Coordination of high fidelity replication with mutagenic translesion synthesis

高保真复制与诱变跨损伤合成的协调

• 批准号: 10063001
• 负责人: Alba Guarne
• 金额: $ 34.98万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-15 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063001
• 关键词: Antibiotic Resistance; Archaea; Bacteria; Binding; Biochemical; Biochemical Genetics; Biological Assay; Biophysics; Bypass; Closure by clamp; Collaborations; Collection; Complex; Coupled; Cryoelectron Microscopy; DNA; DNA Repair; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Escherichia coli; Eukaryota; Failure; Genetic; Genome; Genomic Instability; Goals; Human; Hydrophobicity; In Vitro; Individual; Knowledge; Lesion; Maintenance; Malignant Neoplasms; Modeling; Molecular; Molecular Sieve Chromatography; Mutagenesis; Mutation; Nucleic Acids; Organism; Pathway interactions; Play; Polymerase; Process; Proteins; Research; Roentgen Rays; Role; Slide; Structural Models; Structure; Techniques; Therapeutic; Time; biophysical techniques; clinically significant; complex IV; defined contribution; genetic approach; human disease; in vivo; insight; light scattering; mange; molecular modeling; novel; pathogen; pathogenic microbe; programs; repair function; repaired; replicase; single molecule; stoichiometry

ABSTRACT Failure to properly coordinate DNA replication with repair and potentially mutagenic translesion DNA synthesis (TLS) contributes to mutations that underlie numerous human disease states, including cancers, as well as antibiotic resistance and adaptation of clinically significant microbial pathogens. We use Escherichia coli as a model to define fundamental mechanisms by which organisms mange the actions of their high fidelity replicative DNA polymerase(s) (Pol) with those of low fidelity TLS Pols involved in lesion bypass and mutagenesis. The process by which one Pol replaces another at a replication fork is referred to as `Pol switching'. Sliding clamp proteins (DnaN or β in bacteria; PCNA in eukaryotes and archaea) play essential roles in these switches. The generally accepted `toolbelt' model for Pol switching postulates that two different Pols simultaneously bind separate hydrophobic clefts in the same β clamp to sequentially access the DNA. In stark contrast with the toolbelt model, we recently determined that TLS Pols interact with each other and with the Pol III replicase, and while the mechanistic contributions of these interactions are currently unknown, we nevertheless demonstrated that Pol-Pol interactions are absolutely required for Pol switching in vitro and in vivo. We have also identified several novel Pol-β clamp interactions important for Pol function. In Aim 1, we will exploit our structural model of the Pol III-β clamp-Pol IV complex, as well as a wealth of biochemical, biophysical, single molecule and genetic approaches to define for the first time in molecular detail the specific contributions to switching of discrete Pol III-Pol IV and Pol IV-β clamp interactions. In Aim 2 we will exploit structural insights we have gained regarding the Pol II-β clamp complex to define in molecular detail how the β clamp manages Pol II processivity, proofreading and Pol III-Pol II and Pol II-Pol IV switching. In Aim 3, we will use small angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM), molecular modeling and a combination of biochemical and biophysical approaches to structurally define how the different Pols interact with each other and the β clamp, and how DNA influences these interactions. In addition, we will determine the protein stoichiometry of the different Pol-β clamp and Pol-β clamp-Pol complexes using size exclusion chromatography coupled with multi angle light scattering (SEC-MALS), which Pols interact with each other, and whether or not these interactions are competitive. Taken together, results of these studies will provide unprecedented insight into the molecular mechanisms by which E. coli manages and coordinately regulates the actions of its different Pols during DNA replication, repair and TLS. Finally, our findings may identify critical steps in these higher order regulatory networks that generalize to other bacteria and can be targeted by chemotherapeutics to control replication and mutagenesis.

抽象的 未能与修复和潜在的诱变转移DNA合成正确协调DNA复制 （TLS）促进了众多人类疾病状态（包括癌症）的突变以及 抗生素耐药性和临床上显着的微生物病原体的适应性。我们将大肠杆菌用作 定义有机体来控制其高保真作用的基本机制的模型 复制性DNA聚合酶（S）（pol）具有低忠诚度TLS POL的病变旁路和 诱变。一个POL在复制叉上替换另一个POR的过程称为`p pol 交换'。滑动夹具蛋白（细菌中的DNAN或β；真核生物中的PCNA和古细菌）发挥必不可少的 这些开关中的角色。普遍接受的“工具面”模型用于切换的假设是两个不同的 POLS在相同的β夹具中仅结合单独的疏水性裂缝，以顺序访问DNA。 与工具面模型形成鲜明对比，我们最近确定TLS POLS相互相互作用，并与 Pol III复制酶，尽管这些相互作用的机械贡献目前尚不清楚，但我们 然而，表明POL POL相互作用是绝对必需的 体内。我们还确定了几种对于POL功能很重要的新型POL-β夹具相互作用。在AIM 1中，我们将 利用我们的pol III-β夹具夹IV复合物以及大量生化的结构模型 生物物理，单分子和遗传方法首次定义分子细节 离散Pol III-POL IV和POL IV-β夹具相互作用的切换的贡献。在AIM 2中，我们将利用 我们获得了有关Pol II-β夹具复合物的结构见解，以分子细节定义β 夹具管理POL II的加工性，校对和Pol III-POL II和POL II-POL IV转换。在AIM 3中，我们将 使用小角度X射线散射（SAXS），冷冻电子显微镜（Cryo-EM），分子建模和A 生化和生物物理方法的结合结构上定义了不同的pol相互作用 彼此和β夹具，以及DNA如何影响这些相互作用。此外，我们将确定 使用尺寸排除的不同POL-β夹和Pol-β夹具配合物的蛋白质化学计量法 色谱与多角度光散射（SEC-MALS）相互作用，彼此相互作用， 这些互动是否具有竞争力。综上所述，这些研究的结果将提供 大肠杆菌管理和协调调节的分子机制的前所未有的见解 在DNA复制，修复和TLS期间，其不同的pol的作用。最后，我们的发现可能确定关键 在这些高阶监管网络中，该网络推广到其他细菌，可以针对 化学治疗药以控制复制和诱变。