Regulation of DNA replication and repair

描述（由申请人提供）：本研究计划的长期目标是开发一个综合的机制观，生物体如何协调其复制机制与其他参与DNA修复和损伤耐受的细胞因子的行动。不这样做会导致遗传保真度的丧失，并导致人类疾病。我们实验室和其他实验室的工作已经明确表明，DNA聚合酶（Pol）的持续合成能力（？）或DnaN滑动夹）在这个高度复杂的过程中起着多种重要作用。拟议的研究计划利用综合遗传生化物理生化方法，特别强调如何确定？夹钳协调E.大肠杆菌复制酶，DNA聚合酶III全酶（Pol III HE），具有polB编码的Pol II和dinB编码的Pol IV，其在复制和跨损伤DNA合成（TLS）中起作用，以及具有Hda蛋白，其通过使DnaA起始蛋白失活来调节DNA复制的起始。在下一个进展期间，我们将利用体外试验，以表征相互作用的Pol III HE，Pol II，和Pol IV与各种突变？钳蛋白。作为这项工作的一部分，我们将纯化异二聚体夹蛋白，其在一个亚基中具有单一突变，或在每个亚基中具有不同突变。使用这些突变的钳，我们将解剖的机制（S）？钳介导Pol转换以协调高保真复制与TLS。我们还将利用遗传学方法来定义体内Pol转换的机制，并确定是否有其他细胞因子参与这一至关重要的过程。我们预计，由我们的研究结果支持的Pol开关模型将作为其他生物体（包括人类）中类似开关机制的有价值的范例。此外，由于TLS Pos在生命的所有三个分支中都很好地保守，因此我们的研究结果也将有助于我们理解应激时诱变的潜在机制，从而影响发病机制和抗生素耐药性，以及TLS Pos在体细胞超突变期间有助于免疫球蛋白多样性的机制。我们还将应用我们正在开发的方法来表征Pol转换为Hda蛋白，以确定E.大肠杆菌协调复制与Hda依赖的复制起始调节。如果不能适当地控制启动，可能是致命的。我们将区分Hda功能的不同模型，并将确定Hda和Pol III HE是否同时结合相同的？止血钳我们还将利用遗传和生物化学方法来确定Hda是否可以调节TLS Pol对复制叉的访问，直到需要它们的时候。由于复制错误对诱变有显著贡献，并且由于DNA复制的起始和延伸的协调调节对于基因组稳定性至关重要，因此我们在这些领域的发现也可能为开发新型抗生素确定新的靶点类别。公共卫生相关性：如果不能协调不同复制和修复因子的作用，就会导致遗传保真度的丧失，并导致人类疾病。由于复制和修复的机制是非常保守的从细菌到人类，我们将利用大肠杆菌作为模型系统，以了解不同的复制和修复因子的行为是如何相互协调调节。我们预计，我们的研究结果将作为理解人类类似控制网络的框架，这些事件的复杂性要大得多，因此，将有助于我们理解导致癌症和其他人类疾病的机制。

DESCRIPTION (provided by applicant): The long-term goal of this research program is to develop an integrated mechanistic view of how organisms coordinate the actions of their replication machinery with those of other cellular factors involved in DNA repair and damage tolerance. Failure to do so leads to a loss of genetic fidelity and contributes to human disease. Work from our laboratory and others have demonstrated unambiguously that DNA polymerase (Pol) processivity clamps (? or DnaN sliding clamps) play multiple essential roles in this highly complex process. The proposed research program utilizes an integrated genetic-biochemical-physical biochemical approach, placing particular emphasis on determining how the ? clamp coordinates the actions of the E. coli replicase, DNA polymerase III holoenzyme (Pol III HE), with the polB-encoded Pol II and the dinB-encoded Pol IV, which act in replication and translesion DNA synthesis (TLS), as well as with the Hda protein, which regulates initiation of DNA replication by inactivating the DnaA initiator protein. Over the next progress period, we will utilize in vitro assays to characterize interactions of Pol III HE, Pol II, and Pol IV with various mutant ? clamp proteins. As part of this work, we will purify heterodimeric clamp proteins bearing either a single mutation in one subunit, or different mutations in each subunit. Using these mutant clamps, we will dissect the mechanism(s) by which the ? clamp mediates Pol switching to coordinate high fidelity replication with TLS. We will also utilize genetic approaches to define the mechanism(s) of Pol switching in vivo, and to determine whether additional cellular factors contribute to this critically important process. We anticipate that model(s) for Pol switching supported by our results will serve as a valuable paradigm for similar switch mechanisms in other organisms, including humans. Moreover, since TLS Pols are well conserved throughout all three branches of life, results from our studies will also contribute to our understanding of the mechanisms underlying mutagenesis under times of stress, thereby impacting on pathogenesis and antibiotic resistance, as well as the mechanism(s) by which TLS Pols contribute to immunoglobulin diversity during somatic hypermutation. We will also apply the approaches that we are developing to characterize Pol switching to the Hda protein in order to define the mechanism by which E. coli coordinates replication with Hda-dependent regulation of initiation of replication. Failure to properly regulate initiation can be lethal. We will distinguish between different models for Hda function, and will determine whether Hda and Pol III HE simultaneously bind to the same ? clamp. We will also utilize genetic and biochemical approaches to determine whether Hda acts to regulate access of TLS Pols to the replication fork until such time as they are required. Since replication errors contribute significantly to mutagenesis, and since the coordinate regulation of initiation and elongation of DNA replication is critically important for genome stability, our findings in these areas may also identify new classes of targets for the development of novel antibiotics. PUBLIC HEALTH RELEVANCE: Failure to coordinate the actions of the different replication and repair factors leads to a loss of genetic fidelity and contributes to human disease. Since mechanisms of replication and repair are remarkably well conserved from bacteria to humans, we will utilize Escherichia coli as a model system to understand how the actions of different replication and repair factors are coordinately regulated with each other. We anticipate that our results will serve as a framework for understanding similar control networks in humans, were the complexity of the events is far greater, and as such, will contribute to our understanding of mechanisms contributing to cancer and other human diseases.