Administrative supplement to support investigation into the structural basis of ubiquitin signaling in response to DNA alkylation damage

支持调查泛素信号传导响应 DNA 烷基化损伤的结构基础的行政补充

• 批准号: 10580459
• 负责人: Patrick Lombardi
• 金额: $ 4.96万
• 依托单位: MOUNT ST. MARY'S UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10580459
• 关键词: Address; Administrative Supplement; Affect; Affinity; Alkylating Agents; Alkylation; Binding; Binding Sites; Biochemistry; Biophysics; Calorimetry; Cell Death; Cell physiology; Cells; Cellular biology; Chemistry; Collaborations; Color; Complex; DNA Alkylation; DNA Damage; DNA Modification Process; DNA Repair; DNA Repair Pathway; DNA Replication Inhibition; DNA Sequence Alteration; DNA lesion; Defect; Dependence; Detection; Diagnosis; Disease; Event; Exposure to; First Generation College Students; Genomic Instability; Goals; Grant; Human body; Investigation; Knock-out; Knowledge; Left; Link; Lysine; Measures; Modification; Molecular; Mutate; NMR Spectroscopy; Pathway interactions; Polyubiquitin; Population Biology; Principal Investigator; Process; Proliferating; Property; Proteins; Protomer; Repair Complex; Research; Research Personnel; Research Proposals; Side; Signal Transduction; Site; Source; Specificity; Structure; Students; Therapeutic; Time; Titrations; UBD protein; Ubiquitin; Universities; X-Ray Crystallography; biophysical properties; biophysical techniques; cancer therapy; chemotherapy; cytotoxic; experimental study; lower income families; mutant; novel; nucleobase; prevent; recruit; repair enzyme; repaired; response; structural biology; three dimensional structure; tumor; undergraduate student

Project Summary/Abstract DNA alkylation damage comprises a class of prevalent, harmful nucleobase modifications that occur thousands of times per cell per day in the human body as a result of endogenous and exogenous sources. Left unrepaired, DNA alkylation damage can result in genetic mutations, the inhibition of DNA replication, and cell death. Several DNA repair pathways have evolved to reverse the numerous DNA modifications that result from alkylation damage. While the repair enzymes in these pathways are well studied, much less is known about the upstream signaling events that initiate DNA repair and localize repair complexes to damage sites. It was recently shown that the ALKBH3-ASCC DNA repair complex is recruited to alkylation damage sites by binding chains of the protein ubiquitin that are assembled in proximity to the DNA lesions. The protein ASCC2 is responsible for binding the polyubiquitin chains that localize the ALKBH3-ASCC complex. A vast array of different types of polyubiquitin chains are present in cells, however, and it is unclear how ASCC2 selectively recognizes the K63- linked polyubiquitin chains that signal alkylation damage. The PI proposes to use a combination of structural biology, cell biology, and biophysics to investigate ASCC2’s selectivity for K63-linked polyubiquitin chains and the dependence of ALKBH3-ASCC complex localization on the unique ubiquitin-binding properties of ASCC2. The specific aims of the project are: 1) to identify the novel ASCC2:ubiquitin binding interface that imparts enhanced affinity for polyubiquitin chains, 2) to determine the structural basis of ASCC2’s specificity for binding K63-linked polyubiquitin chains, and 3) to quantify the contribution of ASCC2’s ubiquitin-binding properties to DNA alkylation damage repair. Investigating the outstanding questions associated with DNA alkylation damage repair will allow clinicians to better understand diseases that result from defects in alkylation damage repair pathways and to more effectively deploy alkylating agents as therapeutics, especially for the treatment of cancer. Furthermore, these experiments will also provide valuable research opportunities for students at Mount St. Mary’s University (MSMU), where substantial populations of the biology, chemistry, and biochemistry majors are first-generation college students (16.7%), students of color (42.5%), or students from moderate- or low-income families (27.1 % Pell Grant recipients). Overall, the proposed experiments will address a lack of knowledge in the current understanding of DNA alkylation damage repair while greatly enhancing research opportunities for students at MSMU.

项目概要/摘要 DNA 烷基化损伤包括一类普遍存在的有害核碱基修饰，发生数千次 由于内源性和外源性来源未修复，人体内每个细胞每天的次数， DNA 烷基化损伤可导致基因突变、DNA 复制抑制和细胞死亡。 DNA 修复途径已经发展到可以逆转烷基化引起的众多 DNA 修饰 虽然这些途径中的修复酶已得到充分研究，但对上游的了解却少之又少。 最近显示，启动 DNA 修复并将修复复合物定位到损伤位点的信号事件。 ALKBH3-ASCC DNA 修复复合物通过结合链被招募到烷基化损伤位点 蛋白质 ASCC2 负责在 DNA 损伤附近组装。 结合定位 ALKBH3-ASCC 复合物的多聚泛素链。 然而，细胞中存在多聚泛素链，目前尚不清楚 ASCC2 如何选择性识别 K63- PI 建议使用结构组合来表示烷基化损伤。 生物学、细胞生物学和生物物理学研究 ASCC2 对 K63 连接的多聚泛素链的选择性 ALKBH3-ASCC 复合体定位依赖于 ASCC2 独特的泛素结合特性。 该项目的具体目标是：1）确定新型 ASCC2：泛素结合界面，该界面赋予 增强对多聚泛素链的亲和力，2) 确定 ASCC2 结合特异性的结构基础 K63 连接的多聚泛素链，以及 3) 量化 ASCC2 泛素结合特性对 DNA 烷基化损伤修复研究与 DNA 烷基化损伤相关的突出问题。 修复将使忠实的人们更好地了解由烷基化损伤修复缺陷引起的疾病 途径并更有效地将烷化剂用作治疗剂，特别是用于癌症的治疗。 此外，这些实验也将为圣路易斯山的学生提供宝贵的研究机会。 圣玛丽大学 (MSMU)，该校拥有大量生物学、化学和生物化学专业的学生 第一代大学生（16.7%）、有色人种学生（42.5%）或中低收入学生 总体而言，拟议的实验将解决缺乏知识的问题。 目前对 DNA 烷基化损伤修复的理解，同时大大增加了研究机会 MSMU 的学生。