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Volkmer, Elias (2004): Human checkpoint proteins hRad9, hHus1, and hRad1 form a DNA damage-responsive complex. Dissertation, LMU München: Medizinische Fakultät



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