Intellectual Merit: DNA replication is the process by which all living organisms make copies of their DNA, and it is the foundation of biological inheritance. The synthesis of the new DNA strands is catalyzed by enzymes called DNA polymerases, which use the mother DNA strand as a template to synthesize a new copy. From bacteria to humans, efficient DNA replication requires proteins known as processivity factors to ensure that the polymerase moves rapidly along DNA without dissociating from it. In particular, sliding clamps are oligomeric ring-shaped proteins that encircle DNA, providing an anchor for the DNA polymerase. To load clamps onto DNA, an open clamp loader-clamp complex must form. It is generally assumed that clamps exist as closed rings in solution and that clamp loaders must therefore actively open their interfaces. Very few studies, however, have addressed this problem directly. The dynamics of clamp opening will be investigated with the goal of understanding the mechanisms by which clamp loaders are able to load sliding clamps onto DNA. The fairly static view of clamp loading that has emerged from structural data is intrinsically inadequate to understand the mechanistic details of how these proteins achieve their function. This limitation will be tackled directly by our experimental design, which is based on the measurement and analysis of the spontaneous fluctuations of a small number of molecules. Initially, the solution oligomerization equilibrium dynamics of the processivity clamps of E. coli (a dimer) and S. cerevisiae (a trimer) will be characterized. These proteins are among the most studied sliding clamps, and yet their association affinities and rate constants have not been fully characterized. Then, the conformational dynamics of sliding clamps in solution, bound to the clamp loaders, and bound to DNA will be characterized. Single-molecule fluorescence techniques are particularly well-suited to investigate the structural dynamics of biopolymers, and will be used in this project to characterize the conformational fluctuations in processivity clamps. The successful completion of these studies will provide vital mechanistic insights into how processivity factors work.
Broader Impacts: Graduate students can enrich their educational experience by learning about and participating in all aspects of the synergistic and joint efforts of the Levitus (ASU) and Bloom (UF) labs. ASU students will spend a fraction of each summer at UF to immerse themselves into the molecular biology aspects of the project, while a student from UF will spend time at ASU to learn about single-molecule and other spectroscopic techniques. Students from underrepresented groups will be recruited through a series of existing programs at ASU and UF. A series of activities aimed at increasing the retention and chances of success of minority students and early-career faculty, including mentoring female junior faculty and minority graduate students, will be continued, as well as participation in student research conferences for underrepresented undergraduate students within the STEM disciplines.
