Human checkpoint proteins hRad9, hHus1, and hRad1 form a DNA damage-responsive complex

摘要: Human cells have evolved protective mechanisms such as DNA repair and cell cycle checkpoints in order to promote stability of the genome. Studies on hereditary instability syndromes associated with a higher incidence malignancies like Xeroderma pigmentosum or Nijmegen breakage syndrome demonstrated that genetic defects subsequent dysfunction specific mechanism trigger development cancer. Within last years, investigation gained increasing importance cancer research. Checkpoints are signaling cascades halt response damage, thereby providing time for preventing accumulation alterations. While p53-dependent G1-S checkpoint has been extensively investigated, little is known about other humans G2-M S-phase progression checkpoint. human ataxia telangiectasia (AT) showed AT fail induce several ionizing radiation (IR), indicating gene defect responsible AT-associated cancers. The (ATM) significant sequence homology kinase sprad3 fission yeast Schizosaccharomyces pombe (S. pombe). In S. pombe, spRad3 regulates activation damage. Defects gene, ATM, sensitize organisms radiomimetic drugs, suggesting conservation pathways from well potential role carcinogenesis. The discovery checkpoint-deficient mutants led cloning additional genes their homologs. This group novel includes homologs sprad9 (hRAD9), sphus1 (hHUS1), sprad1 (hRAD1). these required spRad3, one more render sensitive genotoxic agents. Mutations within rad may bring an increased rate mutations genomic shown p53 be inherited predisposition cancer. In view this process carcinogenesis, we undertaken cellular molecular analysis proteins hRad9, hHus1, hRad1 leukemia line K562 keratinocytes. Using antibodies co-immunoprecipitation Western-blot experiments associate biochemical complex similar spRad9-spHus1-spRad1 reported yeast. To generate model system protein function amenable analysis, prepared epitope-tagged expression vectors hRad1, which were transfected into by electroporation, resulting transient protein. By simultaneous transiently expressed recapitulate formation endogenous proteins. Immunoprecipitation studies lysates hRad9-overexpressing revealed hRad9 undergoes post-translational modifications. Co-expression resulted large increase amount highly modified form hHus1 either of, stabilize hRad9. Previously, direct correlation between phosphorylation damage was proposed. study, show phosphorylated interacts well. present results suggest hRad9-hHus1-hRad1 actively participates evolutionarily conserved damage-induced cascade. hRad1 seems possess exonuclease activity. presence putative DNA-metabolizing multimolecular complex, coupled data place spRad9, spHus1, spRad1 early pathway suggests sensor scans genome damaged DNA. Once detected, initiate endonucleolytic processing lesions interactions downstream elements, link unknown recognition components signal-transducing include ATM kinase, implicated enforcing arrest after Potential goals research implementation screening tests identify familial treatment transfer. Another aim checkpoint-based therapy. More than 50% all malignant tumors contain mutated p53, p53-deficient tumor lack induction One emerging hypothesis selective inhibitors compensating would preferentially radiosensitize cells. Thus, provides further targets chemotherapeutic agents will help design future strategies