Mechanisms for the high fidelity of translesion synthesis by Y-family DNA polymerases in human cells

人类细胞中 Y 家族 DNA 聚合酶高保真度跨损伤合成的机制

• 批准号: 10550540
• 负责人: LOUISE PRAKASH
• 金额: $ 43.18万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10550540
• 关键词: ATP phosphohydrolase; Active Sites; Affect; Biochemical; Cell physiology; Cells; Chromosomal Instability; Cryoelectron Microscopy; DNA biosynthesis; DNA lesion; DNA-Directed DNA Polymerase; DNA-dependent ATPase; Excision; Family; Genetic; Genome Stability; Homeostasis; Human; Label; Malignant Neoplasms; Molecular; Mutation; Nucleotides; Phosphodiesterase I; Play; Polymerase; Proteins; Research; Role; WRN gene; Y protein; genome integrity; genome-wide; helicase; prevent; protein function; replicase; restraint; tumorigenesis

ABSTRACT By promoting replication through DNA lesions, translesion synthesis (TLS) DNA polymerases (Pols) play a critical role in preventing chromosomal instability and protecting against tumorigenesis. Unlike replicative Pols, TLS Pols have less constrained active sties and they lack proofreading 3'→5' exonuclease activity. Consequently, purified TLS Pols synthesize DNA opposite DNA lesions with an extremely low fidelity. Despite this, TLS operates in a predominantly error-free manner in normal human cells (not derived from cancers), The overall objective in this project is to identify the cellular processes and mechanisms by which high fidelity is imposed upon TLS by the intrinsically highly error-prone Y-family Pols. Using a combination of genetic, cellular, biochemical, and structural approaches, we will address the following questions: (1) Do the Y-family Pols associate with other protein factors in a multiprotein ensemble and do these proteins have activities that elevate the fidelity of the TLS Pol? (2) What is the protein composition of the entire Y-family Pol ensemble for error-free TLS in human cells? (3) How is the fidelity of TLS modulated by the components of the multiprotein ensemble? (4) What are the molecular underpinnings of action mechanisms via which components of the multiprotein ensemble impose high fidelity on Y-family Pols? To pursue these questions, we have identified a number of protein factors that function in TLS specifically in conjunction with Y-family Pols; included among these proteins are WRN which possesses DNA helicase and 3'→5' exonuclease activities, and WRNIP1 which has a DNA dependent ATPase activity. How these activities contribute to the fidelity of TLS by Y-family Pols opposite different types of DNA lesions will be analyzed in extensive mutational studies that include genome wide sequencing. Using proximity labeling in which TurboID is fused to Polη, we will determine whether there are additional proteins that function in TLS in conjunction with Y-family Pols and whether activities in these proteins affect the fidelity of TLS by these Pols. In biochemical studies with the purified multiprotein ensemble of Polη or Polι, we will ascertain the roles of WRN 3'→5' exonuclease, WRN and WRNIP1 ATPase, and of any other newly identified activities in the high fidelity of TLS by these Pols opposite different types of DNA lesions. From cryo- EM studies with the purified multiprotein ensemble of Polη or Polι, we will determine mechanistically how the components of the multiprotein ensemble modulate the fidelity of these Y-family Pols opposite DNA lesions. Cumulatively, these studies will identify the components of the multiprotein Y-family TLS replicases which carry out high fidelity TLS in human cells. They will reveal the mechanisms by which the various components constrain Y-family Pols' active sites to restrain nucleotide (nt) misincorporation and how WRN's 3'→5' exonuclease activity is coordinated with the TLS Pol for the removal of misinserted nt. These studies will be paradigm shifting and will open new vistas of research into the mechanistic details of TLS Pols' fidelity and they will give impetus to further elaboration of the roles of TLS Pols in genome integrity.

抽象的 通过 DNA 损伤促进复制，跨损伤合成 (TLS) DNA 聚合酶 (Pols) 发挥着 与复制性 Pols 不同，它在防止染色体不稳定和防止肿瘤发生方面发挥着关键作用。 TLS Pols 的活性粒细胞限制较少，并且缺乏校对 3'→5' 核酸外切酶活性。 经测试，纯化的 TLS Pols 合成的 DNA 与 DNA 损伤相反，但保真度极低。 TLS 在正常人类细胞（并非源自癌症）中以基本无错误的方式运行， 该项目的总体目标是确定高保真度的细胞过程和机制。 结合遗传、细胞、 生物化学和结构方法，我们将解决以下问题：（1）Y家族Pols 与多蛋白整体中的其他蛋白质因子相关联，这些蛋白质是否具有提高 TLS Pol 的保真度？ (2) 整个 Y 家族 Pol 整体的蛋白质组成是多少？ 人类细胞中的 TLS？（3）多蛋白整体的成分如何调节 TLS 的保真度？ (4) 多蛋白成分通过哪些作用机制的分子基础是什么？ 为了提出这些问题，我们已经确定了一些 这些蛋白质中包括在 TLS 中专门与 Y 家族 Pols 结合发挥作用的蛋白质因子； 是具有 DNA 解旋酶和 3'→5' 核酸外切酶活性的 WRN，以及具有 DNA 的 WRNIP1 依赖的 ATP 酶活性。这些活性如何有助于 Y 家族 Pols 相反的 TLS 保真度。 不同类型的 DNA 损伤将在包括全基因组范围的突变广泛研究中进行分析 使用 TurboID 与 Polη 融合的邻近标记，我们将确定是否存在。 与 Y 家族 Pols 一起在 TLS 中发挥作用的其他蛋白质以及这些蛋白质是否具有活性 在使用 Polη 或纯化的多蛋白整体进行生化研究时，这些 Pols 会影响 TLS 的保真度。 Polι，我们将确定 WRN 3'→5' 核酸外切酶、WRN 和 WRNIP1 ATPase 以及任何其他新的作用 确定了这些 Pols 在高保真度 TLS 中对不同类型的 DNA 损伤的活性。 使用 Polη 或 Polι 的纯化多蛋白整体进行 EM 研究，我们将从机制上确定如何 多蛋白整体的成分调节这些 Y 家族 Pols 对 DNA 损伤的保真度。 累积起来，这些研究将鉴定多蛋白 Y 家族 TLS 复制品的成分，这些成分 在人体细胞中进行高保真 TLS 他们将揭示各种成分的机制。 限制Y家族Pols的活性位点以抑制核苷酸（nt）错误掺入以及WRN如何从3'→5' 核酸外切酶活性与 TLS Pol 协调，以去除错误插入的 nt。 范式转变，将为 TLS Pols 保真度的机制细节研究开辟新的前景，并且它们 将推动进一步阐述 TLS Pols 在基因组完整性中的作用。