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.

No description available.

No description available.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1981. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Bibliography: leaves 174-182. / by Daniel Jeffrey Capon. / Ph.D.

Thesis. 1976. Ph.D.--Massachusetts Institute of Technology. Dept. of Biology. / Microfiche copy available in Archives and Science. / Vita. / Bibliography: leaves 231-249. / Ph.D.

A crucial factor in determining the accuracy of DNA replication is maintenance of a balanced supply of deoxyribonucleoside triphosphates (dNTPs) at replication forks. Perturbation of dNTP biosynthesis can induce dNTP pool imbalance with deleterious genetic consequences, including increased mutagenesis, recombination, chromosomal abnormalities and cell death. Using the T4 bacteriophage system, I investigated the molecular basis of mutations induced by imbalanced dNTP pools in vivo. Two approaches were adopted to disturb dNTP biosynthesis: 1) using mutations which affect the deoxyribonucleotide biosynthesis pathway; 2) exogenously supplying mutagenic deoxyribonucleoside analogs which are then taken up by cells and are metabolized to dNTPs. The levels of dNTPs under different conditions were measured in crude extracts of phage-infected cells, while mutagenic effects were quantitated by analysis of certain rII mutations, thought to revert to wild type along either GC-to-AT or AT-to-GC transition pathways. The mutation pathways stimulated by dNTP pool perturbations were confirmed by direct DNA sequencing after amplification of template by the polymerase chain reaction (PCR). By replacing phage ribonucleotide (rNDP) reductase with the host, Escherichia coli, rNDP reductase, in phage-infected cells, I examined the mechanism of mutation induced by the thymidine analog 5- bromodeoxyuridine (BrdUrd) in vivo. Although both AT-to-GC and GC-to- AT transition mutations were stimulated many hundred-fold when cells were grown in medium containing 100 μM BrdUrd, GC-to-AT transitions were stimulated predominantly when T4 reductase was active, while ATto- GC transitions were stimulated more when E. coli reductase was active. By examining the control by dNTPs on CDP reduction, I found that the T4 rNDP reductase is substantially inhibited by either BrdUTP or dTTP in crude enzyme extracts. These experimental results are consistent with the hypothesis that mutagenic effects of BrdUrd are based on dNTP perturbations, supporting the model that rNDP reductase is a major determinant of BrdUrd mutagenesis. I also studied the mutator phenotype of one temperature-sensitive conditional lethal mutant, T4 ts LB3, which specifies a thermolabile T4 deoxycytidylate (dCMP) hydroxymethylase. At the sites of different rII mutations, I found 8- to 80-fold stimulation of GC-to-AT transitions induced by ts LB3 at a semipermissive temperature (34° C). Sequence analysis of revertants from the most sensitive gene marker, rII SN103, showed that either cytosine within the mutated triplet can undergo change to either thymidine or adenine, supporting a model in which mutagenesis induced by ts LB3 at a semipermissive temperature is based on dNTP pool perturbations. The putative depletion of hydroxymethyldeoxycytidine triphosphate (hm-dCTP) caused by the temperature-labile dCMP hydroxymethylase presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations. However, no significant dNTP pool abnormalities were observed in extracts from ts LB3 phageinfected cells even when cells were grown at the semi-permissive temperature, suggesting that imbalanced dNTP pools occurred only locally, close to replication forks. These results support a model of dNTP "functional compartmentation", in which DNA replication is fed by a small and rapidly depleted pool, with the bulk of measurable dNTP in a cell representing a replication-inactive pool. To further characterize the mutagenic specificity and DNA site specificity induced by T4 ts LB3, I developed a fast forward mutation approach using thymidine kinase as a marker gene. The studies confirmed that the principal mutagenic effect induced by ts LB3 is C-to- T transition, while C-to-A transversion mutagenesis also occurs. Analysis of DNA sequences around each mutation also suggests that local DNA context influences mutation frequency. / Graduation date: 1991

Deoxyribonucleic acid (DNA) replication in eukaryotic cells requires a highly complex series of protein-DNA interactions. Elucidation of the mechanisms by which DNA replication occurs is vital to the understanding of cellular growth. DNA polymerase alpha is an enzyme with a putative role in the replication of eukaryotic DNA. Modification by phosphorylation and dephosphorylation is one process by which enzymatic activity is regulated. The purpose of this research was to determine if a phosphorylation event could be of significance in the expression of DNA polymerase alpha activity. Evidence will be presented for the regulation of DNA polymerase alpha by phosphorylation. A highly purified DNA polymerase alpha fraction was prepared from Chinese hamster ovary cells. Purification procedure included ion-exchange chromatographies and affinity chromatography. Both the crude DNA polymerase alpha activity and the highly purified DNA polymerase alpha activity were stimulated six-fold by the addition of exogenous bovine cardiac muscle cyclic AMP-dependent protein kinase. Dephosphorylation of the highly purified DNA polymerase alpha fraction by alkaline phosphatase resulted in a concomitant decrease in DNA polymerase alpha activity. An endogenous protein kinase activity was detected in the highly purified DNA polymerase alpha fraction. Incubation of this fraction in a protein kinase reaction mixture including adenosine triphosphate (ATP) could stimulate DNA polymerase alpha activity to twelve-fold that observed in controls with no pre-incubation. The endogenous protein kinase activity in the highly purified DNA polymerase alpha fraction was utilized to indicate (1) the linear increase in DNA polymerase alpha activity with time of phosphorylation which was dependent on the presence of ATP, (2) the linear relationship between γ³²P-ATP incorporation and DNA polymerase alpha activity, and (3) the incorporation of labelled phosphate into DNA polymerase alpha as determined by SDS-PAGE analysis. Finally, the co-purification of the endogenous protein kinase with DNA polymerase alpha is presented. The significance of this research in relation to the heterogeneous nature reported for DNA polymerase alpha is discussed. It is speculated that the phosphorylational regulation of DNA polymerase alpha may play a role in the transformation of cells.

We describe a simple, sensitive, nonradioactive, relatively rapid and relatively inexpensive protocol to measure DNA synthesis in cultured cells by a chemiluminescent bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA). We show that it exhibits similar sensitivity and activity as traditional 3H-thymidine incorporation assays and a commercial chemiluminescent BrdU ELISA kit when tested in commonly used cell lines, such as NIH 3T3 cells, mink lung epithelial cells (Mv1LU), and baby hamster kidney (BHK-21) fibroblasts. This assay also exhibits a wider dynamic range than colorimetric BrdU ELISA methods. Besides being a viable, nonradioactive alternative to 3H-thymidine incorporation assays, our BrdU ELISA is less expensive than a commercial chemiluminescent BrdU ELISA kit.

Chromosomal DNA of A. vinelandii thought to carry multiple gene copies was examined in efforts to visualize its chromosomal structure using electron microscopy. The chromosomal DNA of A. vinelandii may have multiple circular genomic units carrying multiple copies of genes. Three possible branch construction schemes and their replication modes are postulated in this study.