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An attempt has been made to demonstrate an association between the carcinogenic activity of a chemical compound and its capacity to induce DNA damage and chromosome aberrations which may result in mutations and/or neoplastic transformation. Twenty-five 4-nitroquinoline 1-oxide (4NQO) derivatives and five related compounds of 4-nitropyridine 1-oxide (4NPO) of varying carcinogenicity were examined. [Formulae omitted] The induction of DNA damage, chromosome aberrations and clone forming capacity were used as end points. Monolayer cultures of embryonal Syrian-hamster cells and an established line of baby hamster kidney cells (BHK-21) were employed in this study. DNA damage, as measured by the unscheduled incorporation of tritiated thymidine (³H-TdR), was assayed by the autoradiographic procedure. To distinguish DNA repair synthesis from DNA replication synthesis at S-phase, cultured embryonal hamster cells were arrested at G₁ by growing them in an arginine deficient medium (ADM) prior to the application of the various carcinogens. The unscheduled uptake of radioisotope was estimated by counting the number of grains per diploid nucleus of carcinogen treated cells. The highly oncogenic derivatives of 4NQO and 4NPO elicited an elevated level of unscheduled ³H-TdR incorporation in treated cells, while the weakly oncogenic compounds induced only a smaller amount of DNA repair synthesis. The non-oncogenic N-oxides failed to provoke any detectable ³H-TdR uptake. Chromosome aberrations were studied in ADM-arrested cells which were exposed to the various compounds and then triggered into division by transferring them into the regular growth medium. A direct proportionality was observed between the degree of carcinogenicity of a compound and the frequency of induced chromosome aberrations. The clone forming ability of treated cells was employed as a means to compare the cytotoxicity of the 4NQO and 4NPO derivatives. Potent carcinogens were highly cytotoxic; weakly carcinogenic compounds showed only a slight lethal effect and non-oncogenic derivatives did not affect cell survival. This study demonstrated the capacity of carcinogens to induce alterations at the chromosome and DNA level. The possible role of DNA repair and chromosome aberrations in neoplastic transformation was discussed. The use of DNA repair synthes as an economic and relevant tool for identifying mutagens and/or carcinogens has been suggested. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Cell duplication in Escherichia coli involves complex events, coordinated with chromosome replication. Because of the importance of chromosomes in perpetuating the normal cell cycle the initiation of their replication must be coordinated with cellular division. Following initiation, the cell must replicate and segregate its chromosomes, create a site necessary for septation and divide. These events could be coordinated by either; (1) biochemical reactions involving diffusible enzymes, or (2) multienzyme complexes which are localized at the site of DNA replication and cell division. In the latter case, the cyclic events of replication, segregation and cell division may be coordinated by physical-chemica1 or biochemical means. In any case, physical association implies pleiotropic effects. To test this hypothesis, cell division of the initiator mutant of E. coli , isolated by Kohiyama (1968) was studied. The temperature-sensitive initiator mutant E. coli CR 34T83 (ts DnaA) grew normally at 30 C, and at the restrictive temperature (42 C). The DNA replication as measured by radioactive precursor uptake, stopped after approximately 40 minutes and was equivalent to completion of rounds of replication started. Measurement of ribo- and deoxyribonucleotide triphosphate pools by thin-layer chromatography at 30 C and 42 C indicated residual DNA synthesis was not due to a limitation in the DNA precursors. Using a combination of density and differential radioactive labelling for the starts and ends of chromosomes, a