The object is to use SV40 DNA replication as a tool in developing new methods to investigate molecular events of the mammalian cell cycle. An assay has been developed for a "cytoplasmic factor(s)" required to convert viral replicating DNA molecules to mature viral DNA in isolated nuclei. This assay will be used in purifying the factor(s) and determining its composition, stability, molecular weight, isoelectric point and possible enzymatic activities relevant to DNA replication. The factor's mechanism of action and specificity will be explored to purified nuclei under defined conditions by characterizing viral DNA replication in the presence and absence of added factor. These studies utilize pulse-chase techniques in conjunction with a restriction endonuclease to examine details of DNA chain elongation and termination. The factors effect on nuclear stability and RNA synthesis will be determined. Specificity will be examined by comparisons with a similar system using polyoma. Finally, the techniques developed in these experiments will be tested for their application in designing new assays for factors required to initiate DNA replication and factors from mitotic cells responsible for nuclear membrane dissociation and chromatin condensation. Such assays would open the way for future in vitro studies on DNA replication and mitotic induction. These events are fundamental to understanding oncogenesis, a pathological alteration of the cell cycle.
NIH ExPorter
NIH ExPorter