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(PP364) Characterization of DNA-PKcs telomeric end-capping function in mammalian cells.
Williams, Eli*,1, Bailey, Susan1, Jeggo, Penelope2, Ullrich, Robert1, 1 Department of Environmental and Radiological Health Sciences, Fort Collins, CO, USA2 Genome Damage and Stability Centre, Falmer, Brighton, UK
ABSTRACT- Accurate recognition and processing of double-stranded DNA ends, whether they are created endogenously or exogenously, is critical in maintaining genomic stability. One important subset of this cellular function involves telomeres, specialized nucleoprotein structures that function to preserve the linear ends of eukaryotic chromosomes. Recent work has elucidated unexpected links between DNA double strand break (DSB) repair proteins and telomere function. The catalytic subunit of the non-homologous end-joining (NHEJ) protein DNA-dependent protein kinase (DNA-PKcs) interacts with mammalian telomeres via mechanisms that remain poorly understood. This interaction has been phenotypically demonstrated by the generation of telomere fusion events (telomere-to-telomere and telomere-to-DSB) in mouse and human cells deficient in DNA-PKcs. These chromosomal rearrangements inappropriately maintain large blocks of telomeric sequence at the point of fusion, suggesting a role for DNA-PKcs in telomeric end-capping. Our current studies serve to further characterize the mechanism by which DNA-PKcs acts at mammalian telomeres by investigating the nature of the telomere dysfunction. We have utilized immunoFISH, a specialized approach that combines immunofluorescence and fluorescence in situ hybridization techniques, in order to visualize -H2AX foci (DNA damage marker) and telomeric sequence simultaneously. Co-localization of -H2AX foci and telomeres indicates the misidentification and signaling of the telomere as a DSB. This is in agreement with our previous studies which identified telomere-DSB fusions at chromosome translocation breakpoints. Additionally, we have used a specific chemical inhibitor of DNA-PKcs to identify the repair pathway responsible for creating the telomeric fusions. Our results support the hypothesis that DNA-PKcs deficiency produces improperly capped telomeres following replication. Furthermore, these dysfunctional telomeres are identified as DSBs -despite the presence of ample telomeric sequence- triggering a DNA damage response, thus contributing to the instability and tumorigenesis seen in these backgrounds.
Key words: Telomeres, DNA-PKcs, Genomic Instability
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