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

描述(申请人提供)：DNA聚合酶4(POL4)是一种在DNA复制和DNA修复中起关键作用的重要酶。该酶由四个亚基组成：p125和p50，它们构成催化核心，p68和p12，它们为酶提供更高的稳定性，增强的活性，并为其他蛋白质伙伴提供更多的结合位点。以往的实验发现了一种新的DNA损伤反应，在这种反应中，Pol 4的最小亚基p12迅速降解，从而产生一个三亚单位DNA聚合酶4酶(Pol 43)。为了比较POL4和POL43的酶性质，进行了动力学研究。有趣的是，它确定POL43显示出增强的保真度和校对能力，并增加了DNA损伤的停滞。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更全面地了解p12的降解。P12的降解将根据时间和剂量反应来表征，并将确定给定细胞系中每个特定药物的p12半衰期。这将通过Western blotting和密度测定来完成。此外，还将利用缺乏特定DNA损伤反应蛋白的细胞系(如P53或ATM)，以便更全面地了解导致p12降解的信号过程。接下来，细胞将通过血清饥饿或逆流离心洗脱来同步，这样就可以在细胞周期的不同阶段评估p12的水平。在此基础上，通过检测DNA修复速率来评估p12 siRNA缺失对DNA修复的影响，通过清除环丁烷嘧啶二聚体和3H_2AX病灶，用流式细胞仪和免疫荧光显微镜方法测量。预计当p12耗尽时，修复会更快发生。用蛋白酶体抑制剂阻止p12降解的补充实验也将被利用，应该会产生与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以发出其降解信号，将通过siRNA进行分析，以确定这些酶在p12降解中的参与。P12的泛素化仍然不是很清楚，因此鉴定参与p12降解的E3连接酶是非常重要的。该项目保持着强烈的健康重点，因为DNA损伤反应的功能对于维持基因组的完整性至关重要，从而预防许多癌症和其他疾病。 公共卫生相关性：为了预防癌症和限制疾病，人类细胞不断地通过激活DNA损伤反应和复杂的DNA修复机制来修复DNA损伤。我们计划对新发现的DNA损伤反应的一个方面进行深入的表征，即损伤诱导的蛋白质亚基的降解，从而使亲本酶更容易地识别和纠正受损的DNA，从而可能为观察到的亚基降解提供一个适应性和功能性的原因。通过我们对这种反应的完整描述，我们将获得关于这种DNA损伤反应的进一步知识和理解，这可能为许多癌症和其他由遗传不稳定带来的疾病的DNA修复提供重要线索。)

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. )