Project 6: Vpr subversion of DNA repair

P6。摘要 辅助蛋白Vpr促进HIV-1在分裂的T细胞中复制，确保HIV-1通过T细胞的有序进展。 HIV-1的生命周期Vpr最显著的表型是诱导DNA损伤检查点和细胞周期 在G2/M阶段的逮捕，虽然潜在的细节仍然难以捉摸。有令人信服的证据表明Vpr 与几种识别受损DNA的细胞蛋白相互作用，靶向它们进行蛋白酶体降解 通过CRL 4DCAF 1 E3泛素连接酶。虽然很明显，原则上，DNA修复的Vpr拮抗作用最终可以 尽管Vpr对HIV-1有益，但Vpr介导的细胞DNA修复破坏的全部程度尚不清楚。在这 项目，我们建议系统地探索Vpr与DNA修复机制的参与程度， 专注于识别和处理引入HIV-1 cDNA的“损伤标记”的DNA修复途径 在逆转录过程中。我们的目标是发现和验证其他特定的DNA修复蛋白 Vpr靶向的那些，特别是介导DNA损伤检查点和细胞周期阻滞诱导的那些。 此外，我们将从生物化学、生物药理学和结构上分析Vpr与 已经确定的目标HLTF，MUS 81-EME 1和hHR 23 A，以及在此研究中发现的任何新目标。 项目总的来说，我们的研究将确定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，并在结构上表征负责的CRL 4DCAF 1 E3/Vpr/靶复合物。

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.