Maintenance of a steady-state level of DNA supercoiling is essential for diverse biological processes. There is constant competition between the rate of supercoil formation arising from moving transcription or replication forks, and the rat of supercoil disappearance due to collision of negative and positive supercoil waves diffusing along the DNA. Removal of both positive and negative supercoils is the function of type 1B DNA topoisomerase enzymes (Topo 1B). Topo1B forms a freely reversible covalent phosphotyrosine linkage with a single strand of duplex DNA, resulting in a break in the DNA backbone that allows supercoiled DNA to unwind in a controlled fashion using transient enzyme interactions with the downstream rotating DNA portion. Human Topo-DNA covalent complex forms the sole target for the widely used anticancer drugs irinotecan and topotecan, which disrupt supercoil unwinding. Despite the central role of the Topo-DNA complex in regulating superhelical density and in anticancer pharmacology, little is known about how the enzyme senses superhelical density to maintain an optimal steady-state level of supercoiling, and importantly, how small molecules interfere with this process. This proposal aims to elucidate how Topo1B activity responds to discrete changes in DNA supercoiling and to determine the mechanism of action of small molecules that perturb topoisomerase-catalyzed supercoil unwinding.
The specific aims are to use a unique DNA minicircle substrate containing a single Topo1B recognition site, and a variable number of supercoils, to study the effect of supercoiling on binding affinity (via fluorescence anisotropy), and the kinetics of supercoil unwinding (via gel-based observation of discrete supercoiled intermediates). From kinetic simulations of the supercoil unwinding data, rate constants for DNA cleavage, unwinding, and religation will be calculated. These studies will also be extended to explore the role of electrostatic interactions between the enzyme and the rotating portion of the downstream DNA backbone using both methylphosphonate substitutions of the minicircle DNA and mutations of positively charged protein residues that interact with this DNA region. To simulate the effects of bound cellular proteins during DNA rotation, this study will measure the effect of DNA rotational drag on supercoil unwinding by affixing proteins with increasing molecular weight to a specific binding site on the rotating DNA. These same experiments will be expanded to study the mechanism of small molecule inhibitors and their effect on the efficiency of supercoil unwinding.
The importance of Topoisomerase 1B (Topo) in the maintenance of genome structure has made it a valuable clinical drug target. This proposal not only seeks to understand the temporal regulation of supercoil unwinding by Topo, it also hopes to use this information as the basis for understanding how small molecules change the dynamics of supercoil unwinding. Elucidation of the mechanism provides a fundamental basis for the design of novel Topo inhibitors. ! ! PUBLIC HEALTH RELEVANCE: The importance of Topoisomerase 1B (Topo) in the maintenance of genome structure has made it a valuable clinical drug target. This proposal not only seeks to understand the temporal regulation of supercoil unwinding by Topo, it also hopes to use this information as the basis for understanding how small molecules change the dynamics of supercoil unwinding. Elucidation of the mechanism provides a fundamental basis for the design of novel Topo inhibitors.
|Cravens, Shannen L; Schonhoft, Joseph D; Rowland, Meng M et al. (2015) Molecular crowding enhances facilitated diffusion of two human DNA glycosylases. Nucleic Acids Res 43:4087-97|