Type of Document Dissertation Author Kamau, Edwin Author's Email Address email@example.com URN etd-04072005-181452 Title DNA Supercoiling with a Twist Degree Doctor of Philosophy (Ph.D.) Department Biological Sciences Advisory Committee
Advisor Name Title Anne Grove Committee Chair David Donze Committee Member Grover Waldrop Committee Member Patric DiMario Committee Member Graca Vicente Dean's Representative Keywords
- vaccinia topoisomerase i
- dna protein interaction
- dna binding and bending
- high mobility group (hmgb) proteins
- dna supercoiling
Date of Defense 2004-04-04 Availability unrestricted AbstractThe level of torsion in double-stranded DNA regulates base-pair stability and DNA conformation. It is important in initiation and regulation of specific DNA metabolic processes as well as chromatin assembly. High mobility group proteins (HMGB) are architectural proteins whose HMG DNA binding domains confer significant preference for distorted DNA, such as supercoiled DNA and 4-way junctions. HMGB proteins play a role in transiently regulating or conserving DNA torsion. Topoisomerases regulate DNA supercoiling, which has been argued to provide a coherent explanation for the main modes of transcriptional control - stringent control, growth-rate control and growth-phase control during the normal cell growth.
In this study, we have shown that HMO1, a Saccharomyces cerevisiae HMGB protein which is required for normal growth, plasmid maintenance and for regulating the susceptibility of yeast chromatin to nuclease binds linear duplex DNA but has little preference for DNA with altered conformations. Divergent box A binds DNA and contributes structure-specific binding. Unlike most HMGB proteins, HMO1 does not supercoil relaxed DNA in the presence of topoisomerase. Casein Kinase II phosphorylates HMO1, altering its DNA binding properties. We have also shown that deletion of the highly basic C-terminal tail of HMO1 localizes this otherwise both nuclear and cytoplasmic protein only to the cytoplasm. As the C-terminally truncated HMO1 has been reported to rescue the hmo1 knockout phenotype, we conclude that the main function of HMO1 lies in the cytoplasm, and not in the nucleus.
Vaccinia topoisomerase I relaxes supercoiled DNA. We have shown that it interacts with enrofloxacin, a fluoroquinolone antibiotic which otherwise targets DNA gyrase and topoisomerase IV. Enrofloxacin inhibits DNA relaxation by Vaccinia topoisomerase I. When presented with relaxed DNA, the enzyme executes the reverse reaction, supercoiling the DNA. Enrofloxacin does not interfere with the catalytic cleavage site of Vaccinia topoisomerase I or its ability to bind DNA. The mechanistic implication of these observations is that protein-DNA contacts downstream of the cleavage site must contribute to DNA supercoiling, contrary to the free rotation mechanism proposed for DNA relaxation.
Filename Size Approximate Download Time (Hours:Minutes:Seconds)
28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access Kamau_dis.pdf 2.01 Mb 00:09:17 00:04:46 00:04:10 00:02:05 00:00:10
If you have questions or technical problems, please Contact LSU-ETD Support.