Regulation of DNA replication and repair

描述(由申请人提供)：这项研究计划的长期目标是发展一种综合的机制观点，了解生物体如何将其复制机制的行动与参与DNA修复和损伤耐受的其他细胞因子的行动相协调。如果做不到这一点，就会导致基因保真度的丧失，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地证明，DNA聚合酶(POL)过程性钳制(？或DNAN滑动夹具)在这一高度复杂的过程中扮演着多种重要角色。拟议的研究计划利用了遗传-生化-物理生化的综合方法，特别强调如何确定？Clip协调大肠杆菌复制酶DNA聚合酶III全酶(PolIII HE)与polB编码的PolII和编码dinB的PolIV的作用，这些基因参与复制和跨损伤DNA合成(TLS)，以及HDA蛋白，HDA蛋白通过灭活Dna A启动子蛋白来调节DNA复制的启动。在下一个进展阶段，我们将利用体外实验来表征POL III HE、POL II和POL IV与各种突变体？的相互作用。钳制蛋白。作为这项工作的一部分，我们将提纯含有一个亚基单一突变或每个亚基不同突变的异源二聚体钳制蛋白。利用这些突变的钳制，我们将剖析(S)通过什么机制？钳位调节POL切换，以协调与TLS的高保真复制。我们还将利用遗传学方法来确定体内POL转换的机制(S)，并确定其他细胞因素是否参与了这一至关重要的过程。我们预计，我们的结果支持的POL转换模型(S)将为包括人类在内的其他生物中类似的转换机制提供一个有价值的范例。此外，由于TLS POLS在生命的所有三个分支中都被很好地保存，我们的研究结果也将有助于我们理解应激条件下潜在的突变机制，从而影响发病机制和抗生素耐药性，以及TLS POLS在体细胞超突变过程中促进免疫球蛋白多样性的机制(S)。我们还将应用我们正在开发的方法来表征POL切换到HDA蛋白的特征，以便定义大肠杆菌协调复制与依赖HDA的复制启动调节的机制。未能适当地规范启动可能是致命的。我们将区分HDA功能的不同型号，并确定HDA和POLIII HE是否同时绑定到相同的型号？夹子。我们还将利用遗传和生化方法来确定HDA是否采取行动来控制TLS POL对复制叉子的访问，直到需要它们的时候。由于复制错误对突变有重要作用，而且DNA复制的起始和延长的协调调节对于基因组的稳定性至关重要，我们在这些领域的发现也可能为开发新的抗生素确定新的靶点。公共卫生相关性：未能协调不同复制和修复因子的行动会导致遗传保真度的丧失，并导致人类疾病。由于复制和修复机制从细菌到人类都非常保守，我们将利用大肠杆菌作为模型系统来了解不同复制和修复因子的作用是如何相互协调调节的。我们预计，我们的结果将成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，将有助于我们理解癌症和其他人类疾病的致病机制。

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.