Coordination of high fidelity replication with mutagenic translesion synthesis

摘要 未能正确协调DNA复制与修复和潜在致突变性跨损伤DNA合成 (TLS)导致许多人类疾病状态的突变，包括癌症，以及 抗生素耐药性和适应临床上重要的微生物病原体。我们使用大肠杆菌作为 模型来定义生物体管理其高保真度行为的基本机制 复制型DNA聚合酶（Pol）与参与病变旁路的低保真度TLS Pol， 诱变一个Pol在复制分叉处替换另一个Pol的过程称为“Pol 交换滑动钳蛋白（细菌中的DnaN或β;真核生物和古细菌中的PCNA）在细胞增殖中起重要作用。 在这些开关中的作用。普遍接受的Pol切换的“工具带”模型假设两个不同的 Pol同时结合相同β钳中的单独疏水裂缝以顺序地接近DNA。在 与工具带模型形成鲜明对比的是，我们最近确定TLS Pol相互作用， Pol III复制酶，虽然这些相互作用的机制贡献目前尚不清楚，但我们 然而，证明了Pol-Pol相互作用是体外和体内Pol转换所必需的。 vivo.我们还鉴定了几种对Pol功能重要的新型Pol-β钳相互作用。在目标1中，我们 利用我们的Pol III-β clamp-Pol IV复合物的结构模型，以及丰富的生物化学， 生物物理，单分子和遗传方法，以确定第一次在分子细节的具体 这有助于离散Pol III-Pol IV和Pol IV-β钳相互作用的转换。在目标2中，我们将利用 我们已经获得了关于Pol II-β钳复合物的结构见解，以在分子细节上定义β-钳复合物如何在细胞内形成。 箝位管理Pol II持续性、校对以及Pol III-Pol II和Pol II-Pol IV切换。在目标3中，我们 使用小角X射线散射（SAXS），低温电子显微镜（cryo-EM），分子建模和 生物化学和生物物理方法的组合，以在结构上定义不同的Pol如何相互作用 以及DNA如何影响这些相互作用。此外，我们将确定 不同Pol-β夹和Pol-β夹-Pol复合物的蛋白质化学计量，使用尺寸排阻 色谱法与多角度光散射（SEC-MALS）偶联，其中Pol彼此相互作用， 以及这些互动是否具有竞争性。这些研究的结果将提供 对E.大肠杆菌管理和协调调节 其不同Pol在DNA复制、修复和TLS中的作用。最后，我们的研究结果可能会确定关键的 这些更高阶的调控网络中的步骤，概括到其他细菌，可以通过 控制复制和诱变的化学治疗剂。

ABSTRACT Failure to properly coordinate DNA replication with repair and potentially mutagenic translesion DNA synthesis (TLS) contributes to mutations that underlie numerous human disease states, including cancers, as well as antibiotic resistance and adaptation of clinically significant microbial pathogens. We use Escherichia coli as a model to define fundamental mechanisms by which organisms mange the actions of their high fidelity replicative DNA polymerase(s) (Pol) with those of low fidelity TLS Pols involved in lesion bypass and mutagenesis. The process by which one Pol replaces another at a replication fork is referred to as `Pol switching'. Sliding clamp proteins (DnaN or β in bacteria; PCNA in eukaryotes and archaea) play essential roles in these switches. The generally accepted `toolbelt' model for Pol switching postulates that two different Pols simultaneously bind separate hydrophobic clefts in the same β clamp to sequentially access the DNA. In stark contrast with the toolbelt model, we recently determined that TLS Pols interact with each other and with the Pol III replicase, and while the mechanistic contributions of these interactions are currently unknown, we nevertheless demonstrated that Pol-Pol interactions are absolutely required for Pol switching in vitro and in vivo. We have also identified several novel Pol-β clamp interactions important for Pol function. In Aim 1, we will exploit our structural model of the Pol III-β clamp-Pol IV complex, as well as a wealth of biochemical, biophysical, single molecule and genetic approaches to define for the first time in molecular detail the specific contributions to switching of discrete Pol III-Pol IV and Pol IV-β clamp interactions. In Aim 2 we will exploit structural insights we have gained regarding the Pol II-β clamp complex to define in molecular detail how the β clamp manages Pol II processivity, proofreading and Pol III-Pol II and Pol II-Pol IV switching. In Aim 3, we will use small angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM), molecular modeling and a combination of biochemical and biophysical approaches to structurally define how the different Pols interact with each other and the β clamp, and how DNA influences these interactions. In addition, we will determine the protein stoichiometry of the different Pol-β clamp and Pol-β clamp-Pol complexes using size exclusion chromatography coupled with multi angle light scattering (SEC-MALS), which Pols interact with each other, and whether or not these interactions are competitive. Taken together, results of these studies will provide unprecedented insight into the molecular mechanisms by which E. coli manages and coordinately regulates the actions of its different Pols during DNA replication, repair and TLS. Finally, our findings may identify critical steps in these higher order regulatory networks that generalize to other bacteria and can be targeted by chemotherapeutics to control replication and mutagenesis.