Molecular Mechanisms of Y-Family Translesion Polymerase Activity in Bacillus subtilis

枯草芽孢杆菌 Y 家族跨损伤聚合酶活性的分子机制

• 批准号: 10730396
• 负责人: Elizabeth Simmons Thrall
• 金额: $ 49.52万
• 依托单位: FORDHAM UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10730396
• 关键词: Antibiotic Resistance; Bacillus subtilis; Bacteria; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Bypass; Cell Death; Cell Survival; Cells; Closure by clamp; DNA; DNA Damage; DNA Repair; DNA Repair Gene; DNA Replication Damage; DNA Sequence Alteration; DNA biosynthesis; DNA lesion; DNA replication fork; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Defect; Development; Ensure; Escherichia coli; Exclusion; Family; Fluorescence Microscopy; Genetic Materials; Genetic Transcription; Genome Stability; Gram-Negative Bacteria; Gram-Positive Bacteria; Growth; Label; Life; Modeling; Molecular; Mutagenesis; Mutation; Pathway interactions; Play; Polymerase; Process; Proteins; Regulation; Role; SS DNA BP; Site; Slide; Stress; Visualization; Work; cell growth; experimental study; insight; molecular imaging; protein aminoacid sequence; protein protein interaction; recruit; response; single molecule

Cells must efficiently and accurately replicate their genetic material, yet this process is challenged by the presence of unrepaired DNA damage on the template strand. In the DNA damage tolerance pathway translesion synthesis (TLS), specialized translesion polymerases replicate damaged DNA, promoting cell survival under stress. Most TLS polymerases are lower fidelity than replicative DNA polymerases, and thus their activity must be tightly regulated under normal growth conditions to maintain genome stability. Conversely, stress-induced mutagenesis by TLS polymerases can promote cell survival under certain conditions, such as by contributing to the development of antibiotic resistance in bacteria. The gram-negative bacterium E. coli has served as a model species for mechanistic studies of TLS polymerase regulation, but it is not known whether the same principles apply in other bacterial species, including the model gram-positive bacterium B. subtilis, which has two Y-family TLS polymerases, Pol Y1 and Pol Y2. By combining biochemical and microbiological experiments with live-cell single-molecule imaging, we will provide a comprehensive picture of the spatial organization, dynamics, and molecular coordination of the TLS polymerases Pol Y1 and Pol Y2 in B. subtilis. This study will reveal new insights into how DNA replication fidelity is maintained during normal growth and will broaden our understanding of TLS and DNA damage tolerance in bacterial species beyond E. coli. Aim 1: Determine how TLS polymerases respond to replication perturbations in B. subtilis In E. coli, TLS polymerases are excluded from the replication fork during normal cellular growth but selectively enriched in response to replication perturbations. It is not known, however, whether B. subtilis Pol Y1 and Pol Y2 are regulated in a similar manner. We will use live-cell single-molecule imaging to visualize fluorescently- labeled Pol Y1 and Pol Y2 molecules during normal replication and upon DNA damage, allowing us to determine if and how they respond to replication perturbations. Aim 2: Elucidate the role of the DnaN clamp in coordinating Pol Y1 and Pol Y2 activity The bacterial replication processivity factor, the DnaN sliding clamp, interacts with a wide range of proteins involved in DNA replication and repair through a common binding site. Pol Y1 and Pol Y2 contain clamp-binding motifs (CBMs), short peptide sequences that bind to DnaN. We will combine biochemical and microbiological assays with live-cell single-molecule imaging to elucidate the role of DnaN in regulating TLS in B. subtilis. Aim 3: Determine whether and how interactions with SSB play a role in TLS polymerase recruitment Bacterial single-stranded DNA-binding proteins (SSBs) act as a conserved binding platform for DNA replication and repair proteins. In E. coli, the TLS polymerase Pol IV is selectively enriched at stalled replication forks through its interaction with SSB. We will determine whether SSB plays a similar role in TLS polymerase regulation in B. subtilis by combining biochemical binding assays with live-cell single-molecule imaging.

细胞必须高效、准确地复制其遗传物质，但这一过程面临着以下挑战： 模板链上存在未修复的 DNA 损伤。 DNA损伤耐受途径中的跨损伤 合成（TLS），专门的跨损伤聚合酶复制受损的DNA，促进细胞在 压力。大多数 TLS 聚合酶的保真度低于复制 DNA 聚合酶，因此它们的活性必须 在正常生长条件下受到严格调控，以维持基因组稳定性。相反，压力引起的 TLS 聚合酶的诱变可以在某些条件下促进细胞存活，例如通过促进 细菌中抗生素耐药性的发展。革兰氏阴性细菌大肠杆菌已成为模型 用于 TLS 聚合酶调节机制研究的物种，但尚不清楚是否具有相同的原理 适用于其他细菌物种，包括模型革兰氏阳性细菌枯草芽孢杆菌，它有两个 Y 家族 TLS 聚合酶，Pol Y1 和 Pol Y2。通过结合生化和微生物实验 活细胞单分子成像，我们将提供空间组织的全面图片， 枯草芽孢杆菌中 TLS 聚合酶 Pol Y1 和 Pol Y2 的动力学和分子协调。这项研究 将揭示在正常生长过程中如何维持 DNA 复制保真度的新见解，并将拓宽我们的研究范围 了解大肠杆菌以外的细菌物种的 TLS 和 DNA 损伤耐受性。 目标 1：确定 TLS 聚合酶如何响应枯草芽孢杆菌中的复制扰动 在大肠杆菌中，TLS 聚合酶在正常细胞生长过程中被排除在复制叉之外，但有选择地 响应复制扰动而丰富。然而，尚不清楚枯草芽孢杆菌 Pol Y1 和 Pol Y2 以类似的方式进行调节。我们将使用活细胞单分子成像来可视化荧光- 在正常复制期间和 DNA 损伤时标记 Pol Y1 和 Pol Y2 分子，使我们能够确定 它们是否以及如何响应复制扰动。 目标 2：阐明 DnaN 钳在协调 Pol Y1 和 Pol Y2 活性中的作用 细菌复制持续因子（DnaN 滑动夹）与多种蛋白质相互作用 通过共同的结合位点参与 DNA 复制和修复。 Pol Y1 和 Pol Y2 包含钳位结合 基序 (CBM)，与 DnaN 结合的短肽序列。我们将生物化学和微生物学结合起来 活细胞单分子成像分析阐明了 DnaN 在枯草芽孢杆菌中调节 TLS 的作用。 目标 3：确定与 SSB 的相互作用是否以及如何在 TLS 聚合酶招募中发挥作用 细菌单链 DNA 结合蛋白 (SSB) 充当 DNA 复制的保守结合平台 和修复蛋白质。在大肠杆菌中，TLS 聚合酶 Pol IV 在停滞的复制叉处选择性富集 通过与 SSB 的相互作用。我们将确定 SSB 是否在 TLS 聚合酶中发挥类似的作用 通过将生化结合测定与活细胞单分子成像相结合来调节枯草芽孢杆菌。