Polymerase Dynamics at the Replication Fork

复制叉的聚合酶动力学

• 批准号: 1329285
• 负责人: Charles McHenry
• 金额: $ 51万
• 依托单位: University of Colorado at Boulder
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1329285&HistoricalAwards=false
• 关键词: Polymerase; Dynamics; Replication; Fork

Intellectual Merit. DNA replication is a central process important for proliferation of all cell types. Because of highly conserved features, the bacterium Escherichia coli provides a useful prototype to understanding replication of all organisms. DNA replication proceeds bidirectionally on anti-parallel duplex DNA at a branch point called the replication fork. Because of this, one strand is synthesized as short fragments (Okazaki fragments) on one strand (the lagging strand) at the fork. Since the replicase that synthesizes DNA is highly processive, meaning that it can proceed long distances without releasing, there must be a mechanism for triggering the lagging strand replicase to release and recycle to the RNA primer for the next Okazaki fragment. This research seeks to distinguish between two models for Okazaki fragment cycling. The favored hypothesis is that synthesis of a new RNA primer provides a cycling signal sensed by the DnaX complex that loads both a ring-like processivity factor on DNA and chaperones the polymerase component of the replicase onto it. The DnaX complex is also thought to chaperone the polymerase off of the old Okazaki fragment and escort it onto the new primer, concomitant with loading a new processivity factor and hydrolysis of ATP. Macromolecular interactions responsible for this chaperoning reaction will be determined. The results of the research will make a significant contribution to our understanding of the dynamics of DNA replication and the role of this important process in the maintenance of genome stability, not just in bacteria, but also in higher organisms.Broader Impacts. The project will involve graduate and undergraduate students in the research. A laboratory module, incorporating hands-on use of robotics equipment for screening for replication mutants, will be implemented for an undergraduate biochemistry course, and details of the module will be published, so that other universities will be able to use it for their curricula. Such training will provide valuable exposure to equipment and procedures commonly used in pharmaceutical and biotechnology industries, but rarely made available in undergraduate classes, and thus may increase the competitiveness of the students to obtain employment upon graduation. The PI will disseminate fundamental knowledge on bacterial DNA replication to the scientific and lay public through continued contributions to an updatable online encyclopedia article published in collaboration with the American Society for Biochemistry and Molecular Biology.

智力优势。 DNA复制是所有细胞类型增殖的重要中心过程。 由于高度保守的特征，大肠杆菌为理解所有生物体的复制提供了有用的原型。 DNA复制在称为复制叉的分支点在反平行双链DNA上双向进行。 因此，一条链在分叉处的一条链（滞后链）上合成为短片段（冈崎片段）。 由于合成DNA的复制酶是高度进行性的，这意味着它可以长距离进行而不会释放，因此必须有一种机制来触发滞后链复制酶释放并再循环到下一个冈崎片段的RNA引物。 本研究旨在区分冈崎片段循环的两种模式。 一个有利的假设是，一个新的RNA引物的合成提供了一个由DnaX复合物感知的循环信号，该复合物在DNA上加载了一个环状的持续合成因子，并将复制酶的聚合酶成分陪伴在其上。DnaX复合物也被认为是伴随着加载一个新的持续合成因子和ATP的水解，将聚合酶从旧的冈崎片段中分离出来，并将其护送到新的引物上。 将确定负责该陪伴反应的大分子相互作用。 这项研究的结果将为我们理解DNA复制的动力学以及这一重要过程在维持基因组稳定性方面的作用做出重大贡献，不仅在细菌中，而且在高等生物中。 该项目将涉及研究生和本科生的研究。 将在本科生生物化学课程中实施一个实验室单元，其中包括动手使用机器人设备筛选复制突变体，该单元的细节将公布，以便其他大学能够在其课程中使用。这种培训将提供宝贵的机会，让学生接触制药和生物技术行业常用但在本科生班很少提供的设备和程序，从而提高学生毕业后就业的竞争力。 PI将通过与美国生物化学和分子生物学学会合作出版的可更新在线百科全书文章，向科学界和公众传播细菌DNA复制的基础知识。