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.

项目摘要/摘要 DNA烷基化损伤包括一类普遍存在的、发生在数千次的有害的碱基修饰 作为内源性和外源性来源的结果，人体内每天每个细胞的次数。无人修理， DNA烷基化损伤可导致基因突变、DNA复制抑制和细胞死亡。几个 DNA修复途径的进化逆转了烷基化引起的大量DNA修饰 损坏。虽然对这些途径中的修复酶进行了很好的研究，但对上游的修复酶知之甚少 启动DNA修复并将修复复合体定位到损伤位置的信号事件。它最近被放映了 ALKBH3-ASCC DNA修复复合体通过与 在DNA损伤附近组装的蛋白质泛素。ASCC2蛋白负责 结合定位ALKBH3-ASCC复合体的多泛素链。种类繁多的不同类型 然而，细胞中存在多泛素链，目前尚不清楚ASCC2如何选择性识别K63- 连接的多泛素链发出烷基化损伤的信号。PI建议使用结构上的组合 生物学、细胞生物学和生物物理学研究ASCC2‘S对K63连接的多泛素链和 ALKBH3-ASCC复合体的定位依赖于ASCC2独特的泛素结合特性。 该项目的具体目标是：1)确定新的ASCC2：泛素结合界面 增强多泛素链的亲和力，2)确定ASCC2的S结合特异性的结构基础 K63连接的多泛素链，以及3)量化ASCC2的S泛素结合特性对 DNA烷基化损伤修复。调查与DNA烷基化损伤相关的未决问题 修复将使临床医生更好地了解由于烷基化损伤修复缺陷而导致的疾病 目的是为了更有效地利用烷化剂作为治疗药物，特别是癌症的治疗药物。 此外，这些实验还将为圣彼得堡的学生提供宝贵的研究机会。 玛丽大学(MSMU)，那里有大量的生物、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%)或中低收入家庭学生 家庭(27.1%佩尔助学金获得者)。总体而言，拟议的实验将解决在 目前对DNA烷基化损伤修复的理解，同时极大地加强了对 密歇根州立大学的学生。