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 通过通过DNA病变促进复制，透疗合成（TLS）DNA聚合酶（POLS）播放 在预防染色体不稳定性和预防肿瘤发生中的关键作用。与复制pol不同， TLS pol的活动性较少，并且缺乏校对3'→5'外切核酸酶活动。 因此，纯化的TLS pols合成了与DNA病变相对的DNA，其保真度极低。尽管 这，TLS在正常的人类细胞（不是从癌症中得出）以主要无错误的方式工作 该项目的总体目的是确定高保真性的细胞过程和机制 本质上容易出现的Y家庭pol强加于TLS。结合遗传，细胞， 生化和结构方法，我们将解决以下问题：（1）Y-家庭pol 与多蛋白质蛋白合奏中的其他蛋白质因子相关联，并且这些蛋白质具有升高的活性 TLS pol的保真度？ （2）整个Y-家庭POL集合的蛋白质组成是什么，无错误 人类细胞中的TL？ （3）如何通过多蛋白质蛋白的成分调节TLS的保真度？ （4）动作机制的分子基础是多蛋白的成分 合奏对Y-家庭的pol施加高忠诚？要解决这些问题，我们已经确定了许多 在TLS中起作用的蛋白质因子与Y-家庭POL相结合；包括这些蛋白质 是具有DNA解旋酶和3'→5'外切核酸酶活动的WRN，并且具有DNA的WRNIP1 依赖性ATPase活性。这些活动如何促进Y-家庭pol的TLS的保真度 在广泛的突变研究中将分析不同类型的DNA病变，其中包括基因组广泛 测序。使用将涡轮融合到Polη的接近标记，我们将确定是否存在 在TLS中起作用的其他蛋白质与Y-家庭POLS以及这些蛋白质中的活性是否存在 影响这些pol的TLS的保真度。在具有纯化的多蛋白质合奏或POLη或的生化研究中 POLι，我们将确定WRN 3'→5'外丝丝，WRN和WRNIP1 ATPase的作用，以及其他任何新的新的 这些POL与不同类型的DNA病变相反，在TLS高忠诚度中鉴定出活性。来自冷冻 EM通过纯化的polη或polι的多蛋白质集合的研究，我们将从机械上确定如何确定 多蛋白质合奏的成分调节了这些Y-家庭pol的忠诚度相反的DNA病变。 累积地，这些研究将确定多蛋白Y-家庭TLS复制酶的成分，这些复制酶 在人类细胞中执行高保真度TL。他们将揭示各种组成部分的机制 限制Y-家庭的POLS的活性位点，以限制核苷酸（NT）的失误以及WRN的3'→5' 核酸外切酶的活性与TLS POL协调，以去除Missinserted NT。这些研究将是 范式转移，并将为TLS Pols的忠诚的机械细节开辟新的研究远景 将推动进一步阐述TLS POLS在基因组完整性中的作用。