Mechanistic Studies of Replication Initiation in Prokaryotes

项目摘要 我们研究的一个长期目标是确定细胞启动的分子机制， DNA复制。起始代表细胞增殖的中心承诺; 复制可导致遗传不稳定性、DNA损伤和基因拷贝数的变化。从 从生物医学的角度来看，启动是一个关键的途径，应该容易受到治疗干预 用于控制细菌感染和癌症;然而， 他们的活动不足以推动这种努力。 本更新申请集中于细菌中DNA复制的起始。尽管基本 这一过程的生物化学框架已经到位，机械和调节原则， 复制复合物的组装仍然是高度神秘的。在过去的项目期间，我们回答了长- 关于细菌复制解旋酶如何物理加载到DNA上以及ATP如何 周转变构控制这一过程。我们发现了一种新的构象转换机制， 控制引物酶结合的复制解旋酶，引物酶合成短RNA以启动DNA 合成.我们对六聚体解旋酶如何利用ATP驱动核酸 移位以及小分子和伴侣蛋白如何控制这些酶。 我们过去的进展为我们解决涉及启蒙的令人兴奋的新问题铺平了道路。使用结构， 生物化学和单分子方法，我们将解决有关结构的基本问题， 复制体形成早期的组织，起始因子交换的分子手段 伴侣蛋白在适当的时间顺序，和复制解旋酶的能力，导航双链体 核酸路障拟议研究的结果将是主要步骤的机械图 参与将双链体染色体区域转化为延伸复制叉。这些发现将 为DNA复制领域和依赖ATP的机器的更广泛作用定义新的原则， 它还将建立新的见解和方法，以推进药物发现工作， 细菌起始系统

PROJECT SUMMARY A long-term goal of our research is to define the molecular mechanisms underpinning the initiation of cellular DNA replication. Initiation represents a central commitment to cell proliferation; inappropriate onset of replication can lead to genetic instabilities, DNA damage, and changes in gene copy number. From a biomedical perspective, initiation is a keystone pathway that should be susceptible to therapeutic intervention for controlling bacterial infections and cancers; however, a molecular-level understanding of initiation factors and their activities is insufficiently complete to advance such efforts. The present renewal application focuses on the initiation of DNA replication in bacteria. Although a basic biochemical framework for this process is in place, the mechanistic and regulatory principles by which replication complexes are assembled remain highly enigmatic. In the past project period, we answered long- standing questions about how the bacterial replicative helicase is physically loaded onto DNA and how ATP turnover allosterically controls this process. We uncovered a new conformational switching mechanism in the replicative helicase that controls the binding of primase, which synthesizes short RNAs to jump-start DNA synthesis. We developed new insights into how hexameric helicases use ATP to drive nucleic acid translocation and how small molecules and partner proteins can control these enzymes. Our past progress paves the way for us to tackle exciting new problems involving initiation. Using structural, biochemical, and single-molecule approaches, we will address fundamental questions regarding the structural organization of early-stage replisome formation, the molecular means by which initiation factors exchange partner proteins in an appropriate temporal order, and the ability of replicative helicases to navigate duplex nucleic-acid roadblocks. The outcome of the proposed studies will be a mechanistic picture of the major steps involved in converting a duplex chromosomal region into an elongating replication fork. These findings will define new principles for the field of DNA replication and the broader action of ATP-dependent machines and switches; it will also establish new insights and approaches for advancing drug-discovery efforts that target bacterial initiation systems.