人DNA聚合酶δ第四亚基p12依赖泛素化修饰的下调机制分析

Human DNA polymerase δ (Pol δ), a heterotetrameric complex, is one of the major replicative enzymes that is involved in the duplication and repair of chromosomal DNA. Our previous studies indicated that p12 subunit could regulate the enzymatic functions of Pol δ through its rapid depletion that resulted in Pol δ conformation change. But, far less is known about p12 depletion as the regulatory mechanisms and pathways involved in its down regulation after DNA damage. Based on the key roles of ubiquitination process involved in various DNA repair mechanisms, our goals in this application are directed toward dissecting the ubiquitination of p12 as its down regulatory mechanism involved in the integrated Pol δ functions. We propose a systematic investigation of the hypothesis that the down regulation of p12 leads to a reduction in the rate of DNA synthesis by Pol δ that contributes to the overall DNA checkpoint response. Concurrently, an investigation of the ubiquitination of p12 as a mechanism for its down regulation will be pursued, in terms of its linkage with PCNA ubiquitination which initiates a damage avoidance pathway involving a translesion polymerase switch. Two major approaches will be followed. 1). the down regulation of p12 by genotoxic treatments will be studied, as well as the dependence on ATR/Chk1 signaling. The effects of p12 down regulation on subunit structure, sub-cellular localization, and activity of Pol δ will be studied. The ability of p12 levels to modulate the rates of replication fork progression, S-phase progression and thereby cell growth will be investigated. 2). the involvement of hRad18/hRad6 as the enzymes responsible for the ubiquitination of p12 will be investigated. An in vitro reconstitution system will be developed using a model primer-template, which will be used to study the ubiquitination of p12 as an integrated process with that of PCNA as part of the Pol δ-PCNA complex. These two objectives represent a convergent investigation. These studies could contribute significantly to the understanding of human disease, by uncovering novel mechanisms involved in maintaining genomic stability and control of cell proliferation. Deregulation or dysfunction in these two critical areas are among the key elements of the etiology of cancer. Investigation of the discovery that a key protein of the replication complex is down regulated in response to DNA damage has significant implications in regard to its potential contributions to the treatment of human disease, i.e., the development of drug strategies that could be used in cancer therapy or in genetic disease.

由四个亚基构成的真核生物DNA聚合酶Polδ是染色体DNA复制过程中最主要的复制酶。前期研究表明，最小亚基p12通过自身降解改变Polδ的空间结构来调控Polδ酶学功能，但对这种降解的下调机制和途径均不很清楚。基于泛素化修饰过程在多种DNA修复机制中所起的关键作用，本项目拟通过对p12泛素化修饰/下调途径分析为切入点，研究p12下调减缓Polδ-DNA合成速率以及在细胞检查点响应中的角色，阐明p12泛素化修饰在跨损伤合成途径中的作用及相互关系，揭示p12泛素化修饰调控Polδ在由PCNA泛素化引发跨损伤修复中的"损伤避免机制"。通过本项目研究，期望从人类癌症发病的起因揭示：由于泛素修饰系统异常而引起Polδ功能改变，不能确保准确的染色体DNA复制和损伤修复，使得遗传基因组不稳定，进而导致肿瘤的发生，为今后以Polδ/p12为潜在新靶标，设计新型肿瘤诊断、治疗手段来抗击癌症疾病提供理论参考。

人源DNA 聚合酶Pol δ是由四个亚基（p125，p50，p68，p12）构成的异源四聚体复合物（酶），在DNA 的复制和各种损伤修复途径中扮演关键角色。在细胞内经由各种因子诱导产生DNA 损伤的过程中，Pol δ通过最小亚基p12自身降解而改变Pol δ的空间结构来调控Pol δ酶学功能，但对p12这种降解的下调机制和途径均不很清楚。基于泛素化修饰在多种DNA修复机制中所起的特殊重要作用，本基金项目针对p12 亚基降解的现象，从DNA损伤触发p12降解（下调）的特性、细胞内p12水平对DNA合成速率的影响、DNA损伤下的p12泛素化修饰下调机制、以及p12泛素化修饰在跨损伤合成途径中的调控机制等方面开展研究。通过建立基于已知E1查找特异性介导靶蛋白泛素反应E2-E3的方法，确认了RNF8/UbcH5c(E2/E3)是调控p12亚基被多泛素化修饰经蛋白酶体降解的重要途径之一；对应细胞内DNA损伤触发的p12亚基降解，运用二维电泳和MALDI-TOF-MS质谱鉴定技术，共鉴定出34个与DNA损伤响应及修复相关的蛋白参与了协调p12亚基下调以应答DNA的损伤，并选取部分蛋白进行了相应的验证；p12亚基除了依赖于多泛素化修饰-蛋白酶体途径降解以应对DNA损伤外，我们还发现在细胞凋亡过程中可能存在着一个非泛素化修饰依赖的下调机制，即：钙离子激活μ-Calpain途径介导细胞凋亡过程中p12亚基的降解机制；通过生物信息学手段，分析了不同生物聚合酶Pol δ亚基的组成，并从生物进化层面阐述了p12亚基在高等生物进化过程中的重要性；更为重要的是，我们发现了在相同的RNF8/UbcH5c调控系统下p50亚基单泛素化修饰的现象，揭示了p12亚基下调协同p50亚基的泛素化修饰在DNA跨损伤修复途径中可能扮演的重要角色。本项目研究对从人类癌症发病的起因揭示：由于Pol δ功能改变而使得遗传基因组不稳定，进而导致肿瘤发生的机制具有十分重要的科学意义。也为今后以Pol δ/p12为潜在新靶标，设计新型肿瘤诊断、治疗手段来抗击癌症疾病提供理论参考。