Regulation of translesion synthesis by the bacterial replisome

项目摘要 DNA损伤阻碍了复制体的进程。如果这些损伤得不到解决，就会导致细胞死亡。一个 突出的解决机制是跨损伤合成(TLS)，这是一种DNA损伤耐受途径，在其中 容易出错的TLS聚合酶与高保真的复制聚合酶交换以合成通过病变， 使复制体能够越过损伤。虽然TLS聚合酶有助于基因组的稳定， 它们的错误调控被认为与癌症进展和抗菌药的开发有关。 抵抗。这项建议将采用生化和遗传学方法，结合体外培养和细胞培养 基于单分子成像，以了解TLS是如何在细菌复制过程中受到调控的。 目的1：阐明SSB在调节Pol IV介导的TLS中的作用 我们已经证明了TLS聚合酶POLIV被招募到DNA损伤依赖的复制体内 通过与SSB互动的方式。由于SSB结构性地存在于复制体中，这提高了 POLIV如何有选择地瞄准停滞的叉子的问题。为此，我们将致力于阐明这一点。 通过确定SSB的动力学和导致Pol IV招募的分子事件，确定了SSB的机制。 最后，通过使用其他SSB相互作用蛋白(SIPs)的单分子成像，我们将测试是否损伤- 依赖复制体招募是一种一般机制，或者它是否是Pol IV所独有的。 目的2：与SSB的相互作用对于招募TLS聚合酶到它们的作用部位是否重要？ 根据我们的观察，POL IV与SSB的相互作用对其进行TLS的能力至关重要，我们 将决定POL II和SSB之间的相互作用是否对其功能也很重要。为了实现这一目标，我们将 开发选择性地消融POL II-SSB相互作用的突变体。有了这些变种人，我们接下来要问 是否需要POL II-SSB相互作用来将POL II招募到停滞的复制体和TLS。 此外，我们将确定Pol IV-SSB相互作用在突变DNA双链断裂中的作用 修复，一条与适应性突变和抗生素耐药性有关的途径。 目的3：POLIII的构象动力学如何调节TLS聚合酶的访问和合成？ 为了确定Pol IV如何与b2钳结合和解离，我们将使用单分子FRET来 在活跃复制过程中跟踪聚合酶-钳制复合体的构象动力学。此外， 我们将确定模板链上的DNA损伤如何影响这些构象动力学。 目标4：确定影响TLS在分叉点和启动之间竞争的因素 病变下游DNA合成的重排与TLS竞争以解决停滞不前的复制分叉。我们 提供了辅助解旋酶加速再启动动力学的初步细胞和体外数据。在这一目标中 我们将致力于确定能够发挥这种作用的解旋酶及其作用机制。

Project Summary DNA damage blocks the progress of the replisome. Left unresolved these lesions can result in cell death. A prominent resolution mechanism is translesion synthesis (TLS), a DNA damage tolerance pathway in which an error-prone TLS polymerase switches with a high fidelity replicative polymerase to synthesize past the lesion, enabling the replisome to progress past the damage. While TLS polymerases contribute to genome stability, their misregulation has been implicated in both cancer progression and the development of antimicrobial resistance. This proposal will employ biochemical and genetic approaches in combination with in vitro and cell based single-molecule imaging to understand how TLS is regulated during bacterial replication. Aim 1: Elucidate the role of SSB in regulating Pol IV-mediated TLS We have demonstrated that the TLS polymerase Pol IV is recruited to replisomes in a DNA damage dependent manner through interactions with SSB. As SSB is constitutively present in the replisome this raises the question of how Pol IV is selectively targeted to stalled forks. In this aim we will work to elucidate this mechanism by determining the dynamics of SSB and the molecular events that lead to Pol IV recruitment. Finally, by using single-molecule imaging of other SSB-interacting proteins (SIPs), we will test if damage- dependent replisome recruitment is a general mechanism or if it is unique to Pol IV. Aim 2: Is an interaction with SSB important for recruiting TLS polymerases to their site of action? Building on our observations that the interaction of Pol IV with SSB is critical for its ability to carry out TLS, we will determine if an interaction between Pol II and SSB is also important for its function. In this aim we will develop mutants that selectively ablate the Pol II-SSB interaction. With these mutants in hand we will next ask whether the Pol II-SSB interaction is required for the recruitment of Pol II to stalled replisomes and TLS. Furthermore, we will determine the role of the Pol IV-SSB interaction in mutagenic DNA double strand break repair, a pathway implicated in adaptive mutagenesis and antibiotic resistance. Aim 3: How do conformational dynamics of Pol III regulate TLS polymerase access and synthesis? In order to determine how Pol IV binds and dissociates from the b2 clamp, we will use single-molecule FRET to follow the conformational dynamics of the polymerase-clamp complex during active replication. Furthermore, we will determine how DNA lesions on the template strand influence these conformational dynamics. Aim 4: Identify factors that influence the competition between TLS at the fork and repriming Repriming of DNA synthesis downstream of a lesion competes with TLS to resolve stalled replication forks. We present preliminary cellular and in vitro data that accessory helicases accelerate repriming kinetics. In this aim we will work to identify the helicases that can exert this effect and their mechanism of action.