RESUMO
Germline mutations in the human DNA mismatch repair (MMR) genes MSH2 and MLH1 are associated with the inherited cancer disorder Lynch syndrome (LS), also known as hereditary nonpolyposis colorectal cancer or HNPCC. A proportion of MSH2 and MLH1 mutations found in suspected LS patients give rise to single amino acid substitutions. The functional consequences in regard to pathogenicity of many of these variants are unclear. We have examined the functionality of a panel of MLH1 missense mutations found in LS families, by testing the variant proteins in functional assays, addressing subcellular localization, and protein-protein interaction with the dimer partner PMS2 and the MMR-associated exonuclease 1. We show that a significant proportion of examined variant proteins have functional defects in either subcellular localization or protein-protein interactions, which is suspected to lead to the cancer phenotype observed in patients. Moreover, the obtained results correlate well with reported MMR activity and with in silico analysis for a majority of the variants.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/química , Animais , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Células HeLa , Humanos , Camundongos , Endonuclease PMS2 de Reparo de Erro de Pareamento , Modelos Moleculares , Proteína 1 Homóloga a MutL , Proteínas MutL , Mutagênese Sítio-Dirigida , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Homologia Estrutural de ProteínaRESUMO
Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.
Assuntos
Núcleo Celular/enzimologia , Enzimas Reparadoras do DNA/análise , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/análise , Exodesoxirribonucleases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular , Reparo de Erro de Pareamento de DNA , Enzimas Reparadoras do DNA/genética , Exodesoxirribonucleases/genética , Humanos , Carioferinas/metabolismo , Camundongos , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/metabolismo , Sinais de Localização Nuclear , Proteínas Nucleares/metabolismoAssuntos
Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Animais , Reparo do DNA/genética , Enzimas Reparadoras do DNA/deficiência , Exodesoxirribonucleases/deficiência , Humanos , Neoplasias/patologia , Oncogenes/genética , Recombinação Genética/genética , Telômero/genética , Telômero/metabolismoRESUMO
Mlh1 is an essential factor of mismatch repair (MMR) and meiotic recombination. It interacts through its C-terminal region with MutL homologs and proteins involved in DNA repair and replication. In this study, we identified the site of yeast Mlh1 critical for the interaction with Exo1, Ntg2, and Sgs1 proteins, designated as site S2 by reference to the Mlh1/Pms1 heterodimerization site S1. We show that site S2 is also involved in the interaction between human MLH1 and EXO1 or BLM. Binding at this site involves a common motif on Mlh1 partners that we called the MIP-box for the Mlh1 interacting protein box. Direct and specific interactions between yeast Mlh1 and peptides derived from Exo1, Ntg2, and Sgs1 and between human MLH1 and peptide derived from EXO1 and BLM were measured with K(d) values ranging from 8.1 to 17.4 microM. In Saccharomyces cerevisiae, a mutant of Mlh1 targeted at site S2 (Mlh1-E682A) behaves as a hypomorphic form of Exo1. The site S2 in Mlh1 mediates Exo1 recruitment in order to optimize MMR-dependent mutation avoidance. Given the conservation of Mlh1 and Exo1 interaction, it may readily impact Mlh1-dependent functions such as cancer prevention in higher eukaryotes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência Conservada , Reparo de Erro de Pareamento de DNA , Exodesoxirribonucleases/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Calorimetria , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteína 1 Homóloga a MutL , Mutagênese Sítio-Dirigida , Mutação/genética , Peptídeos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Deleção de Sequência , Especificidade da Espécie , Relação Estrutura-Atividade , Técnicas do Sistema de Duplo-HíbridoRESUMO
The heavy metal cadmium (Cd) is a human carcinogen that inhibits DNA repair activities. We show that DNA mismatch repair (MMR)-mediated cell cycle arrest after alkylation damage is suppressed by exposure to Cd and that this effect is reversed by preincubation with excess of zinc (Zn). We show that Cd-mediated inactivation of MMR activity is not caused by disruption of complex formation between the MMR proteins hEXO1-hMutS alpha and hEXO1-hMutL alpha nor does Cd inhibit 5'-exonuclease activity of hEXO1 in vitro. Thus, our studies show that exposure of human cells to Cd suppresses MMR activity, a repair activity known to play an important role in colon cancer and that this effect can be reversed by Zn treatment.