This project focuses on three giant motor proteins, toposiomerases, helicases, and FtsK that move DNA through large distances utilizing the energy of NTP hydrolysis and mechanical strain on DNA. We hope to understand how these proteins perform these vital roles in DNA replication and chromosomal segregation. We will use single DNA molecule enzymology complemented with bulk measures. The action of a single enzyme acting on DNA is measured by the resultant changes in DNA force, torque, and extension. The single DNA molecules can be supercoiled or braided at will to generate substrates for the enzymes. We will measure the rates of enzyme action, processivity, stall force, and chirality in interaction with superhelical DNA. These results will then be compared with bulk measures and measures in vivo. The clear medical relevance stems primarily from two sources. First, topoisomerases are the favored targets of antibiotics such as ciprofloxacin, and anticancer agents, such as etoposide and adriamyin. The understanding of their unusual dominant poisoning of their targets has greatly aided the development of more potent drugs. Second, interference in proper segregation of chromosomes by mutations that affect motor proteins accompanies and exacerbates human diseases, including cancer and premature aging.
| Cost, Gregory J; Cozzarelli, Nicholas R (2007) Directed assembly of DNA molecules via simultaneous ligation and digestion. Biotechniques 42:84, 86-9 |
| Cost, Gregory J; Cozzarelli, Nicholas R (2006) Smc5p promotes faithful chromosome transmission and DNA repair in Saccharomyces cerevisiae. Genetics 172:2185-200 |
| Gore, Jeff; Bryant, Zev; Stone, Michael D et al. (2006) Mechanochemical analysis of DNA gyrase using rotor bead tracking. Nature 439:100-4 |
| Stray, James E; Crisona, Nancy J; Belotserkovskii, Boris P et al. (2005) The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling. J Biol Chem 280:34723-34 |
| Pease, Paul J; Levy, Oren; Cost, Gregory J et al. (2005) Sequence-directed DNA translocation by purified FtsK. Science 307:586-90 |
| Levy, Oren; Ptacin, Jerod L; Pease, Paul J et al. (2005) Identification of oligonucleotide sequences that direct the movement of the Escherichia coli FtsK translocase. Proc Natl Acad Sci U S A 102:17618-23 |
| Hardy, Christine D; Cozzarelli, Nicholas R (2003) Alteration of Escherichia coli topoisomerase IV to novobiocin resistance. Antimicrob Agents Chemother 47:941-7 |
| Khodursky, Arkady B; Bernstein, Jonathan A; Peter, Brian J et al. (2003) Escherichia coli spotted double-strand DNA microarrays: RNA extraction, labeling, hybridization, quality control, and data management. Methods Mol Biol 224:61-78 |
| VanLoock, Margaret S; Alexandrov, Alexander; Yu, Xiong et al. (2002) SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes. Curr Biol 12:472-6 |
| Dekker, N H; Rybenkov, V V; Duguet, M et al. (2002) The mechanism of type IA topoisomerases. Proc Natl Acad Sci U S A 99:12126-31 |
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