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.

细胞必须有效，准确地复制其遗传物质，但此过程受到挑战 在模板链上存在未修复的DNA损伤。在DNA损伤耐度途径中 合成（TLS），专门的跨质聚合酶复制受损的DNA，促进细胞存活下的生存 压力。大多数TLS聚合酶比复制DNA聚合酶低的保真度，因此它们的活性必须 在正常生长条件下受到严格调节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，例如通过有助于 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聚合酶调节的机械研究的物种，但尚不清楚是否有相同的原理 应用于其他细菌种类，包括革兰氏阳性细菌枯草芽孢杆菌，该细菌具有两个Y-家庭 TLS聚合酶，Pol Y1和Pol Y2。通过将生化和微生物学实验与 活电池单分子成像，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的动力学和分子配位。这项研究 将揭示有关在正常增长期间如何保持DNA复制保真度的新见解，并将扩大我们的 对大肠杆菌以外的细菌物种的TLS和DNA损伤耐受的了解。 目标1：确定TLS聚合酶如何响应枯草芽孢杆菌的复制扰动 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中，但有选择地 响应复制扰动而富含。但是，尚不清楚枯草芽孢杆菌pol y1和pol是否 Y2以类似的方式受到调节。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损伤后，标记为Pol Y1和Pol Y2分子，使我们能够确定 如果以及它们如何响应复制扰动。 AIM 2：阐明DNAN夹在协调pol y1和pol y2活性中的作用 细菌复制加工性因子DNAN滑动夹与广泛的蛋白质相互作用 通过常见的结合位点参与DNA复制和修复。 pol y1和pol y2包含夹具结合 基序（CBM），与DNAN结合的短肽序列。我们将结合生化和微生物学 具有活细胞单分子成像的测定，以阐明DNAN在调节枯草芽孢杆菌中的TLS中的作用。 目标3：确定与SSB的相互作用以及如何在TLS聚合酶募集中发挥作用 细菌单链DNA结合蛋白（SSB）充当DNA复制的保守结合平台 并修复蛋白质。在大肠杆菌中，TLS聚合酶pol IV选择性地富集在停滞的复制叉处 通过与SSB的互动。我们将确定SSB是否在TLS聚合酶中起相似的作用 通过将生化结合测定与活细胞单分子成像相结合，在枯草芽孢杆菌中调节。