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

DNA 聚合酶 lambda 的结构和功能与破坏 Watson-Crick 碱基配对的 DNA 损伤相反

基本信息

批准号：
10065004
负责人：
LOUISE PRAKASH
金额：
$ 25.45万
依托单位：
UNIVERSITY OF TEXAS MED BR GALVESTON
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-18 至 2022-11-30
项目状态：
已结题

项目摘要

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.

DNA 聚合酶 (Pol) z 是 B 家族 Pol，在受损 DNA 的广泛复制中发挥着重要作用。 Pol 的生化研究表明，它在 DNA 对面插入核苷酸 (nt) 的效率非常低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) 需要以下连续作用一个插入器和一个延伸器 Pol，通过与不同 DNA 损伤相反的方向延伸合成，Pol 执行在酵母中，Rev1 发挥着不可或缺但非催化作用。作为 Pol 的一个组成部分，它提高了 Pol 从不正确的 nts 相对 DNA 延伸的效率检查发现，Rev1/Pol 复合体促进了高度容易出错的 TLS，从而解释了这一点。与酵母 Pol 功能所需的 Rev1 形成鲜明对比， Rev1 不是正常人类细胞 TLS 中 Pol 功能所必需的，相反，我们在此提供证据表明。 Pol 是人类 (h) Pol 不可或缺的组成部分，并且与 Pol 相配合，hPol 促进了在拟议的研究中，我们将利用 TLS 对抗各种 DNA 损伤的基本无差错模式。结合遗传、细胞、生物化学和结构研究来：阐明 Pol● 在 Pol 中的作用人体细胞中依赖的TLS，确定Pol●在TLS相对DNA中的保真度和作用机制损害 Watson-Crick (W-C) 碱基配对的损伤，并定义了允许 Pol● 的分子机制在目标 1 中，我们将： (a) 分析 Pol● 及其域的要求。针对人类细胞中各种 DNA 损伤的 Pol 依赖性 TLS 和诱变；(b) 检查 Pol● 在小鼠基因组中携带的 cII 基因的紫外线诱导诱变中的作用 (c) 确定 Pol● 结构域将 Pol 定位到紫外线损伤的人类细胞的复制灶中的要求 (d)；检查 Pol● 是否与 UV 损伤的人体细胞中的物理复合物中的 hPol 结合；以及 (e) 确定 Pol● 对紫外线照射的人类细胞中分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 Pol● 在插入核苷酸时的催化效率和保真度相对于N1-甲基腺嘌呤（N1-MeA）和（6-4）TT光产物的3'T和5'T，并且（b）进行预- 稳态动力学研究以确定 Pol● 插入正确 nt 相反的作用机制这些损害 W-C 碱基配对的 DNA 损伤在目标 3 中，我们将 (a) 确定二元和的结构。在存在或不存在传入 dTTP 的情况下，Pol● 的三元复合物与 N1-MeA 模板结合， (b) 分别确定与 (6-4) TT 结合的 Pol● 的二元和三元配合物的结构分别在不存在或存在传入 dATP 的情况下包含光产物的模板，以及 (c) 对结构中被认为对稳定受损结构很重要的残基进行突变分析模板。