A Cyclin E fragment inhibits DNA repair by binding to Ku70 and preventing recruitment of XRCC4 to double-strand breaks

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Cyclin E/Cdk2 is a critical regulator of cell cycle progression from G1 to S phase in mammalian cells that has been found to be deregulated in many types of neoplasms. However, the influence of Cyclin E expression upon apoptosis has not been determined. We found that genotoxic stress leads to a dramatic decrease in Cyclin E levels in hematopoietic tumor cells, coinciding with the timely appearance of its proteolytic fragment, p18-Cyclin E. Overexpression of p18-Cyclin E in a variety of cell types induces apoptosis. By yeast-two-hybrid assays, mass spectrometry analyses, and co-immunoprecipitations we have identified Ku70, a critical component of non-homologous end joining DNA repair (NHEJ) as a new interacting partner of p18-Cyclin E. Neutral comet assays used to determine residual double-strand breaks indicative of an ineffective NHEJ, showed significant difference in both tail length and tail moment in the presence of p18-Cyclin E following irradiation. Moreover, a plasmid reactivation assay indicated that p18-Cyclin E reduced the colony formation, known to be associated with NHEJ activity, to ~10% as compared to ~60% for cell lysates containing wild-type Cyclin E. Gel electrophoretic analyses indicated no detectable end-ligation even in cells that have received the lowest concentration of p18-Cyclin E during transfection. DNA pull-down assays showed that the assembly of Ku70-Ku80 and DNA-PKcs is not affected by the presence of p18-Cyclin E. However the recruitment of XRCC4 and Ligase IV was largely prevented. These data indicate a profound effect of p18-Cyclin E on cellular survival and NHEJ that is dependent on its interaction with Ku70. Mapping the p18-Cyclin E interaction domain of Ku70 using a series of deletion and site-specific mutants of Ku70, revealed that their interface resides at the N-terminus of Ku70, which is different from the Bax binding site located at the C-terminus. Moreover, we found that p18-Cyclin E is degraded by polyubiquitination and subsequent targeting to proteasomal degradation. Furthermore, interaction with Fbw7 (member of the SCF ubiquitin ligase complex) isoforms was determined by co-immunoprecipitation, as p18-Cyclin E carries the C-terminal phosphodegron of wild-type Cyclin E. p18-Cyclin E, as compared to Cyclin E, has a short half-life, as determined by translation inhibition with cycloheximide. However, inhibition of the proteasome leads to increased levels of p18-Cyclin E, but not Cyclin E, that confers a higher sensitivity to apoptotic stimuli. Interaction of Ku70 with p18-Cyclin E and Bax may be unique in linking cell cycle control, DNA repair, and activation of apoptosis following genotoxic stress as well as provide mechanistic insights into the molecular switch between cell death and cell survival depending on the cell type, the nature and severity of the genotoxic challenge.