A Novel Human DNA Damage Response Which Directly Alters DNA Polymerase Delta

一种直接改变 DNA 聚合酶 Delta 的新型人类 DNA 损伤反应

• 批准号: 8475598
• 负责人: Christine Elizabeth LeRoy
• 金额: $ 4.59万
• 依托单位: NEW YORK MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-15 至 2014-05-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8475598
• 关键词: Aphidicolin; Binding Sites; Catalytic Domain; Cell Cycle; Cell Cycle Stage; Cell Line; Cells; Characteristics; Co-Immunoprecipitations; DNA Damage; DNA Polymerase III; DNA Repair; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; Densitometry; Dependence; Disease; Dose; Ensure; Enzymes; Event; Exonuclease; Exposure to; Flow Cytometry; Genetic; Genomics; Goals; Half-Life; Health; Human Cell Line; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Kinetics; Knowledge; Laboratories; Lead; MG132; Maintenance; Malignant Neoplasms; Measures; Methods; Microscopy; Parents; Pathway interactions; Play; Polymerase; Prevention; Process; Property; Protein Kinase; Protein Subunits; Proteins; Pyrimidine Dimers; Research; Role; Serum; Signal Pathway; Signal Transduction; Small Interfering RNA; Staging; Starvation; TNFRSF5 gene; Techniques; Thymidine; Time; Transfection; Ubiquitin; Ubiquitination; Western Blotting; base; cell type; computerized data processing; fighting; human DNA damage; human disease; inhibitor/antagonist; knock-down; multicatalytic endopeptidase complex; novel; prevent; repaired; research study; response; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): DNA polymerase 4 (Pol 4) is an essential enzyme which plays critical roles in both DNA replication and DNA repair. The enzyme consists of four subunits: p125 and p50 which make up the catalytic core, and p68 and p12, which provide the enzyme increased stability, enhanced activity, and additional sites for binding of other protein partners. Previous experiments discovered a novel DNA damage response in which p12, the smallest subunit of Pol 4, was rapidly degraded in response to DNA damage, thereby generating a three-subunit DNA polymerase 4 enzyme (Pol 43). Kinetic studies were performed in order to compare the enzymatic properties of Pol 4 and Pol 43. Interestingly, it was determined Pol 43 demonstrated enhanced fidelity and proofreading abilities, and increased stalling at DNA lesions. Thus, the degradation of p12 in response to DNA damage converts the Pol 4 enzyme into one which can better repair DNA lesions, indicating a potential functional reason for p12 degradation. The long term goals and specific aims of this project focus on obtaining more comprehensive understanding of p12 degradation. The degradation of p12 will be characterized in terms of time- and dose-response, and the p12 half-life for each specific agent in a given cell line will be determined. This will be done through Western blotting and densitometry. In addition, cell lines which are deficient in specific DNA damage response proteins (such as p53 or ATM) will be utilized, such that the signaling processes which lead to p12 degradation can be more comprehensively understood. Next, cells will be synchronized by serum starvation or counterflow centrifugal elutriation such that the levels of p12 can be assessed at various stages in the cell cycle. From there, the effects of siRNA depletion of p12 on DNA repair will be assessed by measuring the rate of DNA repair, through clearance of cyclobutane pyrimidine dimers and 3H2AX foci, measured by flow cytometry and immunofluorescence microscopy methods. It would be expected that when p12 is depleted, repair would occur more quickly. Complementary experiments in which p12 degradation is blocked with proteasomal inhibitors will also be utilized, and should be expected to generate the opposite result from p12 depletion experiments. Finally, a number of potential E3 ligase enzymes which may ubiquitinate p12 to signal its degradation will be analyzed by siRNA in order to determine the involvement of these enzyme in p12 degradation. The ubiquitination of p12 is still not well understood, and thus the identification of the E3 ligase involved in p12 degradation is incredibly important. This project maintains a strong health focus in that the functioning of the DNA damage response is vital to the maintenance of genomic integrity, and thus the prevention of many cancers and other diseases.

描述（由申请人提供）：DNA聚合酶4（Pol 4）是一种在DNA复制和DNA修复中起关键作用的必需酶。该酶由四个亚基组成：构成催化核心的p125和p50，以及p68和p12，它们为酶提供增加的稳定性，增强的活性和用于结合其他蛋白质伴侣的额外位点。先前的实验发现了一种新的DNA损伤反应，其中Pol 4的最小亚基p12在DNA损伤反应中迅速降解，从而产生三亚基DNA聚合酶4酶（Pol 43）。进行动力学研究以比较Pol 4和Pol 43的酶性质。有趣的是，确定Pol 43表现出增强的保真度和校对能力，并增加了DNA损伤处的停滞。因此，响应DNA损伤的p12的降解将Pol 4酶转化为可以更好地修复DNA损伤的酶，这表明p12降解的潜在功能原因。该项目的长期目标和具体目标侧重于更全面地了解p12降解。p12的降解将根据时间和剂量反应来表征，并且将确定给定细胞系中每种特定药剂的p12半衰期。这将通过蛋白质印迹法和密度测定法进行。此外，将利用缺乏特异性DNA损伤反应蛋白（如p53或ATM）的细胞系，从而可以更全面地理解导致p12降解的信号传导过程。接下来，将通过血清饥饿或逆流离心淘析使细胞同步化，使得可以在细胞周期的各个阶段评估p12的水平。从那里，p12的siRNA消耗对DNA修复的影响将通过测量DNA修复的速率来评估，通过环丁烷嘧啶二聚体和3H2AX焦点的清除，通过流式细胞术和免疫荧光显微镜方法测量。预计当p12耗尽时，修复会更快地发生。还将使用蛋白酶体抑制剂阻断p12降解的补充实验，预计将产生与p12耗竭实验相反的结果。最后，一些潜在的E3连接酶，可能泛素化p12的信号，其降解将被分析的siRNA，以确定这些酶在p12降解的参与。p12的泛素化仍然没有得到很好的理解，因此鉴定参与p12降解的E3连接酶非常重要。该项目保持了强烈的健康关注，因为DNA损伤反应的功能对于维持基因组完整性至关重要，从而预防许多癌症和其他疾病。