Cellular Response to Retroviral DNA Integration

描述(由申请人提供)：这项拨款提案将调查细胞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，该系统在整合部位的修复和/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。具体目标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。有人认为，这些蛋白质在整合的修复步骤中发挥作用，该步骤涉及病毒DNA与宿主DNA的5‘端连接，或者在随后可能发生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。5‘端连接的研究采用了S1内切酶处理、Southern印迹分析和Alu-PCR相结合的技术。除了这项技术外，还将使用一种替代技术，该技术依赖于一种专门设计的逆转录病毒突变体，该突变体允许检查5‘端连接事件。然后将用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蛋白是在整合部位聚集并密切参与整合，还是间接参与整合，可能是通过向其他蛋白质发出信号，将使用免疫共沉淀和芯片方法。特异性目标2将使用与特异性目标1类似的方法，研究另一种蛋白--ATR激酶在逆转录病毒DNA整合中的作用。将使用上述技术研究ATR在整合的5‘端连接步骤中的作用。为了表征ATR在逆转录病毒DNA整合中的作用的分子机制，我们将检查ATR的下游靶标。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和芯片将被用来确定ATR是否在整合部位积累。这些研究的结果应该会加深我们对NHEJ和ATR蛋白在促进逆转录病毒DNA整合中所起作用的理解。它们还应该显著提高我们对这些重要细胞通路的功能的理解。

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.