Coordination of DNA replication, repair, and translesion DNA synthesis

描述（由申请人提供）：不能有效地协调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，特别是那些由宿主免疫反应产生的活性氧（ROS）或暴露于抗生素引起的突变，促进了它们对宿主的适应（即病理适应），加剧了治疗。我们研究的长期目标是发展一种综合的机制理解，了解生物体如何协调其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。在过去的10年里，我们的实验室在这项资助的支持下，对我们理解大肠杆菌复制酶与翻译DNA合成DNA聚合酶（TLS Pols）协调作用的机制产生了重大影响。我们的发现成功地挑战了成熟的工具带模型。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，可能促进囊性纤维化气道中这种病原体的持久性。对导致突变的分子机制的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行为的机制的悬而未决的问题。我们将把精力集中在两个关键但尚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。

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.