Regulation of DNA replication and repair

描述（由申请人提供）：本研究项目的长期目标是建立一个综合的机制观点，以了解生物体如何协调其复制机制与其他细胞因子在DNA修复和损伤耐受中的作用。如果不这样做，就会导致遗传保真度的丧失，从而导致人类疾病。我们的实验室和其他人的工作已经明确地证明了DNA聚合酶（Pol）的加工能力夹住(？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的遗传-生物化学-物理生化方法，特别强调确定如何？clamp协调大肠杆菌复制酶，DNA聚合酶III全酶（Pol III HE）与Pol -编码的Pol II和dinb -编码的Pol IV的作用，Pol II和Pol IV在复制和翻译DNA合成（TLS）中起作用，以及与Hda蛋白的作用，Hda蛋白通过使DNA启动蛋白失活来调节DNA复制的起始。在下一个进展阶段，我们将利用体外试验来表征Pol III HE， Pol II和Pol IV与各种突变体的相互作用？夹的蛋白质。作为这项工作的一部分，我们将纯化异二聚体夹蛋白，在一个亚基中携带单个突变，或者在每个亚基中携带不同的突变。使用这些突变钳，我们将剖析？箝位调解Pol切换协调高保真复制与TLS。我们还将利用遗传方法来定义体内Pol转换的机制，并确定是否有其他细胞因素有助于这一至关重要的过程。我们预计，我们的结果支持的Pol开关模型将作为其他生物（包括人类）类似开关机制的有价值的范例。此外，由于TLS pol在所有三个生命分支中都很好地保守，我们的研究结果也将有助于我们理解应激下突变的潜在机制，从而影响发病机制和抗生素耐药性，以及TLS pol在体细胞超突变期间促进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向Hda蛋白的转换，以确定大肠杆菌与Hda依赖性的复制起始调节协调复制的机制。不能适当地调节起始可能是致命的。我们将区分Hda功能的不同模型，并确定Hda和Pol III HE是否同时结合在同一个模型上？夹。我们还将利用遗传和生化方法来确定Hda是否会调节TLS pol对复制分叉的访问，直到需要时为止。由于复制错误对突变有重要贡献，并且由于DNA复制起始和延伸的协调调节对基因组稳定性至关重要，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：不能协调不同复制和修复因子的作用，会导致遗传保真度的丧失，从而导致人类疾病。由于从细菌到人类的复制和修复机制都非常保守，我们将利用大肠杆菌作为模型系统来了解不同的复制和修复因子是如何相互协调调节的。我们预计，我们的结果将作为理解人类类似控制网络的框架，因为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.