Coordination of DNA replication, repair, and translesion DNA synthesis

描述（由申请人提供）：未能有效协调DNA复制与其他细胞过程导致突变和基因组不稳定，导致许多人类疾病状态，包括癌症。人类病原体中的突变，特别是由宿主免疫应答产生的活性氧（ROS）或暴露于抗生素引起的突变，促进它们对宿主的适应（即，病理适应），加重治疗。我们的研究的长期目标是发展一个综合的机制理解生物体如何协调其DNA复制机制的行动与其他细胞因子的DNA修复和损伤耐受性。在过去10年里，我们实验室的工作在这项资助的支持下，对我们理解协调E.大肠杆菌复制酶与跨损伤DNA合成DNA聚合酶（TLS Pos）。我们的研究结果成功地挑战了已经建立的工具带模型.我们还表明，铜绿假单胞菌DNA聚合酶IV（Pol IV）催化的错误有助于突变，可能促进这种病原体在囊性纤维化气道中的持续存在。对导致突变的机制的分子理解对于我们理解基因组不稳定性、人类疾病和病理适应的基础以及开发新疗法的努力至关重要。拟议的研究解决了有关生物体用于管理其不同Pol的行为的机制的未回答的问题。我们将把精力集中在两个关键但研究不足的领域。在之前的支持期间，我们发现特定的E。 coli β-clamp-DNA相互作用是DNA损伤诱导的诱变所必需的，这表明它们赋予Pol开关一个层次顺序，可以用来控制突变率。在目标1中，我们将使用遗传学、生物化学、生物物理学和单分子方法的组合来确定不同的β-钳夹-DNA相互作用对复制保真度和TLS的贡献。在目标2中，我们将使用小角X射线散射（SAXS），尺寸排阻色谱-多角光散射（SEC-MALS），分子模拟和生物化学方法来确定由5种不同的E. coli Pos、clamp和DNA。利用从这些努力中获得的见解，以及遗传、生物化学、生物物理和单分子方法，我们将定义E。coliPos开关。我们还将确定阻碍Pol III合成能力的能力是否与Pol III转换的其他蛋白质共享。这些实验的结果将提供前所未有的深入了解DNA复制，DNA修复和TLS协调调节的分子机制。此外，我们预计，我们的研究结果将确定这些进化上保守的过程中的关键步骤，可以有针对性地控制复制的熟练程度和保真度，以获得治疗效果。

DESCRIPTION (provided by applicant): Failure to efficiently coordinate DNA replication with other cellular processes results in mutations and genome instability, contributing to numerous human disease states, including cancers. Mutations in human pathogens, particularly those caused by reactive oxygen species (ROS) generated by the host immune response, or exposure to antibiotics, promote their adaptation to the host (i.e., pathoadaptation), exacerbating treatment. The long-term goal of our research is to develop an integrated mechanistic understanding of how organisms coordinate the actions of their DNA replication machinery with those of other cellular factors that act in DNA repair and damage tolerance. Work in our lab over the last 10 years supported by this grant has had a major impact on our understanding of mechanisms coordinating the actions of the E. coli replicase with those of translesion DNA synthesis DNA polymerases (TLS Pols). Our findings successfully challenged the well- established tool belt model. We have also shown that errors catalyzed by Pseudomonas aeruginosa DNA polymerase IV (Pol IV) contribute to mutations that likely promote persistence of this pathogen in cystic fibrosis airways. A molecular understanding of the mechanisms that contribute to mutations is crucial to our understanding of the basis for genome instability, human disease, and pathoadaptation, as well as efforts to develop novel therapies. The proposed research addresses unanswered questions regarding mechanisms that organisms use to manage the actions of their diverse Pols. We will focus our efforts in two critical yet understudied areas. During the prior period of support, we discovered that specific E. coli beta-clamp-DNA interactions are required for DNA damage-induced mutagenesis, suggesting they impart a hierarchical order to Pol switches that may be exploited to control mutation rate. In Aim 1, we will determine the contributions of the different beta-clamp-DNA interactions to replication fidelity and TLS using a combination of genetic, biochemical, biophysical, and single molecule approaches. In Aim 2, we will use small angle X-ray scattering (SAXS), size exclusion chromatography-multi angle light scattering (SEC-MALS), molecular modeling, and biochemical approaches to structurally define complexes consisting of the 5 different E. coli Pols, clamp, and DNA. Using insights gained from these efforts, together with genetic, biochemical, biophysical, and single molecule approaches, we will define the mechanisms by which E. coli Pols switch. We will also determine whether an ability to impede Pol III processivity is shared by other proteins that switch with Pol III. Results from these experiments will provide unprecedented insight into the molecular mechanisms underlying coordinate regulation of DNA replication, DNA repair, and TLS. Furthermore, we anticipate that our results will identify critical steps in these evolutionarily conserved processes that can be targeted to control proficiency and fidelity of replication for therapeutic gain.