The biological functions of DNA, including replication, transcription, recombination and repair, require unwinding and rewinding of the intertwined duplex. DNA topoisomerases are the essential enzymes to facilitate these processes. Besides their ubiquitous presence in all cells, and their critical biological functions, DNA topoisomerases are the targets of some of the clinically important antibiotics and anti-cancer drugs. Therefore, functional and mechanistic studies of these enzymes provide useful and relevant insight into the molecular basis of cancer chemotherapy. The long-term goal of our research program is on the function and mechanism of this family of enzymes. The proposed studies will focus on a type IA toposiomerase, topoisomerase Ilia, and a type II enzyme, topoisomerase II, both of which are essential for viability. We will have five specific aims in this proposal, with first two on the biological functions, and the remaining three on the mechanistic aspects.
Aim1 is to elucidate the biological functions of topoisomerase Ilia. We have generated a number of top3a mutants in Drosophila, and several top3a transgenic lines. The top3a mutants have a range of phenotypes including lethality, reduced fertility, and shortened lifespan, suggesting the potential functions of top3a in a wide range of biological functions.
Aim 2 is to probe the genetic interactions between recQ4 and fop3a. RecQ4 belongs to a family of DNA helicases with critical functions in recombination/repair, and is responsible for a genome instability disease in humans, Rothmund Thomson syndrome. We have generated a number of recQ4 mutants in Drosophila, and their interactions with top3a mutants will be investigated.
Aim 3 is to analyze the biochemical reactions of R-loop or D-loop substrates catalyzed with topo Ilia and topo Nip.
Aim 4 is to investigate the mechanistic details of the resolution of a novel double Holliday junction substrate by topo III and a recQ helicase, Bloom syndrome protein.
Aim 5 is to study the topoisomerase II mechanism by probing the kinetics of opening and closure of DNA gate mediated by this enzyme. We have generated a novel oligonucleotide-based substrate with two fluorophores such that DNA gate dynamics can be monitored by FRET. Single molecule experiments will provide kinetic information on this important step in topo II strand passage reaction. This will also allow us to examine the mechanistic basis of a number of anti-cancer drugs that affect the equilibrium of DNA cleavage and religation.
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