Project 6: RNA and DNA alkylation repair

Project 6 项目摘要 烷基化疗法在临床上每天都使用，但缺乏预测性的机理知识限制了 努力宣传和促进其使用。项目6寻求这一基础知识，并以ALKBH 3和 ASCC烷基化修复蛋白和复合物与恶性肿瘤有关。ALKBH 3是一种直接 作用于RNA和ssDNA的损伤逆转脱烷基酶，ASCC是一种多功能的异源三聚体 解旋酶（ASCC 1、ASCC 2和ASCC 3亚基）。目前尚不清楚这两种蛋白质与癌症有关的原因 恶性肿瘤和它们的细胞靶点。项目6的中心假设是基于最近的研究结果， ALKBH 3-ASCC介导的DNA和RNA的烷基化修复对于 在某些癌细胞中的烷基化损伤反应。项目6将确定烷化剂的细胞靶点， ASCC招募到烷化剂诱导的病灶如何与其他DNA过程协调，并定义 信息和生物相关的结构和组件。为了实现这一目标，项目6将利用 SBDR计划项目中可用的资源，并将直接与项目1、2、3和5合作， 以及与SCB和EMB核心，并提供与所有项目的主题协同作用。 Project 6团队由Nima Mosammaparast博士（华盛顿大学）领导，他在 这些发现包括某些癌细胞对ALKBH 3和ASCC 3的依赖性， 烷化剂诱导的病灶，并确定这些病灶与典型的DNA断裂无关 修复蛋白质，但延长转录复合物。Roopa Thapar（医学博士安德森）将带来她的RNA 专业知识，做核磁共振和生物物理分析。Susan Tsutakawa（LBNL）将进行小角X射线散射 （SAXS）和晶体学。项目1的合作者袁和将进行冷冻电子显微镜（CryoEM）。SBDR PI John Tainer将确保与其他项目和计划目标的协调。 总体项目6目标1使用细胞生物学和生物化学来鉴定功能相关的ASCC-ALKBH 3 组装，并测试DNA和RNA修复活动及其与其他修复活动的交叉假设。 细胞中的通路。目标2采用结构生物学（NMR、晶体学、SAXS和CryoEM）和生物物理学 以表征告知机制并使突变验证成为可能的活性位点和界面。与我们 用于生产和表征ASCC蛋白和复合物的初步数据、稳健的测定和系统 在细胞和体外，我们准备将RNA和DNA烷基化修复添加到SBDR 4中。我们会定义生物学- 驱动的结构、机制和功能分离突变导致范式转移烷基化癌症 研究和提供强有力的工具来检查癌症的烷基化疗法。我们的成绩会直接提高 SBDR能够告知癌症研究人员RNA和DNA烷基化反应对癌症的影响 研究、治疗和患者护理决策。预期结果将填补SBDR的空白， DNA和RNA损伤反应的机制知识可能会影响癌症患者的护理。

PROJECT 6 PROJECT SUMMARY Alkylation therapy is used every day in the clinic but the absence of predictive mechanistic knowledge limits efforts to inform and advance its use. Project 6 seeks this foundational knowledge and targets ALKBH3 and ASCC alkylation repair proteins and complexes that have been linked to cancer malignancy. ALKBH3 is a direct damage reversal dealkylase that works on RNA and ssDNA, and ASCC is a multifunctional, heterotrimeric helicase (ASCC1, ASCC2, and ASCC3 subunits). It is not known why these two proteins are linked to cancer malignancy and what their cellular targets are. The central hypothesis for Project 6, based on recent findings by the Project 6 team, is that ALKBH3-ASCC-mediated alkylation repair of both DNA and RNA is critical for alkylation damage responses in some cancer cells. Project 6 will determine cellular targets of alkylating agents, how ASCC recruitment to alkylating agent-induced foci are coordinated with other DNA processes, and define informative and biologically-relevant structures and assemblies. To achieve this, Project 6 will take advantage of the resources available in the SBDR Program Project and will directly collaborate with Projects 1, 2, 3 and 5, plus with the SCB and EMB Cores, and provide thematic synergy with all Projects. The Project 6 team is led by Dr. Nima Mosammaparast (Washington University), who has made groundbreaking discoveries including the dependence of certain cancer cells on ALKBH3 and ASCC3, the first observation of alkylating agent-induced foci, and the determination that these foci are not associated with typical DNA break repair proteins but with elongating transcription complexes. Roopa Thapar (MD Anderson) will bring her RNA expertise and do NMR and biophysical analysis. Susan Tsutakawa (LBNL) will do Small Angle X-ray Scattering (SAXS) and crystallography. Yuan He, Project 1 collaborator, will do Cryo-Electron Microscopy (CryoEM). SBDR PI John Tainer will insure coordination with other Projects and Program goals. Overall Project 6 Aim 1 uses cell biology and biochemistry to identify functionally-relevant ASCC-ALKBH3 assemblies and to test hypotheses on DNA and RNA repair activities and their intersection with other repair pathways in cells. Aim 2 employs structural biology (NMR, crystallography, SAXS, and CryoEM) and biophysics to characterize active sites and interfaces that inform mechanisms and enable mutational validations. With our preliminary data, robust assays, and systems for producing and characterizing ASCC proteins and complexes in cells and in vitro, we are poised to add RNA and DNA alkylation repair to SBDR4. We will define biology- driven structures, mechanisms, and separation-of-function mutations to paradigm shift alkylation cancer research and provide powerful tools to examine alkylating therapies in cancer. Our results will directly improve SBDR’s ability to inform cancer researchers about RNA and DNA alkylation responses with impacts to cancer research, therapy and patient care decisions. The expected results will fill gaps in SBDR for a predictive mechanistic knowledge of DNA and RNA damage responses likely to impact cancer patient care.