RESUMEN
Hereditary non-polyposis colorectal cancer (HNPCC; OMIM 120435-6) is a cancer-susceptibility syndrome linked to inherited defects in human mismatch repair (MMR) genes. Germline missense human MLH1 (hMLH1) mutations are frequently detected in HNPCC (ref. 3), making functional characterization of mutations in hMLH1 critical to the development of genetic testing for HNPCC. Here, we describe a new method for detecting mutations in hMLH1 using a dominant mutator effect of hMLH1 cDNA expressed in Saccharomyces cerevisiae. The majority of hMLH1 missense mutations identified in HNPCC patients abolish the dominant mutator effect. Furthermore, PCR amplification of hMLH1 cDNA from mRNA from a HNPCC patient, followed by in vivo recombination into a gap expression vector, allowed detection of a heterozygous loss-of-function missense mutation in hMLH1 using this method. This functional assay offers a simple method for detecting and evaluating pathogenic mutations in hMLH1.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Mutación/fisiología , Proteínas de Neoplasias/genética , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras , Reparación del ADN/genética , Genes Dominantes , Variación Genética/genética , Vectores Genéticos/genética , Humanos , Homólogo 1 de la Proteína MutL , Mutagénesis , Proteínas NuclearesRESUMEN
Mutations in the S. cerevisiae RAD27 (also called RTH1 or YKL510) gene result in a strong mutator phenotype. In this study we show that the majority of the resulting mutations have a structure in which sequences ranging from 5-108 bp flanked by direct repeats of 3-12 bp are duplicated. Such mutations have not been previously detected at high frequency in the mutation spectra of mutator strains. Epistasis analysis indicates that RAD27 does not play a major role in MSH2-dependent mismatch repair. Mutations in RAD27 cause increased rates of mitotic crossing over and are lethal in combination with mutations in RAD51 and RAD52. These observations suggest that the majority of replication errors that accumulate in rad27 strains are processed by double-strand break repair, while a smaller percentage are processed by a mutagenic repair pathway. The duplication mutations seen in rad27 mutants occur both in human tumors and as germline mutations in inherited human diseases.
Asunto(s)
Reparación del ADN , Genes Fúngicos , Mutación , Proteínas Quinasas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Secuencia de Bases , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Intercambio Genético , Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Mutación de Línea Germinal , Humanos , Mitosis , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS , Neoplasias/genética , Fenotipo , Recombinasa Rad51 , Proteína Recombinante y Reparadora de ADN Rad52 , Recombinación Genética , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.
Asunto(s)
Disparidad de Par Base , Reparación del ADN , ADN/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe , Transglutaminasas , Secuencia de Bases , ADN/química , Cartilla de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genotipo , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS , Reacción en Cadena de la Polimerasa , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/efectos de la radiación , Rayos UltravioletaRESUMEN
Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein. In this study the role of MSH2, MSH3, and MSH6 in mismatch repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of msh2, msh3, and msh6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins. The results indicate that S. cerevisiae has two pathways of MSH2-dependent mismatch repair: one that recognized single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3. The redundancy of MSH3 and MSH6 explains the greater prevalence of hmsh2 mutations in HNPCC families and suggests how the role of hmsh3 and hmsh6 mutations in cancer susceptibility could be analyzed.
Asunto(s)
Adenosina Trifosfatasas , Reparación del ADN/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Alelos , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Cartilla de ADN/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Bases de Datos Factuales , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Modelos Genéticos , Datos de Secuencia Molecular , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga de MutS , Fenotipo , Filogenia , Mutación Puntual/genética , Pruebas de Precipitina , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Análisis de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5'-3' exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.