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

跨损伤合成(TLS)DNA聚合酶(POL)通过DNA损伤促进复制。生化的， 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。虽然有大量关于TLS POLS的结构和功能的信息，以及 多条路径，其主要调节无差错的TLS并且利用一个TLS极或 关于两个POL的顺序作用，没有关于这些TLS过程是如何被调节的信息。基座 根据我们在这里提供的证据，我们提出ATR激酶在正常人类细胞中的作用：(A)促进 通过多条途径主要是无错误的TLS；(B)与复制体一起促进TLS停滞 并稳定在DNA损伤部位；以及(C)通过两个POL的顺序作用来协调TLS，其中一个POL POL插入与DNA损伤相反的核苷酸，另一个POL则扩展合成。在目标1中，我们将 开展研究以确定与ATR熟练细胞不同，TLS通过多个、主要是 无错误途径，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。在目标2中， 确定ATR通过DNA损伤与 复制叉处的复制体(RF)在DNA损伤部位停滞，我们将分析TLS在ATR中的方式 缺陷细胞不同于ATR熟练细胞。对于这些研究，我们将：(A)通过以下方式确定程度 与ATR熟练者相比，ATR缺陷细胞中哪种RF通过紫外线损伤的进展受到抑制 细胞；(B)确定尽管TLS在ATR熟练细胞中与复制体一起工作，但 复制体在ATR缺陷细胞中解体；随后，TLS出现在间隙中，需要PrimPol才能启动 DNA合成和CRL4Cdt2泛素连接酶对增殖细胞核抗原泛素化的影响；(C)确定 环丁烷嘧啶二聚体(CPDS)上的POL发生时动力学较慢，且在ATR缺乏时水平降低 与ATR熟练细胞中的比较；以及(D)确定RF崩溃是否由复制体引起 ATR缺陷细胞DNA损伤部位的拆解导致染色体畸变率大幅上升和 细胞凋亡性死亡在目标3中，我们将分析ATR介导的TLS Pol磷酸化在 形成TLS POL系综，使得依赖于两个POL的TLS的两个不同步骤出现在一个步骤中 物理实体，而不是独立的。 有证据表明，与ATR熟练细胞相比，ATR中TLS依赖于DNA损伤进行复制 缺陷细胞以高度突变的方式运作，出现在缝隙中，传导方式不同，而且更少 而且，在没有ATR的情况下，染色体不稳定和细胞凋亡的发生率极高， 对于通过抑制DNA损伤诱导的突变、染色体不稳定、 和凋亡细胞死亡，ATR在TLS中的作用将有助于基因组的稳定和细胞的动态平衡。

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.