Project 6: Vpr subversion of DNA repair

P6。摘要 辅助蛋白VPR在T细胞分裂中促进HIV-1的复制，确保通过 艾滋病毒-1的生命周期。最显著的vpr表型是诱导dna损伤检查点和细胞周期。 在G2/M阶段被逮捕，尽管潜在的细节仍然难以捉摸。有令人信服的证据表明VPR 与识别受损DNA的几种细胞蛋白相互作用，以蛋白酶体降解为靶点 通过CRL4DCAF1 E3泛素连接酶。虽然很明显，在原则上，VPR对DNA修复的拮抗最终可以 VPR对HIV-1有利，但VPR对细胞DNA修复的完全颠覆程度尚不清楚。在这 项目中，我们建议系统地探索VPR与DNA修复机制的接触程度， 关注识别和处理损伤标记的DNA修复途径，引入HIV-1cDNA 在逆转录过程中。我们的目标是发现和验证其他特异的DNA修复蛋白(S)，这些蛋白是 VPR的靶标，特别是那些介导诱导DNA损伤检查点和细胞周期停滞的基因。 此外，我们将从生物化学、生物物理和结构上分析VPR和 已确定的目标HLTf、MUS81-EME1和hHR23A，以及在本报告中发现的任何新目标(S 项目。总体而言，我们的研究将定义VPR诱导的细胞DNA修复变化的程度，确定特定的 VPR靶向的DNA修复途径/蛋白质，并从结构上表征负责的CRL4DCAF1 E3/VPR/靶复合体。

P6. Abstract The accessory protein Vpr facilitates HIV-1 replication in dividing T cells, ensuring orderly progression through the HIV-1 life cycle. The most remarkable Vpr phenotype is induction of DNA damage checkpoint and cell cycle arrest in G2/M phase, although the underlying details are the still elusive. There is compelling evidence that Vpr interacts with several cellular proteins that recognize damaged DNA, targeting them for proteasomal degradation via CRL4DCAF1 E3 ubiquitin ligase. While evident that, in principle, Vpr antagonism of DNA repair ultimately can be of benefit to HIV-1, the full extent of Vpr-mediated subversion of cellular DNA repair is not known. In this project, we propose to systematically explore the extent of Vpr's engagement with the DNA repair machinery, focusing on DNA repair pathways that recognize and process “marks of damage”, introduced into HIV-1 cDNA during reverse transcription. We aim to discover and validate other specific DNA repair protein(s) that are targeted by Vpr, in particular those mediating the induction of DNA damage checkpoint and cell cycle arrest. Further, we will biochemically, biophysically and structurally analyze the interactions between Vpr and the already identified targets HLTF, MUS81-EME1, and hHR23A, as well as any new target(s) discovered in this project. Overall, our studies will define the extent of Vpr-induced changes in cellular DNA repair, identify specific DNA repair pathways/proteins targeted by Vpr, and structurally characterize the responsible CRL4DCAF1 E3/Vpr/target complexes.