Regulation of the DNA damage Response

描述(申请人提供)：真核细胞面临着对其基因组完整性的持续挑战，复杂的过程已经演变成对DMA损伤的反应。细胞停止分裂、修复损伤，在某些情况下还会发生凋亡，如果不能完成这些过程，可能会导致基因组不稳定和癌症。事实上，这些过程中的缺陷形成了许多癌症易感疾病的分子基础。DMA损伤耐受机制是DMA损伤反应的另一个方面，它允许细胞在存在聚合酶阻断损伤的情况下继续复制，将损伤的修复留到以后的时间。这项建议的总体目标是了解调控DMA损伤耐受性的分子机制。耐受性的一种形式涉及在损伤部位从高保真复制聚合酶切换到跨损伤合成(TLS)聚合酶。这种转换被认为涉及复制滑动钳位在停滞的复制叉处的单素化。这种形式的耐受性容易出错，因为TLS聚合酶的保真度低于复制聚合酶。第二种无错误的DMA损伤容忍度也涉及到增殖细胞核抗原的泛素化。为了了解DNA损伤耐受性和TLS的调控机制，我们将：(1)确定单链DMA调节增殖细胞核抗原泛素化的机制；(2)研究增殖细胞核抗原泛素化与复制和TLS聚合酶之间的关系；(3)确定ATR介导的检查点在TLS和DNA损伤诱导的突变中的作用。我们将使用一种来自非洲爪哇卵的无细胞系统来探索这些问题，该系统概括了DNA损伤耐受性的许多方面。这些提取物为分析调节TLS和损伤修复的复杂途径提供了一个易于处理的系统。这项建议中描述的实验应该有助于我们理解细胞用来调控DNA损伤耐受过程的机制。由于这些过程中的缺陷会导致基因组完整性的丧失、癌症和细胞死亡，这些研究将为癌症的发展提供有价值的见解，并最终可能为癌症的治疗或检测指出新的方法。

DESCRIPTION (provided by applicant): Eukaryotic cells face constant challenges to the integrity of their genome, and sophisticated processes have evolved to respond to DMA damage. Cells arrest cell division, repair damage, and in some cases undergo apoptosis, and failure to carry out these processes can lead to genomic instability and cancer. In fact, defects in these processes form the molecular basis for many cancer-prone disorders. DMA damage tolerance mechanisms are another aspect of the DMA damage response that allow the cell to continue replication in the presence of polymerase-blocking lesions, leaving repair of the damage for a later time. The overall goal of this proposal is to understand the molecular mechanisms that regulate DMA damage tolerance. One form of tolerance involves switching from high-fidelity replicative polymerases to translesion synthesis (TLS) polymerases at the site of damage. This switch is thought to involve monoubiquitination of the replicative sliding clamp PCNA at the stalled replication fork. This form of tolerance is error prone because the TLS polymerases are of lower fidelity than the replicative polymerases. A second, error-free form of DMA damage tolerance also involves the ubiquitination of PCNA. In order to understand the mechanisms regulating DMA damage tolerance and TLS, we will: (1) Determine the mechanism by which single-stranded DMA regulates the ubiquitination of PCNA; (2) Investigate the relationship between PCNA ubiquitination and the switch between replicative and TLS polymerases at the replication fork; and (3) Determine the role of the ATR-mediated checkpoint in TLS and DNA-damage induced mutagenesis. We will explore these questions using a cell free system derived from the eggs of Xenopus laevis that recapitulates many aspects of DNA damage tolerance. These extracts provide a tractable system for analyzing the complex pathways regulating TLS and damage repair. The experiments described in this proposal should help us understand the mechanisms used by cells to regulate DNA damage tolerance process. Because defects in these processes lead to loss of genomic integrity, cancer and cell death, these studies will provide valuable insight into how cancer develops and may ultimately point the way to new approaches for the treatment or detection of cancer.