DNA topoisomerases are ubiquitous enzymes that are found in both prokaryotes and eukaryotes. Mechanistically, they cause DNA strands to pass through broken DNA-protein bridges in a concerted breakage and rejoining reaction in the DNA backbone. Although the precise function of many of these enzymes is not clear, their participation in replication, transcription, recombination and transposition has been suggested. This proposal is designed to investigate the role in replication and recombination. T4 DNA topoisomerase (a type II enzyme) consists of three non-identical subunits which are the products of T4 genes 39, 52 and 60. We propose to use recombinant DNA technology to clone these genes into high expression vectors such that individual components can be isolated and purified. The purified subunits will be examined individually and in combination, in order to determine if they can carry out any or part of the topoisomerization reactions characteristic of the complete enzyme. The site specific interactions of the T4 topoisomerase as well as its component subunits with DNA origins of replication will be examined using its ability to protect the target sites against digestion by DNaseI as assays. The genes for all three subunits, will be sequenced. Furthermore, its role in an excision event will be evaluated through the development of an in vitro system capable of resolving a duplication in the origin region of pBR322 DNA, using either the purified 39-protein alone or in conjunction with other cellular components as suggested by the in vivo process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM021960-10A1
Application #
3270825
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-02-01
Project End
1989-11-30
Budget Start
1984-12-01
Budget End
1985-11-30
Support Year
10
Fiscal Year
1985
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Huang, W M; Libbey, J L; van der Hoeven, P et al. (1998) Bipolar localization of Bacillus subtilis topoisomerase IV, an enzyme required for chromosome segregation. Proc Natl Acad Sci U S A 95:4652-7
Huang, W M (1996) Bacterial diversity based on type II DNA topoisomerase genes. Annu Rev Genet 30:79-107
Bailey, C C; Younkins, R; Huang, W M et al. (1996) Characterization of genes encoding topoisomerase IV of Mycoplasma genitalium. Gene 168:77-80
Casjens, S; Delange, M; Ley 3rd, H L et al. (1995) Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order. J Bacteriol 177:2769-80
Dew-Jager, K; Yu, W Q; Huang, W M (1995) The recA gene of Borrelia burgdorferi. Gene 167:137-40
Takiff, H E; Salazar, L; Guerrero, C et al. (1994) Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations. Antimicrob Agents Chemother 38:773-80
Belland, R J; Morrison, S G; Ison, C et al. (1994) Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone-resistant isolates. Mol Microbiol 14:371-80
Samuels, D S; Marconi, R T; Huang, W M et al. (1994) gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi. J Bacteriol 176:3072-5
Casjens, S; Huang, W M (1993) Linear chromosomal physical and genetic map of Borrelia burgdorferi, the Lyme disease agent. Mol Microbiol 8:967-80
Huang, W M; Ao, S Z; Casjens, S et al. (1988) A persistent untranslated sequence within bacteriophage T4 DNA topoisomerase gene 60. Science 239:1005-12

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