Structure and function of DNA polymerase lambda opposite DNA lesions which disrupt Watson-Crick base pairing

DNA聚合酶（Polymerase，Pol）是一种B家族的聚合酶，在损伤DNA的复制过程中起重要作用。广泛 用Poly的生物化学研究表明，它在插入与DNA相反的核苷酸（nt）时效率很低， 损伤，但在从正确或不正确的nt相对DNA损伤延伸合成方面非常有效。 由于通过多种DNA损伤的跨损伤DNA合成（TLS）需要以下的顺序作用： 一个插入器和一个扩展器Pol，通过扩展与不同DNA损伤相反的合成， 复制受损DNA的关键任务。在酵母中，Rev 1起着不可或缺的非催化作用 作为Pol引物的一个组分，它增加了Pol引物从不正确的nt对端DNA延伸的效率 病变因此，Rev 1/Pol编译器复合体促进了高度易错的TLS，从而导致了 酵母中的损伤诱导突变。与Rev 1对酵母Pol功能的要求形成鲜明对比， 在正常人类细胞中，TLS中的Poll功能不需要Rev 1。相反，我们在这里提供的证据表明， （h）Pol蛋白是人体内不可或缺的组成部分，与Pol蛋白一起，hPol蛋白促进了 TLS的主要无错误模式与各种DNA损伤相反。在拟议的研究中，我们将利用 结合遗传、细胞、生物化学和结构研究，阐明Pol在Pol中的作用 依赖性TLS在人类细胞中的作用，确定了TLS相对DNA中Poll的保真度和作用机制 损害Watson-Crick（W-C）碱基配对的病变，并定义了允许Pol 处理这种DNA损伤的活性位点。在目标1中，我们将：（a）分析政策及其领域的需求 对于Pol β依赖性TLS和人细胞中与多种DNA损伤相对的诱变;（B）检查 （c）确定在小鼠基因组中携带的cII基因的UV诱导的诱变中， 在UV损伤的人细胞中，需要Pol β结构域将Pol β定位到复制焦点中;（d） 检查在UV损伤的人细胞中的物理复合物中Pol β是否与hPol β缔合;和（e） 确定Pol对UV照射的人细胞中叉进展的影响。在目标2中，我们将（a）使用 稳态动力学分析，以确定聚合物在插入核苷酸中的催化效率和保真度 相反的N1-甲基腺嘌呤（N1-MeA）和（6-4）TT光产物的3 'T和5' T，和（B）进行预处理， 稳态动力学研究，以确定插入正确的nt对端的Poll的作用机制 这些DNA损伤损害了W-C碱基配对。在目标3中，我们将（a）确定二元的结构， 在不存在或存在引入的dTTP的情况下，与N1-MeA模板结合的Pol β的三元复合物， （B）测定了与（6-4）TT键合的Poly的二元和三元复合物的结构 分别在不存在或存在引入的dATP的情况下，包含光产物的模板，和（c） 对被认为对稳定受损结构重要的结构残基进行突变分析， template.模板

DNA polymerase (Pol) , a B-family Pol, plays an important role in replication of damaged DNA. Extensive biochemical studies with Pol have shown that it is very inefficient at inserting nucleotides (nts) opposite DNA lesions but is highly efficient at extending synthesis from the correct or incorrect nt opposite DNA lesions. Since translesion DNA synthesis (TLS) through a large variety of DNA lesions requires the sequential action of an inserter and an extender Pol, by extending synthesis opposite from diverse DNA lesions, Pol performs a critical task in the replication of damaged DNA. In yeast, Rev1 performs an indispensable but non-catalytic role as a component of Pol and it increases Pol’s efficiency for extension from incorrect nts opposite DNA lesions. Consequently, the Rev1/Pol complex promotes highly error-prone TLS and thereby accounts for damage induced mutagenesis in yeast. In striking contrast to the requirement of Rev1 for yeast Pol function, Rev1 is not required for Pol function in TLS in normal human cells. Instead, we provide evidence here that Pol acts as an indispensable component of human (h) Pol and in concert with Pol, hPol promotes a predominantly error-free mode of TLS opposite various DNA lesions. In the proposed studies, we will utilize a combination of genetic, cellular, biochemical, and structural studies to: elucidate the role of Pol in Pol dependent TLS in human cells, determine the fidelity and action mechanism of Pol in TLS opposite DNA lesions which impair Watson-Crick (W-C) base pairing, and define the molecular mechanisms that allow Pol’s active site to handle such DNA lesions. In Aim 1, we will: (a) analyze the requirement of Pol and its domains for Pol-dependent TLS and mutagenesis opposite a variety of DNA lesions in human cells; (b) examine the role of Pol in UV induced mutagenesis in the cII gene carried in the mouse genome; (c) determine the requirement of Pol domains for localization of Polinto replication foci in UV damaged human cells; (d) examine whether Pol associates with hPol in a physical complex in UV damaged human cells; and (e) determine the effects of Pol on fork progression in UV irradiated human cells. In Aim 2, we will (a) use steady-state kinetic analyses to determine the catalytic efficiency and fidelity of Pol in inserting nucleotides opposite N1-methyladenine (N1-MeA) and the 3’T and 5’T of a (6-4) TT photoproduct, and (b) carry out pre- steady-state kinetic studies to determine the action mechanism of Pol for inserting the correct nt opposite these DNA lesions which impair W-C base pairing. In Aim 3, we will (a) determine the structures of binary and ternary complexes of Pol bound to N1-MeA template in the absence or presence of an incoming dTTP, respectively, (b) determine the structures of binary and ternary complexes of Pol bound to the (6-4) TT photoproduct-containing templates in the absence or presence of an incoming dATP, respectively, and (c) carry out mutational analyses of residues deemed from the structures as important for stabilizing the damaged template.