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.

转录合成（TLS）DNA聚合酶（Pos）通过DNA损伤促进复制。生物化学， 结构和遗传学研究表明，TLS Pol在复制过程中发挥着高度专业化的作用， DNA损伤尽管有大量关于TLS Pol的结构和功能的信息， 介导主要无错误TLS并利用一个TLS Pol或 由于两个Pol的顺序动作，没有关于如何调节这些TLS过程的信息。基于 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是：（a）促进 通过多个途径主要无错误的TLS;（B）与停止的复制体一起促进TLS 并稳定在DNA损伤位点;和（c）在协调TLS的顺序作用的两个波尔，其中一个 Pol在DNA损伤的对面插入一个核苷酸，另一个Pol从那里延伸合成。在目标1中，我们 进行研究，以确定与ATR熟练细胞不同的是，TLS主要通过多个 无错误途径，ATR缺陷细胞中的TLS通过单一的高度易错途径起作用。目标2： 确定ATR通过DNA损伤结合 复制叉处的复制体（RFs）在DNA损伤位点停滞，我们将分析ATR中TLS的方式， 缺乏ATR的细胞不同于ATR熟练的细胞。就这些研究而言，我们会：（a）以下列方法厘定有关程度： 与ATR熟练的细胞相比，ATR缺陷细胞中通过UV损伤的RF进展受到抑制 （B）确定TLS在ATR熟练细胞中与复制体结合工作， 复制体在ATR缺陷细胞中解体;随后，TLS发生在缺口中，需要PrimPol启动 DNA合成和用于PCNA泛素化的CRL 4Cdt 2泛素连接酶;（c）确定是否积累了 环丁烷嘧啶二聚体（CPD）的聚合动力学较慢，在ATR缺陷型中的水平降低。 与ATR熟练细胞中的RF塌陷进行比较;以及（d）确定是否由复制体引起的RF塌陷 在ATR缺陷细胞中DNA损伤位点的解体赋予染色体畸变的大幅升高， 细胞凋亡在目的3中，我们将分析ATR介导的TLS Pol磷酸化在细胞凋亡中的作用。 形成TLS Pol系综，使得依赖于两个Pol的TLS的两个不同步骤同时发生， 物理实体，而不是独立的实体。 与ATR熟练细胞相反，TLS依赖于ATR中DNA损伤的复制的证据 缺陷细胞以高度致突变的方式运作，发生在间隙中，以不同的方式进行， 有效地，并且染色体不稳定性和凋亡性细胞死亡在缺乏ATR的情况下高度升高， 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内稳态。