From bacteria to humans, efficient DNA replication requires sliding clamps and clamp loaders to increase processivity, making the replication of an entire genome an attainable task. 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. Our main objective in this application is to investigate the dynamics of clamp opening with the goal of understanding the mechanisms by which clamp loaders are able to load sliding clamps onto DNA. Our rationale for these studies is that 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. The process of loading a clamp onto DNA involves a series of dynamic transient interactions whose elucidation has been hindered by the difficulties associated with detecting rare transient conformations experimentally. Our experimental design, which is based on the measurement and analysis of the spontaneous fluctuations of a small number of molecules, tackles this critical need directly. Based on published research and our preliminary results, we hypothesize that the existence of open conformations is more general than previously thought, and that clamps exist in a dynamic equilibrium between closed and open conformations. We further hypothesize that the role of the clamp loader is to selectively stabilize clamps in the open conformation, effectively trapping clamps that have opened spontaneously. The Levitus (ASU) and Bloom (UF) laboratories have worked in collaboration for over a year and generated the preliminary data that motivated the research proposed in this application. We are well-prepared to pursue these studies since, in addition to our strong preliminary data, this collaboration combines the expertise of the PI with cutting-edge single-molecule spectroscopic methods with the expertise of the coPI in the biochemistry of DNA replication. The first specific aim of the proposed project is to characterize the solution oligomerization equilibrium dynamics of the processivity clamps of E. coli (a dimer) and S. cerevisiae (a trimer). These proteins are among the most studied sliding clamps, and yet their association affinities and rate constants have not been fully characterized. The second specific aim is to characterize the conformational dynamics of sliding clamps in solution, bound to the clamp loaders, and bound to DNA. 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.
|Effective start/end date||6/1/12 → 5/31/17|
- NSF: Directorate for Biological Sciences (BIO): $481,368.00
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