preferred place for reinitiation of new replication cycles was shown. It was shown that DNA replication at 42 C terminated at a fixed region of the chromosome, and was identical to the 150 μg/ml chloramphenicol sensitive step involved in the process of initiation of chromosome replication in E. coli. A cessation of cellular division was noted by measurement of cell growth by Coulter Counter, at a shift from 30 C to 42 C, resulting in filamentous growth. Upon a return to 30 C, the cells resume division after approximately 15 - 20 min. The pleiotropic behaviour, that is, the cessation of cell division and initiation of DNA replication was a result of a point mutation in the gene DnaA, coding for a membrane bound protein involved in initiation. This mutation was mapped by transduction and was located at the isoleucine-valine region of the E. coli map. When this gene was transduced to different strains of E. coli K(12) the same pleiotropy was observed. This pleiotropy could be uncoupled, however, at 30 C by inhibitors of DNA synthesis or initiation. During recovery at 30 C from growth under 42 C, expression of cell division was proportional to cell equivalents generated at the restrictive temperature. RNA and protein synthesis, for 10 minutes during the recovery period, was obligatory for initiation of new rounds of replication, but not for the expression of cell division. A cell division "potential" protein was present under the restrictive growth condition. This "potential" was made at a derepressed rate and underwent a rapid degradation if kept at 42 C. At any given time, when returning from 42 C to 30 C, this "potential" allowed expression cell division based on DNA/mass or normal cell equivalents generated at 42 C. The half-life for decay of the division "potential" was estimated to be 1.4 minutes. The results were interpreted, in terms of an enzyme complex, which is common to the initiation of DNA replication and cellular division. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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Fenn, Dickson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 74-81). / Abstract also in Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Acknowledgement --- p.iii / Table of Contents --- p.v / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations and Symbols --- p.xv / Abstract --- p.xvii / Chapter 1.Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Oxidation and Hyperoxidation of Guanine --- p.1 / Chapter 1.2. --- DNA Replication --- p.2 / Chapter 1.3 --- Mutagenesis --- p.3 / Chapter 1.4 --- Literature Survey on Spiroiminodihydantoin (Sp) --- p.4 / Chapter 1.5 --- Purpose of This Work --- p.5 / Chapter 1.6 --- DNA Structure --- p.6 / Chapter 1.6.1 --- Nomenclature --- p.6 / Chapter 1.6.2 --- Torsion Angles --- p.6 / Chapter 1.6.3 --- Sugar Pucker Conformation --- p.7 / Chapter 1.6.4 --- Secondary Structures of DNA --- p.8 / Chapter 2.Chapter Two: --- Materials and Methodology --- p.10 / Chapter 2.1 --- Sample Design --- p.10 / Chapter 2.2 --- Sample Preparation --- p.11 / Chapter 2.2.1 --- DNA Synthesis and Purification --- p.11 / Chapter 2.2.2 --- HPLC Separation --- p.11 / Chapter 2.2.3 --- NMR Samples Preparation --- p.12 / Chapter 2.3 --- NMR Analysis --- p.12 / Chapter 2.3.1 --- Resonance Assignment --- p.14 / Chapter 2.3.1.1 --- Proton --- p.14 / Chapter 2.3.1.2 --- Phosphorous --- p.16 / Chapter 2.3.2 --- Sugar Pucker Conformation --- p.17 / Chapter 2.3.3 --- Backbone Conformation --- p.18 / Chapter 2.4 --- UV Melting Analysis --- p.19 / Chapter 3.Chapter Three: --- "HPLC, NMR and UV Results" --- p.21 / Chapter 3.1 --- HPLC Separation of Sp Diastereoisomers --- p.21 / Chapter 3.2 --- NMR Resonance Assignments --- p.24 / Chapter 3.2.1 --- 5'-GG Sample --- p.24 / Chapter 3.2.2 --- 5'-G(oG) Sample --- p.26 / Chapter 3.2.3 --- 5'-G(Sp) Sample --- p.29 / Chapter 3.2.4 --- 5'-T(oG) Sample --- p.31 / Chapter 3.2.5 --- 5'-T(Sp) Sample --- p.34 / Chapter 3.3 --- Sugar Pucker Conformation --- p.38 / Chapter 3.4 --- Backbone Conformation --- p.41 / Chapter 3.5 --- UV Melting --- p.43 / Chapter 4.Chapter Four: --- Effect of Spiroiminodihydantoin and 7-hydro-8-oxoguanine on Primer-Template Structures --- p.44 / Chapter 4.1 --- Overview --- p.42 / Chapter 4.2 --- NMR Investigations of the Primer-Template Models --- p.45 / Chapter 4.2.1 --- Incorporation of a dCTP Opposite a 5'-GG Template --- p.45 / Chapter 4.2.2 --- Incorporation of a dCTP Opposite a 5'-G(oG) Template --- p.46 / Chapter 4.2.3 --- Incorporation of a dCTP Opposite a 5'-G(Sp) Template --- p.48 / Chapter 4.2.4 --- Incorporation of a dATP Opposite a 5'-T(oG) Template --- p.50 / Chapter 4.2.5 --- Incorporation of a dATP Opposite a 5'-T(Sp) Template --- p.51 / Chapter 4.3 --- Effect of Sp and oG on Primer-Template Structures --- p.52 / Chapter 4.3.1 --- Misaligned Structure with a Sp-Bulge --- p.52 / Chapter 4.3.2 --- C·oG Base Pair in 5'-G(oG) --- p.54 / Chapter 4.3.3 --- Biological Implications --- p.54 / Chapter 5. --- Chapter Five: Preliminary Structural Calculations on Primer- Template Structures --- p.56 / Chapter 5.1 --- Experimental Restraints Extraction --- p.56 / Chapter 5.2 --- Experimental Restraints Distribution --- p.58 / Chapter 5.3 --- Structural Calculations --- p.60 / Chapter 5.4 --- Structural Results --- p.62 / Chapter 5.4.1 --- 5'-GG --- p.63 / Chapter 5.4.2 --- 5'-G(oG) --- p.64 / Chapter 5.4.3 --- 5'-T(oG) --- p.65 / Chapter 5.4.4 --- 5'-T(SpR) with 5'-T(Spl) Restraints --- p.66 / Chapter 5.4.5 --- 5'-T(SpR) with 5'-T(Sp2) Restraints --- p.67 / Chapter 5.4.6 --- 5'-T(SpS) with 5'-T(Spl) Restraints --- p.68 / Chapter 5.4.7 --- 5'-T(SpS) with 5'-T(Sp2) Restraints --- p.69 / Chapter 5.6 --- Structural Analysis --- p.70 / Chapter 6. --- Chapter Six: Conclusions and Future Work --- p.72 / Appendix --- p.73 / References --- p.74

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains ix, 172 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

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Successful replication of the genome during each cell cycle requires that every replication fork merge with its opposing fork. However, lesions in the template DNA or protein-DNA barriers often impede replication forks and threaten the timely completion of genome duplication. If a fork encounters a replication fork barrier (RFB), it can be subject to a variety of fates. In some cases the replisome is maintained in a manner such that it can resume DNA synthesis when the barrier is removed. Alternatively the stalled fork is simply held in a competent state to merge with the opposing fork when it arrives. However, fork stalling can also precipitate dissociation of the replisome (fork collapse) or even fork breakage. If this happens the recombination machinery can intervene to restore DNA integrity and restart replication, albeit with a risk of causing deleterious genetic change if ectopic homologous sequences are recombined. I have exploited a site-specific RFB in fission yeast termed RTS1 to investigate the consequences of perturbing a single replication fork. RTS1 is a polar RFB (i.e. it blocks fork progression in a unidirectional fashion), enabling replication to be completed by the opposing fork. Despite this, fork blockage at RTS1 triggers a strong recombinational response that is able to restart DNA synthesis, which at least initially is highly error prone. Here, I present my work in establishing a live cell imaging approach to visualizing the recombinational response at the RTS1 RFB, demonstrating that the majority of cells initiate recombination-dependent replication (RDR). RDR begins within a few minutes of fork blockage and is only curtailed by the arrival of the opposing fork. It depends on the Rad52 protein, which remains associated with the restarted fork and whose presence correlates with its infidelity. I also illustrate the significance of various genetic factors, including Rad51, the Rad51 mediators, Fml1 helicase, Rad54 translocase, Pfh1 sweepase, and Cds1 checkpoint kinase, in modulating Rad52 localization and block-induced recombination at the RTS1 RFB.