Polymerase Dynamics at the Replication Fork

智力上的功绩。DNA复制是所有类型细胞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的复制提供了一个有用的原型。DNA复制在称为复制分叉的分支点的反平行双链DNA上双向进行。正因为如此，一条链被合成为短片段(冈崎片段)，在分叉处的一条链(滞后链)上。由于合成DNA的复制酶是高度进行性的，这意味着它可以在不释放的情况下进行很长的距离，因此必须有一种机制来触发滞后的链复制酶释放并循环到下一个Okazaki片段的RNA引物。这项研究试图区分冈崎碎片自行车的两种模式。最受欢迎的假设是，合成一种新的RNA引物提供了一个循环信号，该信号被DNAX复合体感受到，该复合体既将环状加工因子加载到DNA上，又将复制酶的聚合酶成分伴侣加载到DNA上。DNAX复合体还被认为陪伴旧的Okazaki片段上的聚合酶，并将其护送到新的引物上，同时伴随着加载新的加工性因子和ATP的水解。负责这一陪伴反应的大分子相互作用将被确定。这项研究的结果将有助于我们理解DNA复制的动态以及这一重要过程在维持基因组稳定中的作用，不仅在细菌中，而且在高等生物体中也是如此。该项目将包括研究生和本科生参与研究。将在本科生生物化学课程中实施实验室模块，其中包括实际使用机器人设备筛选复制突变体，并将公布该模块的细节，以便其他大学能够在其课程中使用该模块。这样的培训将提供宝贵的机会，接触制药和生物技术行业中常用的设备和程序，但很少在本科课堂上提供，因此可能会增加学生毕业后就业的竞争力。该协会将通过与美国生物化学和分子生物学协会合作出版的一篇可更新的在线百科全书文章的持续投稿，向科学和公众传播关于细菌DNA复制的基本知识。

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.