No description available.

The involvement of topoisomerase in DNA replication in a prokaryotic and a eukaryotic system was examined. The mechanism of in vitro DNA replication by an isolated replicative enzyme complex was also investigated. In bacteriophage T4 there is evidence that the type II topoisomerase coded for by the phage is involved in the initiation of replicative growing points. We have looked at the topological structure of the replicating T4 nucleoid by sedimentation of the DNA in neutral sucrose gradients containing various amounts of ethidium bromide. It was determined that at no time after infection does the replicating T4 DNA contain any large amount of negative superhelicity. The absence of the phage topoisomerase did not affect the topology of the nucleoid. It was therefore concluded that the role of the T4 topoisomerase in initiating DNA synthesis in T4 was not exerted at the level of the general topology of the replicating T4 DNA. An isolation procedure for the T4 topoisomerase for pursuance of further studies was also described. New mammalian topoisomerases were shown to be stimulated by epidermal growth factor (EGF) in two cultured fibroblast cell lines. Topoisomerase activity was seen first in the cell cytoplasm and subsequently in the nucleus. The peak of topoisomerase activity in the nucleus corresponded to the peak of EGF-induced DNA synthesis in the cells. At least a part of the topoisomerase activity stimulated by EGF was shown to be due to a type II topoisomerase by the ATP-dependence of the activity. The topoisomerase activity in the cytoplasm was shown to exist in a non-soluble, sedimentable form. Further characterization of the topoisomerase containing complex isolated from the cytoplasm was carried out. The complex was seen to be non-membrane bound and complex. DNA polymerase and nucleoside diphosphate kinase activities were also demonstrated to be contained within the complex. It was shown that this cytoplasmic complex was capable of in vitro DNA replication. Many parameters of the in vitro DNA replication reaction were examined. The process was seen to mimic in vivo replication in several ways. The complex was shown to not only be able to elongate DNA but to initiate replication through the creation of a replication bubble.

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

No description available.

The lexA41 (formerly tsl-l) mutant was previously isolated as a UV-resistant, temperature-sensitive derivative of its UV-sensitive lexA3(Ind⁻) parent. Cells exhibit a so-called "split-phenotype", a phenomenon in which only a subset of the SOS responses can be detected physiologically following inducing treatments. In this work, lexA41 has been cloned and sequenced; the mutant gene retains the lexA3 mutation (Gly to Asp at position 85) and has a second mutation, lexA41 (Ala to Thr at position 132). LexA41 protein is not cleaved by the RecA protein-catalyzed pathway in vivo, but the mutant protein is degraded by the Lon protease at both 32° and 42°C. β-galactosidase activities of lac fusions to thirteen different SOS promoters were measured at 30° and 42° to determine levels of expression and were found to vary considerably. LexA41 protein is deficient in repressor function. The temperature sensitive phenotype is due to increased expression of sulA, which encodes a division inhibitor, at 42°. Excision repair genes, including uvrA, uvrB and uvrD, are constitutively expressed at 30° accounting for the UV resistance of the lexA41 mutant, but the SOS mutagenesis operon, umuDC, is not adequately derepressed explaining the failure to induce mutagenesis in this background. This differential expression of SOS genes gives a plausible explanation of the "split-phenotype" associated with lexA41. In another set of experiments, I have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam⁻). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD, and dinF) were found to express higher levels of (beta)-galactosidase in dam⁻ strains than in isogenic dam⁺ strains. The attempted construction of dam⁻ strains that were also mutant in one of several SOS genes indicated that viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for viability of dam⁻ strains.

No description available.

No description available.