This grant proposal will investigate the role of cellular DNA repair proteins in retroviral DNA integration. The working hypothesis for these studies is: retroviral DNA integration triggers specific cellular DNA repair systems that play a role in the repair and/or chromatin remodeling at integration sites. This host response is required for the successful integration of retroviral DNA into host DNA.
Specific Aim 1 will analyze the contribution of cellular non-homologous end joining (NHEJ) proteins to retroviral DNA integration. It is proposed that these proteins play a role in the repair step of integration that involves the 5'-end joining of viral DNA to the host DNA, or in the chromatin remodeling that may follow this process. These steps are investigated in normal cells and in cells deficient in NHEJ proteins. The investigation of 5'-end joining employs techniques that combine S1 endonuclease treatment with the Southern blot analysis and Alu-PCR. In addition to this technique, an alternative technique will be used that relies on a specifically designed retroviral mutant that allows examination of the 5'-end joining event. Chromatin remodeling will then be examined by advanced immunofluorescence methods combined with FISH (immuno-FISH), and by chromatin immunoprecipitation (ChIP). To determine if NHEJ proteins accumulate at integration sites and participate closely in integration, or are involved in integration indirectly, possibly by signaling to other proteins, co-immunoprecipitation and ChIP methods will be used.
Specific Aim 2 will employ similar methods as Specific Aim 1, to investigate the role of another protein, the ATR kinase, in retroviral DNA integration. The role of ATR in the 5'-end joining step of integration will be investigated using techniques described above. To characterize the molecular mechanisms that underlie ATR function in retroviral DNA integration, downstream targets of ATR will be examined. Western blotting will determine if modifications of these target proteins by ATR are triggered by retroviral DNA integration. In addition immuno-FISH and ChIP will be used to determine if ATR accumulates at integration sites. Results from these studies should deepen our understanding of the role that NHEJ and ATR proteins play to facilitate retroviral DNA integration. They should also significantly enhance our understanding of the function of these important cellular pathways. ? ?
