RESUMO
Bloom's syndrome (BS) is a rare genetic disorder characterised by genomic instability and cancer susceptibility. BLM, the gene mutated in BS, encodes a member of the RecQ family of DNA helicases. Here, we identify hMLH1, which is involved in mismatch repair (MMR) and recombination, as a protein that directly interacts with BLM both in vivo and in vitro, and that the two proteins co-localise to discrete nuclear foci. The interaction between BLM and hMLH1 appears to have been evolutionarily conserved, as Sgs1p, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Mlh1p. However, cell extracts derived from BS patients show no obvious defects in MMR compared to wild-type- and BLM-complemented BS cell extracts. We conclude that the hMLH1-BLM interaction is not essential for post-replicative MMR, but, more likely, is required for some aspect of genetic recombination.
Assuntos
Adenosina Trifosfatases/metabolismo , Pareamento Incorreto de Bases , Síndrome de Bloom , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Neoplasias/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Adaptadoras de Transdução de Sinal , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Far-Western Blotting , Proteínas de Transporte , Linhagem Celular , Núcleo Celular/metabolismo , Sequência Conservada , DNA Helicases/química , DNA Helicases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Proteína 1 Homóloga a MutL , Mutação/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas Nucleares/metabolismo , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , RecQ Helicases , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Conversion of diploidy to haploidy is a method that allows the generation of stable murine/human hybrid cell lines carrying selected human chromosomes in only a single copy. In this setting, it is possible to detect genetic mutations with greater sensitivity and reliability than in diploid cells. Using this method, we were able to identify mutations in the human mismatch repair (MMR) gene hMSH2 in hereditary nonpolyposis colon cancer families, which have escaped detection by the conventional methods. In this report, we show that such hybrid cell lines can also be a valuable tool in the study of the mutated MMR proteins, in particular the variants found in hereditary nonpolyposis colon cancer families that carry missense mutations and where it is unclear whether they predispose to colon cancer. This analysis is made possible by the fact that the human hMSH2 protein is able to complement the MMR defect in the host murine cell line.
Assuntos
Cromossomos Humanos Par 2/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Alelos , Animais , Pareamento Incorreto de Bases , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Inativação Gênica , Humanos , Células Híbridas , Camundongos , Proteína 2 Homóloga a MutS , Mutação de Sentido IncorretoRESUMO
Hepatitis C virus (HCV) infection is a major health problem that leads to cirrhosis and hepatocellular carcinoma in a substantial number of infected individuals, estimated to be 100-200 million worldwide. Unfortunately, immunotherapy or other effective treatments for HCV infection are not yet available, and interferon administration has limited efficacy. Different approaches to HCV therapy are being explored, and these include inhibition of the viral proteinase, helicase, and RNA-dependent RNA polymerase and development of a vaccine. Here we present the design of selective inhibitors with nanomolar potencies of HCV NS3 proteinase based on eglin c. These eglin c mutants were generated by reshaping the inhibitor active site-binding loop, and the results emphasize the role played by residues P5-P4' in enzyme recognition. In addition, alanine scanning experiments provide evidence that the N terminus of eglin c also contributes to NS3 binding. These eglin inhibitors offer a unique tool for accurately assessing the requirements for effective inhibition of the enzymatic activity of NS3 and at the same time can be considered lead compounds for the identification of other NS3 inhibitors in targeted design efforts.