Impact of ATR's role in translesion synthesis on prevention of DNA damage induced mutagenesis and chromosomal instability

ATR 在跨损伤合成中的作用对预防 DNA 损伤诱导的突变和染色体不稳定性的影响

• 批准号: 10634852
• 负责人: SATYA PRAKASH
• 金额: $ 32万
• 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: 2023-03-22 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10634852
• 关键词: Active Sites; Aging; Apoptotic; BRCT Domain; Base Excision Repairs; Base Pairing; Biochemical; Carcinogens; Cell Death; Cell physiology; Cells; Chromosomal Instability; Chromosome abnormality; Complex; DNA; DNA Adduction; DNA Adducts; DNA Damage; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Directed DNA Polymerase; Genetic; Genetic study; Genome Stability; Genomic Instability; Genomics; Growth; Homeostasis; Human; Impairment; Kinetics; Lesion; Link; Mediating; Molecular Conformation; Mutagenesis; Mutation; Nucleotide Excision Repair; Nucleotides; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Polymerase; Prevention; Process; Purines; Pyrimidine Dimers; Reaction; Role; Seckel syndrome; Site; Specificity; Structure; UV induced; Ubiquitination; adduct; adverse outcome; irradiation; novel; nucleotide metabolism; pollutant; prevent; reconstitution; replication stress; restraint; scaffold; tumorigenesis; ubiquitin ligase; ultraviolet lesions

Translesion synthesis (TLS) DNA polymerases (Pols) promote replication through DNA lesions. Biochemical, structural, and genetic studies have indicated that TLS Pols play highly specialized roles in replicating through DNA lesions. Although there is a great deal of information on the structure and function of TLS Pols, and on the multiplicity of pathways that mediate predominantly error-free TLS and which utilize the action of one TLS Pol or the sequential action of two Pols, there is no information on how these TLS processes are regulated. Based upon the evidence we provide here, we propose a role for ATR kinase in normal human cells in (a) promoting predominantly error-free TLS via multiple pathways; (b) promoting TLS in conjunction with the replisome stalled and stabilized at DNA lesion sites; and (c) in coordinating TLS by the sequential action of two Pols in which one Pol inserts a nucleotide opposite the DNA lesion and another Pol extends synthesis therefrom. In Aim 1, we will carry out studies to establish that unlike in ATR proficient cells where TLS operates via multiple, predominantly error-free pathways, TLS in ATR deficient cells operates via a single highly error-prone pathway. In Aim 2, to establish that ATR promotes proficient TLS dependent replication through DNA lesions in conjunction with the replisome at replication forks (RFs) stalled at DNA lesion sites, we will analyze the ways in which TLS in ATR deficient cells differs from that in ATR proficient cells. For these studies, we will: (a) determine the extent by which RF progression through UV lesions is inhibited in ATR deficient cells compared to that in ATR proficient cells; (b) determine that whereas TLS operates in conjunction with replisome in ATR proficient cells, the replisome disassembles in ATR deficient cells; subsequently, TLS occurs in gaps, requires PrimPol for initiating DNA synthesis, and CRL4Cdt2 ubiquitin ligase for PCNA ubiquitination; (c) determine whether accumulation of Pol at cyclobutane pyrimidine dimers (CPDs) occurs with slower kinetics and at reduced levels in ATR deficient cells compared to that in ATR proficient cells; and (d) determine whether RF collapse resulting from replisome disassembly at DNA lesion sites in ATR deficient cells confers a large elevation in chromosomal aberrations and apoptotic cell death. In Aim 3, we will analyze the role of ATR mediated phosphorylation of TLS Pols in the formation of a TLS Pol ensemble so that the two different steps of TLS dependent upon two Pols occur in one physical entity, rather than independently. The evidence that in contrast to ATR proficient cells, TLS dependent replication through DNA lesions in ATR deficient cells operates in highly mutagenic ways, occurs in gaps, is conducted differently and much less efficiently, and that chromosomal instability and apoptotic cell death are highly elevated in the absence of ATR, will be important for establishing that by restraining DNA damage induced mutagenesis, chromosomal instability, and apoptotic cells death, ATR’s role in TLS would contribute to genome stability and cellular homeostasis.

跨损伤合成 (TLS) DNA 聚合酶 (Pols) 通过 DNA 损伤促进复制。 结构和遗传学研究表明，TLS Pols 在复制过程中发挥着高度专业化的作用 尽管有大量关于 TLS Pols 的结构和功能以及关于 DNA 损伤的信息。 多种途径主要介导无差错 TLS，并利用一种 TLS Pol 或 两个 Pols 的顺序操作，没有关于如何监管​​这些 TLS 进程的信息。 根据我们在此提供的证据，我们提出 ATR 激酶在正常人类细胞中的作用：(a) 促进 (b) 与停止的复制体一起推广 TLS (c) 通过两个 Pols 的连续作用来协调 TLS，其中一个 Pol 在 DNA 损伤对面插入一个核苷酸，另一个 Pol 从中延伸合成。在目标 1 中，我们将。 进行研究以确定与 ATR 熟练细胞不同，在 ATR 熟练细胞中 TLS 通过多个（主要是 在目标 2 中，ATR 缺陷细胞中的 TLS 通过单一的高度容易出错的途径进行操作。 确定 ATR 通过 DNA 损伤与 复制叉 (RF) 处的复制体在 DNA 损伤位点停滞，我们将分析 ATR 中 TLS 的方式 缺陷细胞与 ATR 熟练细胞不同，对于这些研究，我们将： (a) 通过以下方式确定程度。 与 ATR 充分的细胞相比，ATR 缺陷细胞中通过 UV 损伤的 RF 进展受到抑制 (b) 确定虽然 TLS 与 ATR 熟练细胞中的复制体一起发挥作用， 复制体在 ATR 缺陷细胞中分解；随后，TLS 出现在间隙中，需要 PrimPol 来启动 DNA合成和CRL4Cdt2泛素连接酶用于PCNA泛素化(c)确定是否积累； 环丁烷嘧啶二聚体 (CPD) 的 Pol 在 ATR 缺陷中以较慢的动力学发生且水平降低 细胞与 ATR 熟练细胞进行比较；以及 (d) 确定 RF 崩溃是否由复制体引起 ATR 缺陷细胞中 DNA 损伤位点的分解导致染色体畸变大幅升高 在目标 3 中，我们将分析 ATR 介导的 TLS Pols 磷酸化在细胞凋亡中的作用。 形成 TLS Pol 系综，以便依赖于两个 Pols 的 TLS 的两个不同步骤发生在一个 物理实体，而不是独立的。 与 ATR 熟练细胞相比，ATR 中通过 DNA 损伤进行 TLS 依赖复制的证据 有缺陷的细胞以高度诱变的方式运作，出现在间隙中，进行方式不同，而且更少 有效地，并且在缺乏 ATR 的情况下染色体不稳定性和细胞凋亡显着升高， 对于确定通过抑制 DNA 损伤诱导的突变、染色体不稳定性、 和凋亡细胞死亡，ATR 在 TLS 中的作用将有助于基因组稳定性和细胞稳态。