DNA replication is carried out by macromolecular structures called ?replication forks?, which are loaded at discrete sites called ?origins?. DNA Replication termination (?termination?) occurs when two replication forks from adjacent origins converge head-on upon the same stretch of DNA and ~60,000 termination events occur each cell cycle in a typical human cell. Termination involves completion of DNA synthesis, unlinking of daughter chromosomes, and unloading of replication proteins. Failure to terminate DNA replication leads to chromosome missegregation and/or activation of error-prone DNA repair pathways that elevate the mutation rate of DNA replication. Therefore, termination defects are expected to cause genomic instability, which is a hallmark of cancer cells. Furthermore, termination involves Topoisomerase II and p97, which are targeted during cancer chemotherapy. Overall, it is critical that we understand how termination works. However, termination is poorly-characterized, in large part because termination cannot be monitored in cells. This technical limitation arises because termination is asynchronous and not localized to specific genomic loci, owing to stochastic origin usage and variable replication fork rates. To overcome this limitation, I recently developed a biochemical approach to monitor synchronous, localized termination in Xenopus egg extracts. This approach allows for extensive mechanistic dissection of termination events and includes the full complement of proteins required for termination, including proteins that are critically important but not yet discovered. This approach led to a biochemical model for termination, which involves rapid convergence of replication forks in contrast to the stalling that was previously-thought to occur. I also generated a large proteomic dataset of potential regulators of termination. My lab will leverage these expertise to address three broad questions about replication termination: 1. How does topological stress influence termination? 2. How does termination regulate subsequent cell cycle events? 3. What is the full set of proteins required for replication termination? Overall, this work will identify new mechanisms and proteins involved in termination, and expand our view of termination to include new stages that occur before and after completion of DNA synthesis. This work provides a foundation for some of the long-term goals of my lab, which are to: 1. Understand the biochemical problems posed by termination and the solutions employed by cells 2. Determine how termination shapes the cell cycle and impacts genomic stability 3. Reconstitute the termination of DNA replication in vertebrates
Subversion of DNA replication is an essential part of oncogenesis, and mutations in DNA replication proteins cause a variety of diseases (from cancer, to drwarfism, and even neurodegeneration), so it is critical for human health that we gain a complete understanding of how DNA replication works. This proposal will study the final stage of DNA replication, which is called ?termination?, to reveal new steps involved in this process and new proteins involved. It is particularly critical to understand termination because it is targeted during cancer chemotherapy (by Etoposide in the clinic and CB-5083 in clinical trials) but remains the most poorly- understood stage of DNA replication.
