Coordination of DNA replication, repair, and translesion DNA synthesis

描述(由申请人提供)：未能有效地协调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。人类病原体的突变，特别是由宿主免疫反应或接触抗生素产生的活性氧物种(ROS)引起的突变，促进了它们对宿主的适应(即病理适应)，从而加剧了治疗。我们研究的长期目标是对生物体如何协调其DNA复制机制与其他细胞因子在DNA修复和损伤耐受中的作用形成一个完整的机制理解。我们实验室在过去10年中由这笔赠款支持的工作对我们理解协调大肠杆菌复制酶和跨病变DNA合成DNA聚合酶(TLS POL)的作用的机制产生了重大影响。我们的发现成功地挑战了公认的工具带模型。我们还表明，由铜绿假单胞菌DNA聚合酶IV(POL IV)催化的错误有助于突变，这些突变可能促进这种病原体在囊性纤维化呼吸道中的持久性。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、人类疾病和病理适应的基础以及开发新的治疗方法至关重要。这项拟议的研究解决了有关生物体用来管理其不同POL的行动的机制的悬而未决的问题。我们将把我们的努力集中在两个关键但未得到充分研究的领域。在之前的支持期间，我们发现特定的E. 在DNA损伤诱导的突变过程中，Coli的β-夹子-DNA相互作用是必需的，这表明它们赋予POL开关一个分层的顺序，可以被用来控制突变率。在目标1中，我们将结合遗传、生化、生物物理和单分子方法，确定不同的β-钳-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)、尺寸排除层析-多角光散射(SEC-MALS)、分子建模和生物化学方法来定义由5种不同的大肠杆菌POL、夹子和DNA组成的复合体的结构。利用从这些努力中获得的见解，以及遗传、生化、生物物理和单分子方法，我们将定义大肠杆菌POLS转换的机制。我们还将确定是否有能力阻止Pol III的处理能力被其他与Pol III转换的蛋白质所共享。这些实验的结果将为DNA复制、DNA修复和TLS的协调调控提供前所未有的分子机制。此外，我们预计，我们的结果将确定在这些进化保守的过程中的关键步骤，这些步骤可以有针对性地控制复制的熟练程度和保真度，以获得治疗收益。

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.