Decoupling of DNA unwinding and synthesis in the replisome induces genome instability

长期以来，教科书上关于DNA复制的观点是，复制体是一个稳定的多蛋白质复合体，在前导链和后导链上无缝协调耦合合成。然而，最近的证据表明一个更动态的复制体，其中单个蛋白质成分在复制过程中交换，创造了DNA解绕，引物和合成的随机过程。一个更动态的复制体将有助于应对DNA复制进程中的各种障碍或挑战。尽管如此，必须保持复制叉上的酶协调，否则一种活性可能占主导地位，产生过量和危险的复制中间体。该项目将确定复制体蛋白质之间的分子接触，这些蛋白质的作用是协调和控制DNA的合成解绕。本科生和研究生将在先进的酶动力学技术，精确的基因操作，和新的生化和细胞分析训练。科学推广计划将鼓励当地小学生通过“DNA日”探索自己基因组的奇迹。根据定义，复制体的进展依赖于六聚解旋酶的DNA双解绕活性来创建单链模板。一旦它被解开，聚合酶全酶迅速重新合成双链DNA。该项目将整合复杂的体外生物化学实验与体内基因组编辑和相关细胞分析，以表征和量化维持复制体偶联所需的多组分分子接触。这项研究还将确定和区分耦合动力学酶的过程，从那些直接通过多亚基复制体接触连接。由此产生的数据、方法和发现将被广泛传播，并将为复制体控制和多种酶活性协调机制提供更深入的见解，这些机制是适当的基因组维持所必需的。这项研究由生物科学理事会分子和细胞生物科学部的遗传机制项目资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The long-held textbook viewpoint on DNA replication is that the replisome is a stable multiprotein complex assembly that seamlessly coordinates coupled synthesis on the leading and lagging strands. However, recent evidence indicates a more dynamic replisome, where individual protein components exchange during replication, creating stochastic processes of DNA unwinding, priming, and synthesis. A more dynamic replisome would be useful in responding to various roadblocks or challenges to DNA replication progression. Still, enzymatic coordination at the replication fork must be maintained, otherwise one activity may dominate, creating excess and dangerous replication intermediates. This project will determine the molecular contacts between replisome proteins that act to coordinate and control DNA unwinding with synthesis. Undergraduate and graduate students will be trained in techniques of advanced enzyme kinetics, precise genetic manipulations, and novel biochemical and cellular assays. Scientific outreach programs will encourage local elementary school students to explore the wonders of their own genome through "DNA Days". By definition, the advancement of the replisome is dependent on the DNA duplex unwinding activity of the hexameric helicase to create single-strand templates. Once it is unwound, the polymerase holoenzyme rapidly resynthesizes duplex DNA. This project will integrate complex in vitro biochemistry experiments with in vivo genomic editing and correlated cellular assays to characterize and quantify multicomponent molecular contacts required to maintain replisome coupling. This research will also determine and differentiate coupled kinetic enzymatic processes from those that are linked directly through multisubunit replisome contacts. The resulting data, methods, and findings will be broadly distributed and will provide deeper insights into the mechanisms for replisome control and coordination of multiple enzymatic activities necessary for proper genome maintenance.This research is funded by the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences in the Directorate of Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.