Studies on Mre11-ku interaction and its modulation by ionizing radiations

The Mre11-Rad50-Nbs1 (MRN) protein complex is a well known sensor of DNA damage, functioning in signaling pathways that activate cell cycle checkpoints. The function of Mre11 is also known to be important for homologous recombination repair (HRR), and recent studies have shown that this protein may function both in HRR, as well as in the classical pathway of non homologous end joining that utilizes in addition to DNA-PK (D-NHEJ) also Ku and the Ligase IV/XRCC4 complex. Indeed, in yeast, in addition to Ku and Ligase IV homologs, also the MRN homologs are implicated in NHEJ. The Ku70/80 heterodimer is among the first proteins that recognize and bind to DNA ends. The crystal structure of Ku reveals an asymmetric ring conformation allowing the threading of the DNA and possibly facilitating rejoining. After completion of end joining, however, the protein will remain trapped on the DNA. This mode of action generates the question as to how trapped Ku is released from the DNA, and as to whether trapped protein serves somehow to the development of a full-blown DNA damage response. Hitherto, only one report suggests a ubiquitin-dependent removal and degradation of Ku80 from DNA, which is independent of the completion of NHEJ. There are no reports published to date investigating a functional role for the trapped Ku protein in the ensuing DNA damage response (DDR) signalling. The present thesis tests the hypothesis that trapped Ku contributes to DDR signalling by somehow facilitating the recruitment of the MRN complex to the site of the DSB. In support of this hypothesis, we demonstrate here that the constitutive interaction between Mre11 and Ku is enhanced after exposure to IR and that this enhancement is dose dependent. The interaction between Mre11 and Ku is studied by immunoprecipitation (IP) from nuclear extracts using an anti-Ku70 antibody while detecting Mre11 by Western blotting. IP studies are carried out using a human alveolar basal epithelium carcinoma cell line (A549), as well as CHO cells and a Ku80 deficient mutant (xrs6). As expected, Ku deficient cells show no detectable interaction between Mre11 and Ku70 and validate thus the specificity of the assay. An interaction between Mre11 and Ku is clearly visible in A549 cells and is markedly enhanced after exposure to 2 and 4Gy of X-rays. The interaction is resistant to EtBr and RNAase suggesting that it is not mediated by DNA or RNA. Rad50 and Nbs1 are also co-precipitated with Mre11, suggesting that the entire MRN complex is involved in the interaction. Notably, in addition to MRN, other DNA


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