Abstract 2474: DDB2 and XPC, two proteins involved in NER, influence ATM recruitment to the UV damage site and regulate downstream signaling pathway

Abstract

Abstract DNA damage has been linked to aging, cellular senescence and cancer. Cells respond to DNA damage by activating the cell cycle checkpoint pathway, which allows cells time to repair their DNA. In case of extensive damage, cells are eliminated through apoptosis. The molecular link between the damage recognition, repair, and the cell cycle checkpoints, is poorly understood. Two PI3 kinases, ATR and ATM, are central to checkpoint activation in response to DNA damage. ATR is principally activated by UV irradiation whereas ATM responds to DNA breaks and processed intermediates generated during NER and functions downstream of ATR. Once activated, ATR and ATM phosphorylate and activate Chk1 and Chk2 protein kinases, respectively, which in turn promote cell cycle checkpoint arrest. Available information about the events of initial checkpoint activation and ensuing signaling cascade upon UV irradiation portrays a conflicting picture. One group supports the direct binding of ATR and ATM to the UV damaged DNA without lesion processing. However, other studies support the requirement of lesion processing and ATR activation by upstream NER factors. Our studies revealed that ATR and ATM co-localize to the damage site along with DDB2 and XPC. Furthermore, ATR and ATM co-localize with XPC in G1-arrested cells showing specific function in response to UV damage which is not due to the DNA replication defect. In addition, by co-immunoprecipitation assay we revealed that both ATR and ATM physically interact with DDB2 and XPC. We revealed that ATM recruitment to the UV damage site is affected in DDB2- and XPC-deficient cells, indicating that these proteins promote ATM recruitment. As expected, ATM-mediated H2AX phosphorylation is lower in DDB2-and XPC-deficient cells. Conversely, ATM-deficiency does not influence DDB2 and XPC recruitment to the UV damage site as demonstrated in ATM knock-down or A-T (ATM-deficient) cells. To determine the defect in cell cycle checkpoint, phosphorylation of Chk1 and Chk2 in DDB2-and XPC-deficient cells was determined and compared to that of control cells. Studies by others show that UV-induced Chk1 phosphorylation is defective in both DDB2- and XPC-deficient cells suggesting a role of DDB2 and XPC in the recruitment of ATR. Interestingly, we observed that UV-induced Chk2 phosphorylation was reduced dramatically in the absence of DDB2, but moderately in the absence of XPC. Therefore, DDB2 and XPC play a conserved role in coordinating repair with cellular checkpoints. Both ATM and Chk2 are putative oncogenes and are involved in oncogenic transformation. Thus, the observations provide a foundation for developing new therapeutic targets and diagnostic tools for the treatment and prevention of cancer. (Supported by NIH grants CA93413, ES2388 and ES12991). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2474. doi:10.1158/1538-7445.AM2011-2474