Cellular Response to Retroviral DNA Integration

描述（由申请人提供）：这项拨款提案将研究细胞DNA修复蛋白在逆转录病毒DNA整合中的作用。这些研究的工作假设是：逆转录病毒DNA整合触发特定的细胞DNA修复系统，在整合位点的修复和/或染色质重塑中发挥作用。这种宿主反应是逆转录病毒DNA成功整合到宿主DNA中所必需的。特异性目的1将分析细胞非同源末端连接（NHEJ）蛋白对逆转录病毒DNA整合的贡献。有人提出，这些蛋白质在整合的修复步骤中发挥作用，包括病毒DNA与宿主DNA的5'端连接，或者在该过程之后的染色质重塑中发挥作用。这些步骤在正常细胞和缺乏NHEJ蛋白的细胞中进行了研究。对5'端连接的研究采用了S1核酸内切酶处理与Southern blot分析和Alu-PCR相结合的技术。除了这项技术之外，还将使用另一种技术，这种技术依赖于一种专门设计的逆转录病毒突变体，可以检查5'端连接事件。然后将通过先进的免疫荧光方法结合FISH（免疫-FISH）和染色质免疫沉淀（ChIP）来检查染色质重塑。为了确定NHEJ蛋白是否在整合位点积累并密切参与整合，或者通过向其他蛋白发出信号间接参与整合，将使用共免疫沉淀和ChIP方法。特异性Aim 2将采用与特异性Aim 1类似的方法，研究另一种蛋白质ATR激酶在逆转录病毒DNA整合中的作用。ATR在整合的5'端连接步骤中的作用将使用上述技术进行研究。为了描述ATR在逆转录病毒DNA整合中的分子机制，我们将研究ATR的下游靶点。Western blotting将确定ATR对这些靶蛋白的修饰是否由逆转录病毒DNA整合触发。此外，免疫fish和ChIP将用于确定ATR是否在整合位点积聚。这些研究的结果应该加深我们对NHEJ和ATR蛋白在促进逆转录病毒DNA整合中的作用的理解。它们也应该显著增强我们对这些重要细胞通路功能的理解。

DESCRIPTION (provided by applicant): 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.