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: 8445519
• 负责人: Christine Elizabeth LeRoy
• 金额: $ 4.72万
• 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 2016-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8445519
• 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) 是一种必需酶，在 DNA 复制和 DNA 修复中发挥着关键作用。该酶由四个亚基组成：p125 和 p50 构成催化核心，p68 和 p12 为酶提供更高的稳定性、增强的活性以及用于结合其他蛋白质伙伴的额外位点。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 损伤时迅速降解，从而产生三亚基 DNA 聚合酶 4 酶 (Pol 43)。进行动力学研究是为了比较 Pol 4 和 Pol 43 的酶特性。有趣的是，我们确定 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，p12 响应 DNA 损伤的降解将 Pol 4 酶转化为能够更好修复 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获得对 p12 降解的更全面的了解。 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试剂的 p12 半衰期。这将通过蛋白质印迹和光密度测定来完成。此外，将利用缺乏特定DNA损伤反应蛋白（例如p53或ATM）的细胞系，以便可以更全面地了解导致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洗使细胞同步化，以便可以在细胞周期的各个阶段评估 p12 的水平。从这里开始，将通过测量 DNA 修复率、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）来评估 p12 的 siRNA 消耗对 DNA 修复的影响。预计当 p12 耗尽时，修复会发生得更快。还将利用蛋白酶体抑制剂阻断 p12 降解的补充实验，并且预计会产生与 p12 消耗实验相反的结果。最后，将通过 siRNA 分析许多可能泛素化 p12 以发出其降解信号的潜在 E3 连接酶，以确定这些酶参与 p12 降解。 p12 的泛素化尚不清楚，因此鉴定参与 p12 降解的 E3 连接酶非常重要。该项目始终高度关注健康问题，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。