RESUMO
Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.
Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo de Erro de Pareamento de DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/fisiologia , Exodesoxirribonucleases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , DNA/genética , DNA/metabolismo , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/efeitos da radiação , Replicação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/efeitos da radiação , Células HeLa , Humanos , Lasers/efeitos adversos , Camundongos , Proteína 1 Homóloga a MutL , Proteína 3 Homóloga a MutS , Células NIH 3T3 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Transporte Proteico/genética , Transporte Proteico/efeitos da radiação , RecQ Helicases/genética , RecQ Helicases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/efeitos da radiação , Fase S , Helicase da Síndrome de WernerRESUMO
So far 18 MLH3 germline mutations/variants have been identified in familial colorectal cancer cases. Sixteen of these variants are amino acid substitutions of which the pathogenic nature is still unclear. These substitutions are known as unclassified variants or UVs. To clarify a possible role for eight of these MLH3 UVs identified in suspected Lynch syndrome patients, we performed several biochemical tests. We determined the protein expression and stability, protein localization and interaction of the mutant MLH3 proteins with wildtype MLH1. All eight MLH3 UVs gave protein expression levels comparable with wildtype MLH3. Furthermore, the UV-containing proteins, in contrast to previous studies, were all localized normally in the nucleus and they interacted normally with wildtype MLH1. Our different biochemical assays yielded no evidence that the eight MLH3 UVs tested are the cause of hereditary colorectal cancer, including Lynch syndrome.
