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

细胞必须高效而准确地复制它们的遗传物质，但这一过程受到 模板链上存在未修复的DNA损伤。在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型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，以保持基因组的稳定。相反，压力引起的 TLS聚合酶的突变在某些条件下可以促进细胞存活，例如通过促进 细菌中抗生素耐药性的发展。革兰氏阴性杆菌E.Coli就是一个榜样。 物种对TLS聚合酶的调节机制进行了研究，但尚不清楚是否存在相同的原理 适用于其他细菌物种，包括模式革兰氏阳性细菌枯草杆菌，它有两个Y家族 TLS聚合酶，Pol Y1和Pol Y2。通过将生化和微生物学实验与 活细胞单分子成像，我们将提供空间组织的全面图像， 枯草杆菌中TLS聚合酶Pol Y1和Pol Y2的动力学和分子配位。本研究 将揭示在正常生长过程中如何保持DNA复制保真度的新见解，并将拓宽我们的 了解除大肠杆菌外细菌的TLS和DNA损伤耐受性。 目的1：确定枯草杆菌中TLS聚合酶对复制扰动的反应 在大肠杆菌中，TLS聚合酶在正常细胞生长过程中被从复制叉中排除，但是有选择性的 丰富了对复制扰动的响应。然而，目前尚不清楚枯草杆菌Pol Y1和Pol Y2的监管方式与此类似。我们将使用活细胞单分子成像来显示荧光- 在正常复制和DNA损伤时标记的Pol Y1和Pol Y2分子，使我们能够确定 他们是否以及如何应对复制扰动。 目的2：阐明DNAN钳在协调Pol Y1和Pol Y2活性中的作用 细菌的复制过程因子，DNAN滑动夹，与广泛的蛋白质相互作用 通过一个共同的结合位点参与DNA复制和修复。Pol Y1和Pol Y2包含夹紧装订 基序(CBM)，与DNAN结合的短肽序列。我们将把生化和微生物学结合起来 用活细胞单分子成像分析来阐明DNAN在调节枯草杆菌TLS中的作用。 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)是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地浓缩 通过它与SSB的互动。我们将确定SSB是否在TLS聚合酶中扮演类似的角色 生化结合分析与活细胞单分子成像相结合对枯草杆菌的调控。

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.