Intellectual Merit: DNA replication has to be coordinated with cell division to ensure it occurs once and only once per cell cycle to maintain genomic stability. However, which signaling pathway(s) or molecule(s) serve as a link between such cell division events and DNA replication remains unknown. Although involvement of Polo-like kinase 1 (Plk1), a regulator of many cell division events, has been implicated in DNA replication, no direct Plk1 substrates have yet been identified for this process. The research goal of this project is to understand how Plk1 regulates DNA replication. This will be accomplished by analyzing how Plk1-mediated phosphorylation of specific proteins involved in initiation of DNA replication regulates DNA replication. Although increasing evidence supports a role of Plk1 in DNA replication, the more significant issues are how Plk1 exerts its function on proteins implicated in DNA replication and the nature of the signals that couple Plk1 to these events. This research will bridge this knowledge gap. The extent to which different aspects of DNA replication are regulated by Plk1 activity toward its substrates will be determined by an array of molecular and cellular techniques. By focusing on the role of Plk1 in DNA replication, the project challenges the traditional view that Plk1 functions only on cell division events. Because DNA replication is a significant process that occurs in every organism and Polo kinase is highly conserved across species, the research will have a broader impact in understanding DNA replication in other organisms. Broader Impact: This project will be conducted primarily by students, making undergraduate and graduate education a major focus. Using experiments designed to address research problems as the core for training, significant integration of research and education will be accomplished. Collaborating with various undergraduate research programs at Purdue, three students will be trained during a typical 10-week module. Thus, at least 9 African American students from Jackson State University will be trained. A Problem-Based Learning (PBL) approach will be implemented to train students in the laboratory and to stimulate scientific interest. The use of problems to introduce concepts will also provide a natural mechanism to highlight the interconnections among disciplines. The education plan will also have an impact on career choices of undergraduate populations.
DNA replication has to be coordinated with cell division to ensure it occurs once and only once per cell cycle to maintain genomic stability. However, which signaling pathway(s) or molecule(s) serves as a link between mitotic events and DNA replication in the subsequent S phase remains unknown. Although involvement of Polo-like kinase 1 (Plk1), a critical regulator of many mitotic events, has been suggested in DNA replication, no direct Plk1 substrates in this process have been identified yet. The research goal of this project is to understand how Plk1 regulates DNA replication. This has been accomplished by achieving two specific aims: (i) analyze how Plk1 phosphorylation of Orc2 regulates DNA replication; and (ii) determine the molecular steps of DNA replication regulated by Plk1-dependent phosphorylation of Hbo1 (histone acetyltransferase binding to Orc1). Although increasing evidence supports a role of Plk1 in DNA replication, the more significant issues are how Plk1 exerts its function on the S-phase machinery and the nature of the signals that couple Plk1 to S phase. Data generated from the proposed study bridged this knowledge gap. The extent to which different aspects of DNA replication are regulated by Plk1-associated kinase activity toward its substrates has been determined by a broad array of molecular and cellular techniques. By focusing on the role of Plk1 in DNA replication, the proposed work challenges the traditional view that Plk1 functions only on mitotic events. The identification of the mechanism by which Plk1 links mitotic events with DNA replication will impact the understanding of how two critical cell cycle events are coordinated to maintain genomic stability, thus opening up a completely new research direction. Because DNA replication is a fundamentally significant process that occurs in every organism and Polo kinase is highly conserved across species, the work has a broader impact in understanding DNA replication in other organisms. This research project has been conducted primarily by students, making undergraduate and graduate education a major focus of this research program. Using experiments designed to address research problems as the core for training, the PI explored the efficacy of integrating education with research through the training program. Collaborating with various undergraduate research programs and the Summer Research Opportunities Program (SROP) at Purdue, thirteen students have been trained during a typical 10-week session module. The PI implemented a Problem-Based Learning (PBL) approach to train students in the lab to stimulate scientific interest. The use of problems to introduce concepts also provides us with a natural mechanism to highlight the interconnections among disciplines. Thus, the processes and objectives essential to PBL align with many fundamental aspects of undergraduate research, making the training model to be used by many other labs and schools. The education and reseach experience had a significant impact on career choices of undergraduate populations.
