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: 8127402
• 负责人: Christine Elizabeth LeRoy
• 金额: $ 2.6万
• 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/8127402
• 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. PUBLIC HEALTH RELEVANCE: In order to prevent cancer and limit disease, human cells are continuously fighting to repair DNA damage by activation of the DNA damage response and through intricate DNA repair mechanisms. We plan to perform in- depth characterization of a newly discovered aspect of the DNA damage response, namely, the damage- induced degradation of a protein subunit, which thereby allows the parent enzyme to more easily identify and correct damaged DNA, thus likely providing an adaptive and functional reason for the observed subunit degradation. Through our complete characterization of the response, we will obtain further knowledge and understanding about this DNA damage response, which may provide important clues to DNA repair in many cancers and other diseases brought about by genetic instability. )

描述（申请人提供）：DNA聚合酶4 （Pol 4）是一种重要的酶，在DNA复制和DNA修复中起着关键作用。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和活性，并为其他蛋白质伴侣提供额外的结合位点。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 （Pol 43）。为了比较Pol 4和Pol 43的酶学性质，进行了动力学研究。有趣的是，Pol 43显示出更高的保真度和校对能力，并增加了DNA损伤的延迟。因此，p12对DNA损伤的降解将Pol 4酶转化为能够更好地修复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，并且将确定每种特定药物在给定细胞系中的p12半衰期。这将通过免疫印迹和密度测定来完成。此外，将利用缺乏特定DNA损伤反应蛋白（如p53或ATM）的细胞系，从而更全面地了解导致p12降解的信号传导过程。接下来，细胞将通过血清饥饿或逆流离心洗脱同步，这样就可以在细胞周期的各个阶段评估p12的水平。在此基础上，通过流式细胞术和免疫荧光显微镜方法测量环丁烷嘧啶二聚体和3H2AX病灶的清除，通过测量DNA修复率来评估siRNA缺失p12对DNA修复的影响。可以预期，当p12耗尽时，修复会更快地发生。还将利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消耗实验相反的结果。最后，我们将通过siRNA分析一些可能泛素化p12以表明其降解的潜在E3连接酶，以确定这些酶在p12降解中的作用。p12的泛素化仍然没有被很好地理解，因此鉴定参与p12降解的E3连接酶是非常重要的。该项目以健康为重点，因为DNA损伤反应的功能对维持基因组完整性至关重要，因此对预防许多癌症和其他疾病至关重要。