Bypass of DNA lesions by translesion synthesis in yeast

描述（由申请人提供）：翻译合成（TLS） DNA聚合酶（pol）通过介导DNA病变的绕过来帮助维持复制叉的连续性。为了阐明包括人类在内的真核生物DNA加合物的复制旁路机制，本研究将利用酵母酿酒酵母进行研究。由于DNA修复和病变旁路过程在酵母和人类之间高度保守，从这些研究中获得的知识将直接适用于理解人类细胞中的TLS机制。在Aim 1中，将使用遗传和生化相结合的方法研究DNA损伤诱导的检查点激酶介导的Rev1磷酸化在pol6依赖性TLS中的作用。特别是，我们将验证Rev1磷酸化通过Pol6提高TLS效率的假设。在Aim 2中，将对我们纯化的新型4亚基Pol6进行研究，该亚基由Rev3， Rev7， Pol31和Pol32亚基组成。具体而言，我们将检查其病变旁路特性，并确定其是否在物理和功能上与PCNA相互作用。在Aim 3中，我们将定义Slx4的作用，我们最近将其确定为pol6依赖性TLS的一个组成部分，并与Rev1结合形成Rev1-Slx4复合物。将进行遗传和生化研究，以确定Rev1-Slx4复合物调节Pol6功能的方式。在Aim 4中，研究将验证Rad6-Rad18-Rad5复合物作为TLS中的结构元件，促进TLS pol及其相关蛋白因子在DNA损伤处的组装的假设。在Aim 5中，将通过携带在质粒上的位点特异性DNA损伤来检测TLS，以确定不同蛋白质复合物对TLS反应的贡献。特别地，我们将验证这样一个假设，即除了在TLS Pol的组装中具有结构作用外，Rad6-Rad18-Rad5复合体还在TLS中充当中介，其中它协调TLS Pol与复制Pol的动作。这些研究还将分析PCNA泛素化以及TLS pol中PCNA结合和泛素结合结构域的作用。通过帮助确保复制叉的连续性，并通过在正常细胞氧化反应中产生的自由基和活性氧的攻击以及暴露于化学和环境致癌物中产生的大量DNA加合物来促进无错误复制，TLS过程为最大限度地减少DNA损伤的致突变效应和抑制人类致癌作用提供了重要手段。例如，人类体内Pol7的失活会导致皮肤癌的高度升高。这些建议的研究与癌症病因学高度相关，因为它们对于阐明TLS在人类细胞中的机制，揭示TLS过程对基因组完整性和预防DNA病变的致突变和致癌后果的贡献非常重要。公共卫生相关性：DNA损伤是由于细胞氧化反应引起的自由基和活性氧的攻击以及暴露于化学和环境致癌物而在人体细胞中产生的。翻译合成DNA聚合酶通过多种DNA加合物促进复制，在维持基因组完整性和保真度方面发挥重要作用。拟议的研究对于阐明控制人类细胞中DNA病变复制旁路的机制以及描述这些过程对避免突变和致癌的贡献将是重要的。

DESCRIPTION (provided by applicant): Translesion synthesis (TLS) DNA polymerases (Pols) help maintain the continuity of the replication fork by mediating the bypass of DNA lesions. To elucidate the mechanisms that govern replicative bypass of DNA adducts in eukaryotes including in humans, the proposed studies will utilize the yeast Saccharomyces cerevisiae. Since DNA repair and lesion bypass processes have been highly conserved between yeast and humans, the knowledge gained from these studies will be directly applicable to the understanding of TLS mechanisms in human cells. In Aim 1, the role of DNA damage-induced checkpoint kinase-mediated Rev1 phosphorylation in Pol6-dependent TLS will be studied using a combined genetic and biochemical approach. In particular, the hypothesis that Rev1 phosphorylation enhances the efficiency of TLS by Pol6 will be tested. In Aim 2, studies will be done with the novel 4-subunit Pol6, comprised of Rev3, Rev7, Pol31, and Pol32 subunits, that we have purified. Specifically, we will examine its lesion bypass properties and will determine whether it physically and functionally interacts with PCNA. In Aim 3, the role of Slx4, which we have recently identified as a component of Pol6-dependent TLS and which associates with Rev1 to form the Rev1-Slx4 complex, will be defined. Genetic and biochemical studies will be conducted to determine the manner in which the Rev1-Slx4 complex modulates Pol6 function. In Aim 4, studies will carried out to test the hypothesis that the Rad6-Rad18-Rad5 complex functions as a structural element in TLS to promote the assembly of TLS Pols and their associated protein factors at DNA lesions. In Aim 5, TLS through site-specific DNA lesions carried on a plasmid will be examined so as to determine the contributions of different protein complexes to the TLS reaction. In particular, we will test the hypothesis that in addition to its structural role in the assembly of TLS Pols, the Rad6-Rad18-Rad5 complex functions as a mediator in TLS wherein it coordinates the actions of the TLS Pols with that of the replicative Pol. The roles of PCNA ubiquitination and of the PCNA binding and ubiquitin binding domains present in TLS Pols will also be analyzed in these studies. By helping ensure the continuity of the replication fork and by promoting error-free replication through a large variety of DNA adducts that result from the attack of free radicals and reactive oxygen species generated during normal cellular oxidative reactions and from exposure to chemical and environmental carcinogens, the TLS processes provide an important means for minimizing the mutagenic effects of DNA damage and for the suppression of carcinogenesis in humans. For instance, the inactivation of Pol7 in humans causes highly elevated levels of skin cancers. The proposed studies are highly relevant for cancer etiology, as they will be important for elucidating the mechanisms of TLS in human cells and for revealing the contributions that TLS processes make to genomic integrity and to the prevention of the mutagenic and carcinogenic consequences of DNA lesions. PUBLIC HEALTH RELEVANCE: DNA lesions are generated in human cells from attack of free radicals and reactive oxygen species resulting from cellular oxidative reactions and from exposure to chemical and environmental carcinogens. By promoting replication through a diverse array of DNA adducts, translesion synthesis DNA polymerases play important roles in maintaining genomic integrity and fidelity. The proposed studies will be important for the elucidation of mechanisms that govern the replicative bypass of DNA lesions in human cells and for delineating the contributions of these processes to the avoidance of mutagenesis and carcinogenesis.