<p>Quinolone treatment induces stabilized cleavage complexes (SCCs), consisting of a covalent gyrase-DNA complex, and processing of these complexes is thought to cause double-strand breaks and chromosome fragmentation. SCCs are required but not sufficient for cytotoxicity; the mechanism that converts SCCs to double-strand breaks is not clearly understood. Evidence of chromosome fragmentation due to quinolones comes from indirect measures such as sedimentation analysis of nucleoids and measurements of lysis viscosity. This work outlines a method that combines agarose plugs, conditional lysis and field inversion gel electrophoresis to allow direct visualization of chromosomal fragmentation resulting from quinolone treatment. We are able to distinguish between latent breaks within the stabilized cleavage complex and irreversible breaks that result from downstream processing.</p><p>When seeking to understand the genetic requirements for quinolone-induced SOS response, we found that a dnaQ mutant has a specific defect in SOS induction following nalidixic acid. The product of dnaQ is the ε subunit of DNA polymerase III, which provides 3' → 5' exonuclease activity. In addition to the nalidixic acid-specific SOS defect, δdnaQ has multiple phenotypes: slow growth, high mutation frequency, and constitutive SOS. We propose that ε has a role in the quinolone response beyond the normal proofreading function of the subunit in the polymerase III core. Using a unique transposon mutagenesis system, we created a library of dnaQ mutants with 15 base pair insertions that were scored phenotypically. We identified mutants that separated the various phenotypes, arguing strongly that ε has multiple functions. The isolation of a stable dnaQ mutant with SOS phenotypes allows the study of this function without confounding results from spurious mutations throughout the chromosome. We also isolated a novel class of SOS "hyper-inducible" mutants. Additionally, my findings with weak and strong β-clamp binding mutants provides the first in vivo characterization of these ε mutants and gives insight into the SOS response following nalidixic acid treatment.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/8773 |
| Date | January 2014 |
| Creators | Whatley, Zakiya Nicole |
| Contributors | Kreuzer, Kenneth N |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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