Mechanistic Studies of Replication Initiation in Prokaryotes

原核生物复制起始的机制研究

• 批准号: 10606469
• 负责人: JAMES M BERGER
• 金额: $ 40.81万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-05-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10606469
• 关键词: Acceleration; Anti-Bacterial Agents; Bacteria; Bacterial Infections; Binding; Biochemical; Biological Assay; Cell Proliferation; Cell Survival; Complex; DNA; DNA Damage; DNA Primase; DNA biosynthesis; DNA replication fork; Enzymes; Gene Dosage; Genetic; Goals; Guanine Nucleotide Exchange Factors; Instruction; Malignant Neoplasms; Molecular; Molecular Conformation; Nucleic Acids; Outcome; Pathway interactions; Peptide Initiation Factors; Predisposition; Process; Prokaryotic Cells; Proteins; RNA; Replication Initiation; Research; System; Therapeutic Intervention; Work; drug discovery; helicase; insight; molecular modeling; single molecule; small molecule

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. I I RELEVANCE (See instructions): The initiation of DNA replication is central to cell proliferation and survival. Initiation pathways should be susceptible to therapeutic intervention against bacterial infections and cancers; however, limitations in our understanding of basic initiation mechanisms impede this goal. The proposed work will provide key molecular models for understanding initiation proteins and activities and will establish new assays and approaches for accelerating antibacterial discovery through efforts that target these factors.

我们研究的一个长期目标是确定启动的分子机制， 细胞DNA复制启动代表细胞增殖的中心承诺;不适当的 复制的开始可导致遗传不稳定性、DNA损伤和基因拷贝数的变化。 从生物医学的角度来看，启动是一个关键的途径，应该是敏感的治疗 控制细菌感染和癌症的干预;然而， 启动因素及其活动不够完整，无法推动这些努力。 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 关于细菌复制解旋酶如何物理加载到DNA上的长期问题， ATP周转如何变构控制这一过程。我们发现了一种新的构象转换 复制解旋酶中控制引物酶结合的机制，引物酶合成短RNA 启动DNA合成我们对六聚体解旋酶如何利用ATP驱动 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，我们将解决基本问题 关于早期复制体形成的结构组织， 起始因子以适当的时间顺序交换伴侣蛋白，并且复制因子的复制能力与蛋白质的表达有关。 解旋酶来导航双链核酸路障。拟议研究的结果将是 将双链体染色体区域转化为双链体染色体区域的主要步骤的机械图 伸长的复制叉。这些发现将为DNA复制领域定义新的原则， 依赖ATP的机器和开关的更广泛的行动;它还将建立新的见解， 用于推进靶向细菌起始系统的药物发现工作的方法。 我我 相关性（参见说明）： DNA复制的起始对细胞增殖和存活至关重要。启动途径应该是 对细菌感染和癌症的治疗干预敏感;然而，我们的局限性 对基本启动机制的理解阻碍了这一目标的实现。这项工作将提供关键 了解起始蛋白和活性的分子模型，并将建立新的测定方法， 通过针对这些因素的努力加速抗菌发现的方法。