Elevated Temperatures ◽

Temperature Sensitive ◽

Mutant Form ◽

Appreciable Effect ◽

Missense Mutations ◽

Dna Mismatch

ABSTRACT The β clamp is an essential replication sliding clamp required for processive DNA synthesis. The β clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of β clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the β clamp, a common site occupied by proteins that bind the β clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis β clamp separate the role of the β clamp in DNA replication from its role in MMR.

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Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Nucleic Acids Research ◽

10.1093/nar/gkl1166 ◽

2007 ◽

Vol 35 (8) ◽

pp. 2609-2619 ◽

Cited By ~ 22

Author(s):

Nina Østergaard Knudsen ◽

Finn Cilius Nielsen ◽

Lena Vinther ◽

Ronni Bertelsen ◽

Steen Holten-Andersen ◽

...

Keyword(s):

Mismatch Repair ◽

Nuclear Localization ◽

Dna Mismatch Repair ◽

Dna Mismatch ◽

Human Dna ◽

Repair Protein ◽

Exonuclease 1 ◽

Mismatch Repair Protein

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Role of DNA Mismatch Repair Defects in the Pathogenesis of Human Cancer

Journal of Clinical Oncology ◽

10.1200/jco.2003.04.060 ◽

2003 ◽

Vol 21 (6) ◽

pp. 1174-1179 ◽

Cited By ~ 451

Author(s):

Päivi Peltomäki

Keyword(s):

Mismatch Repair ◽

Dna Mismatch Repair ◽

Human Cancer ◽

Hereditary Nonpolyposis Colon Cancer ◽

Dna Mismatch ◽

Uterine Endometrium ◽

Hereditary Nonpolyposis ◽

Colon And Rectum ◽

Cancer Syndromes

The DNA mismatch repair (MMR) system is necessary for the maintenance of genomic stability. In a broad sense, all main functions of the MMR system, including the correction of biosynthetic errors, DNA damage surveillance, and prevention of recombination between nonidentical sequences serve this important purpose. Failure to accomplish these functions may lead to cancer. It is therefore not surprising that inherited defects in the MMR system underlie one of the most prevalent cancer syndromes in humans, hereditary nonpolyposis colon cancer (HNPCC). In addition, acquired defects of the same system may account for 15% to 25%, or even a higher percentage, of sporadic cancers of different organs of the “HNPCC spectrum,” including the colon and rectum, uterine endometrium, stomach, and ovaries. Recent studies indicate that the MMR genes may be involved in the pathogenesis of even a broader spectrum of tumors in one way or another. An updated review of the different features of the human MMR system will be provided, with the emphasis on their implications in cancer development.

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Trifluridine to enhance cytotoxicity by DNA mismatch repair deficiency and subsequent MBD4 frameshift mutation in colorectal cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2016.34.4_suppl.608 ◽

2016 ◽

Vol 34 (4_suppl) ◽

pp. 608-608 ◽

Cited By ~ 3

Author(s):

Satoshi Suzuki ◽

Moriya Iwaizumi ◽

Yasushi Hamaya ◽

Kosuke Takagaki ◽

Satoshi Osawa ◽

...

Keyword(s):

Colorectal Cancer ◽

Mismatch Repair ◽

Frameshift Mutation ◽

Dna Mismatch Repair ◽

Clonogenic Assay ◽

Dna Mismatch ◽

Cell Clones ◽

Human Colorectal Cancer Cell ◽

Mmr Deficiency ◽

Colorectal Cancer Patients

608 Background: TAS-102 is composed of trifluridine (FTD) and tipiracil hydrochloride and was shown to prolong survival of patients with refractory metastatic colorectal cancer (CRC). FTD is the active antitumor component of TAS102 and its metabolite TF-TTP chemically resembles 5FU-derived metabolite FdUTP in that both of them are misincorporated into DNA and lead to cytotoxicity. Several groups have reported that stage II-III colorectal cancer patients with tumors that lost DNA mismatch repair (MMR) function do not derive a benefit from 5-FU based chemotherapy. Although FTD is reported to be misincorporated into DNA, it is not known if MMR deficient CRC cells have chemoresistance for FTD. Methods: We first utilized human colorectal cancer cell lines HCT-116 (hMLH1-deficient cells) and HCT116+ch3 (hMLH1-retained cells), and compared cytotoxicity for FTD by clonogenic assay. To further analyze if 5FU refractory CRC cells have chemosensitivity for FTD, we established 5FU refractory HCT116 cells by continuous 5FU treatment for 10 month and analyzed cytotoxicity for FTD. Finally we constructed an expression plasmid of truncated DNA grycosylase MBD4, (MBD4tru) by frameshift mutation with MMR deficiency and stably transfected the construct into HCT116 CRC cells and selected HCT116MBD4tru cell clones. The HCT116MBD4tru cells were treated with FTD and analyzed for cytotoxicity by clonogenic assay. Results: In hMLH1-deficient cells, the number of colony was reduced by FTD treatment to a same degree of hMLH1-proficient cells whereas the number of colony by 5FU treatment is higher in hMLH1-deficitent cells than hMLH1-retained cells (p< 0.05). In 5FU refractory cells, treatment of FTD showed cytotoxicity to the same degree of non-5FU refractory cells. Interestingly, HCT116MBD4tru cells led to cytotoxicity with a higher sensitivity than control cells (p< 0.05). Conclusions: FTD induces cytotoxisity independently of MMR status as well as under 5FU refractory condition, and MBD4 frameshift mutation by MMR deficiency enhances FTD sensitivity. These results suggest that FTD may be useful for patients with MSI-H/MBD4 mutant CRC as well as for those with 5FU refractory CRC.

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Characterization of Pathogenic Human MSH2 Missense Mutations Using Yeast as a Model System: A Laboratory Course in Molecular Biology

Cell Biology Education ◽

10.1187/cbe.03-08-0006 ◽

2004 ◽

Vol 3 (1) ◽

pp. 31-48 ◽

Cited By ~ 10

Author(s):

Alison E. Gammie ◽

Naz Erdeniz

Keyword(s):

Molecular Biology ◽

Mismatch Repair ◽

Recombinant Dna ◽

Dna Mismatch Repair ◽

Genetic Material ◽

Molecular Techniques ◽

Original Research ◽

Missense Mutations ◽

Laboratory Course ◽

Dna Mismatch

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.

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