Regulation of DNA replication and repair

这项研究计划的长期目标是发展一个综合的机械观点， 生物体协调其复制机制与其他细胞因子的作用， DNA修复和损伤耐受如果不这样做，就会导致遗传保真度的丧失，并导致人类 疾病此外，Y-家族DNA聚合酶功能的不适当调节被认为是导致 导致癌症的突变我们实验室和其他机构的工作已经明确表明， DNA聚合酶持续合成钳（或DnaN滑动钳）在这个复合体中起着至关重要的作用 过程拟议的研究计划利用综合遗传<$生化<$物理生化 方法，特别强调如何确定钳帮助协调的行动， E.大肠杆菌复制酶，DNA聚合酶III全酶（Pol III），具有dinB编码的Pol IV，其作用于 跨损伤DNA合成（TLS），并与Hda蛋白，这有助于调节DNA复制的启动 通过使DnaA起始蛋白失活。我们将利用体外测定来表征Pol III和 Pol IV与各种突变的钳蛋白。作为这项工作的一部分，我们将纯化异二聚体夹蛋白 在一个亚基中携带单一突变，或在每个亚基中携带不同突变。利用这些突变体 钳，我们将剖析的机制，其中钳介导的转换之间的Pol III和Pol IV， 使用TLS协调高保真复制。我们将用基因分析来补充这些研究。我们 预计我们的研究结果支持的聚合酶转换模型将作为一个有价值的 在包括人类在内的其他生物体中也存在类似的开关机制。此外，由于Y系列Pos 在生命的所有三个分支中都非常好地保存着，我们的研究结果也将有助于 这对我们理解应激条件下的诱变机制有重要意义， 影响发病机制和抗生素耐药性，以及有助于免疫球蛋白 在体细胞超突变过程中通过易错复制而产生的多样性。我们还将采用我们 已经开发出表征聚合酶切换到Hda蛋白，以了解的作用， 在协调复制与Hda依赖的复制起始调节中的钳位。未能正确 调控起始导致过度复制、基因组不稳定，并且可能是致命的。我们将区分 Hda功能的不同模型，并将确定Hda和Pol III是否同时结合相同的 夹子。我们还将确定Hda是否限制Y系列Pol访问复制分叉， 这是TLS所需的时间。最后，由于复制错误对诱变有重要作用， 而且由于DNA复制起始和延伸的协调调节对于 基因组的稳定性，我们在这些领域的发现也可能确定新的目标类别的发展， 新型抗生素

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. Furthermore, inappropriate regulation of Y-family DNA polymerase function is proposed to contribute to mutations that lead to cancer. Work from our laboratory and others have demonstrated unambiguously that DNA polymerase processivity clamps (¿ or DnaN sliding clamps) play critically important roles in this complex process. The proposed research program utilizes an integrated genetic¿biochemical¿physical biochemical approach, placing particular emphasis on determining how the ¿ clamp helps to coordinate the actions of the E. coli replicase, DNA polymerase III holoenzyme (Pol III), with the dinB-encoded Pol IV, which acts in translesion DNA synthesis (TLS), and with the Hda protein, which helps to regulate initiation of DNA replication by inactivating the DnaA initiator protein. We will utilize in vitro assays to characterize interactions of Pol III 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 by which the ¿ clamp mediates a switch between Pol III and Pol IV to coordinate high fidelity replication with TLS. We will complement these studies with genetic analyses. We anticipate that the model(s) for polymerase switching supported by our results will serve as a valuable paradigm for similar switch mechanisms in other organisms, including humans. In addition, since Y-family Pols are remarkably well conserved throughout all three branches of life, results from our studies will also contribute significantly to our understanding of mechanisms underlying mutagenesis under times of stress, thereby impacting on pathogenesis and antibiotic resistance, as well as mechanisms contributing to immunoglobulin diversity by error-prone replication during somatic hypermutation. We will also apply the approaches that we have developed to characterize polymerase switching to Hda protein in order to understand the role of the ¿ clamp in coordinating replication with Hda-dependent regulation of initiation of replication. Failure to properly regulate initiation leads to over-replication, genome instability, and can be lethal. We will distinguish between different models for Hda function, and will determine whether Hda and Pol III simultaneously bind to the same ¿ clamp. We will also determine whether Hda acts to limit access of Y-family Pols to the replication fork until such time as they are required for TLS. Finally, 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.