All ETDs from UAB

Advisory Committee Chair

Patrick Higgins

Advisory Committee Members

David Bedwell

Peter Prevelige Jr

Susan Ruppert

Tim Townes

Document Type


Date of Award


Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine


A gyrase mutant strain (gyrB652) has severe growth defect at 42°C. The survival at high temperature is dependent on DNA repair proteins. The purified mutant gyrase was assayed for supercoiling acitivity and quinolone-induced DNA cleavage activity. The results suggested that the mutant enzyme is not temperature-sensitive, but has slower kinetics than the wild type gyrase. The DNA supercoiling levels at multiple chromosomal loci in exponential phase cells were monitored with γδ resolvase-mediated recombination assay. The wild type cells showed homogenous supercoiling levels throughout most of the chromosome. But the superhelicity at dif locus was significantly lower than the other loci. This feature of dif is dependent on gyrase acitivity, as even more dramatic decrease in the superhelicity was discovered in gyrB652 and other gyrase mutant strains. We proposed that replication fork convergence at the terminus results in the low supercoiling level at dif. dif was the only locus that showed low supercoiling level in the terminus region. It raised the possibility that dif is a preferred replication termination site. Mutations in the ParC subunit of Topo IV lead to slightly reduced chromosomal supercoiling levels. But the overall supercoiling status mimicked the wild type strain, with homogenous supercoiling levels throughout most of the chromosome, and a iii moderately lower level at dif. This suggests that Topo IV doesn’t play a major role in resolving the supercoiling tension created by replication and transcription. The heterogeneity of chromosomal supercoiling levels increased in gyrase mutants. Gyrase mutants tend to have lower supercoiling level around the terminus region. But the extent of influence varied with each mutant. A mild gyrase mutant (gyrA209) showed low supercoiling levels only around the terminus. While a decreasing gradient of supercoiling was observed in gyrB652 strain along the replichores. Besides, the deepest supercoiling drop occurred in the origin half of the replichores in gyrB652 strain. A correlation between the supercoiling change and replication fork stall in gyrB652 strain was postulated. The temperature-sensitive phenotype of gyrB652 might be induced by extensive replication fork arrest.



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