Each cell of Salmonella typhimurium possesses a chemosensory system, which detects gradients, and a flagellum, which propels it along the gradient. The flagellum comprises a 6000 rpm rotary motor, located in the cell's inner membrane, land a propeller, which is a helically shaped filament made up of flagellin subunits. Reversal of the direction of motor rotation causes the cell to tumble and therefore to reorient, a process abetted by a change in the pitch and hand of the helical filament. The chemosensory system controls the path of the cell by modulating the frequency of motor reversal as a function of changes in concentration of chemical attractants and repellents. We are interested in seeing the three-dimensional structure of the flagellar components, especially the motor and the filament, in determining their compositions, in identifying the genes which encode for these structures, in understanding the mechanism by which these structures function, and in determining how the cells assemble such structures. We propose to (1) isolate and characterize a more complete motor complex than has heretofore been studied; (2) determine the three-dimensional structure of the motor complex to 20 Angstroms or better, using cryoelectron microscopy; (3) attempt to correlate structure and function by analyzing images of the motor complex from mutant cells; (4) determine the three-dimensional structure of the filament to 10 Angstroms resolution by cryoelectron microscopy in order to visualize the domains of the flagellin subunit; (5) compare the 10 Angstroms maps of filaments in different structural states in order to determine the mechanism by which the filament adopts and changes its shape; and (5) alter the amino acid sequence of the flagellin subunit to probe the mechanism of flagellar filament assembly. topoisomerases include the elucidation of the mechanism of excision of rDNA rings from the chromosomal cluster when the total cellular level of DNA topoisomerases is low; the idea that the supercoiling of the DNA template by transcription is the underlying cause of instability of genomic clusters of rDNA and other repetitive sequences will be tested. Genetic and biochemical approaches, including the design of sequence-specific supercoiling enzymes, will be used to test whether there are specific anchoring points that divide the chromosomes into topologically separate domains, and whether DNA topoisomerase II has a role in the organization of such loops. Experiments will also be carried out to test further the dispensability of DNA topoisomerases in the initiation of replication in eukaryotes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Harvard University
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Azarova, Anna M; Lyu, Yi Lisa; Lin, Chao-Po et al. (2007) Roles of DNA topoisomerase II isozymes in chemotherapy and secondary malignancies. Proc Natl Acad Sci U S A 104:11014-9
Kwan, Kelvin Y; Greenwald, Rebecca J; Mohanty, Subhasis et al. (2007) Development of autoimmunity in mice lacking DNA topoisomerase 3beta. Proc Natl Acad Sci U S A 104:9242-7
Stupina, Vera A; Wang, James C (2005) Viability of Escherichia coli topA mutants lacking DNA topoisomerase I. J Biol Chem 280:355-60
Jiang, Xiaofeng (2005) Random mutagenesis of the B'A' core domain of yeast DNA topoisomerase II and large-scale screens of mutants resistant to the anticancer drug etoposide. Biochem Biophys Res Commun 327:597-603
Stupina, Vera A; Wang, James C (2004) DNA axial rotation and the merge of oppositely supercoiled DNA domains in Escherichia coli: effects of DNA bends. Proc Natl Acad Sci U S A 101:8608-13
Lyu, Yi Lisa; Wang, James C (2003) Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIbeta. Proc Natl Acad Sci U S A 100:7123-8
Kwan, Kelvin Y; Moens, Peter B; Wang, James C (2003) Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase III beta. Proc Natl Acad Sci U S A 100:2526-31
Wang, Yong; Lynch, A Simon; Chen, Sue-Jane et al. (2002) On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant. J Biol Chem 277:1203-9
Wang, Yong; Lyu, Yi Lisa; Wang, James C (2002) Dual localization of human DNA topoisomerase IIIalpha to mitochondria and nucleus. Proc Natl Acad Sci U S A 99:12114-9
Kwan, K Y; Wang, J C (2001) Mice lacking DNA topoisomerase IIIbeta develop to maturity but show a reduced mean lifespan. Proc Natl Acad Sci U S A 98:5717-21

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