Accurate and efficient DNA replication is critical for the survival of all organisms. The overall goal of this project is to understand how the molecular dynamics of the processivity clamp regulate its interactions that are central to efficient DNA replication. The focus of this proposal is on the Escherichia coli beta clamp, which is a ring- shaped dimeric protein that encircles DNA and provides a binding platform for other proteins to access DNA. Processivity clamps also increase the efficiency of DNA replication by tethering DNA polymerases to DNA and increasing their processivity. Appropriate loading of the beta clamp is critical for regulation of events at the replication fork and for efficient DNA replication. Our central hypothesis, which is supported by our preliminary data, is that trapping of specific transiently sampled beta clamp conformations by the clamp loader underlies loading of beta onto DNA and determines the efficiency of this process. To test this hypothesis, in aim 1, we will design variants of the beta clamp with altered stability or asymmetry and determine the thermal stability and oligomeric state of these altered clamps. We will also probe the interactions of the altered clamps with the clamp loader.
In aim 2, we will determine the dynamics of the beta clamp alone and in complex with clamp loader by Transverse Relaxation Optimized SpectroscopY (TROSY) NMR relaxation and hydrogen exchange measurements.
In aim 3, we will determine the biological functions of the designed clamp protein variants, including their ability to interact with and be loaded onto DNA by the clamp loader, and to function in processive DNA replication. Correlating the dynamics of the beta clamp with its proficiency in these different activities will provide important insights into the relationship between clamp dynamics and function. Moreover, this work will provide fundamental insights into the roles of protein dynamics in regulating protein-protein interactions.
The proposed research is relevant to public health because it will lead to a better understanding of the fundamental process of bacterial DNA replication as regulated by the beta processivity clamp protein, which is expected to increase the likelihood that new antibiotics could be developed to target the essential activities of the beta clamp. Antibiotic resistance is a growing public health threat and new antibiotics are critically needed. Therefore the proposed research is relevant to the part of the NIH mission that pertains to the development of fundamental knowledge that will reduce the burden of human disease.