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

细胞必须有效和准确地复制它们的遗传物质，然而这一过程受到了生物学家的挑战。 模板链上存在未修复的DNA损伤。在DNA损伤耐受途径中， 合成（TLS），专门的translesion聚合酶复制受损的DNA，促进细胞存活下 应力大多数TLS聚合酶比复制型DNA聚合酶的保真度低，因此它们的活性必须是稳定的。 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 通过TLS聚合酶的诱变可以在某些条件下促进细胞存活，例如通过促进细胞的增殖。 细菌对抗生素耐药性的发展。革兰氏阴性菌E.大肠杆菌作为一个模型， 种进行TLS聚合酶调节的机制研究，但不知道是否相同的原理 适用于其他细菌物种，包括模式革兰氏阳性菌B。枯草芽孢杆菌，其具有两个Y-家族 TLS聚合酶，Pol Yl和Pol Y2。通过结合生物化学和微生物实验， 活细胞单分子成像，我们将提供一个全面的图片的空间组织， 动力学以及B中TLS聚合酶Pol Y1和Pol Y2的分子配位。枯草杆菌。本研究 将揭示在正常生长过程中DNA复制保真度是如何维持的新见解，并将拓宽我们的研究领域。 了解TLS和DNA损伤耐受性在细菌物种超越E。杆菌 目的1：确定TLS聚合酶如何响应B中的复制扰动。枯草 在大肠在正常细胞生长期间，TLS聚合酶被排除在复制叉之外，但选择性地 对复制扰动的反应而富集。然而，不知道B。枯草杆菌Pol Y1和Pol Y2也以类似的方式进行调节。我们将使用活细胞单分子成像来显示荧光- 标记的Pol Y1和Pol Y2分子在正常复制过程中和DNA损伤后，使我们能够确定 它们是否以及如何对复制扰动作出反应。 目的2：阐明DnaN夹在协调Pol Y1和Pol Y2活性中的作用 细菌复制持续性因子，DnaN滑动钳，与广泛的蛋白质相互作用 通过共同的结合位点参与DNA复制和修复。Pol Yl和Pol Y2含有夹式结合 基序（CBM），与DnaN结合的短肽序列。我们将结合联合收割机生物化学和微生物 用活细胞单分子成像分析来阐明DnaN在调节B中TLS中的作用。枯草杆菌。 目的3：确定与SSB的相互作用是否以及如何在TLS聚合酶募集中发挥作用 细菌单链DNA结合蛋白（SSBs）是DNA复制的保守结合平台 和修复蛋白质。在大肠在大肠杆菌中，TLS聚合酶Pol IV在停滞的复制叉处选择性富集 通过与SSB的互动。我们将确定SSB是否在TLS聚合酶中发挥类似的作用 B中的调节。枯草芽孢杆菌通过结合生物化学结合试验与活细胞单分子成像。

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.