| Enzymes involved in eukaryotic DNA replication were studied in developing embryonic chicken brains (ECB) and in human breast carcinoma cells (BRCC) during apoptosis. A systematic study was undertaken to search for the presence of a DNA helicase in complex with the known replication enzyme DNA polymerase-alpha (pol-alpha). Antibodies against human helicases (I, IIA, IIB, III, IV) were used in ELISA to test for the presence or absence of helicases in the replication complex (RC) isolated from ECB or BRCC. In the present study we characterize pol-alpha and DNA helicase-III (hel-III) in ECB during brain development and in BRCC's going through the stages of apoptosis. In both ECB and in human breast carcinoma cells, it was determined that hel-III forms a complex with pol-alpha. In ECB, the pol-alpha/hel-III complex was shown to be developmentally regulated. In the 11th day after fertilization, the pol-alpha/hel-III complex is present whereas by the later stages of development, the complex is no longer detectable. The level of enzymatic activity of both pol-alpha and hel-III was shown to decrease with embryonic aging.; A novel DNA helicase assay, the radioactive oligonucleotide in membrane filtration effluent (ROME) assay, was developed. The ROME assay uses radiolabeled calf thymus DNA as the enzyme substrate. The oligonucleotide helicase product is then separated by membrane filtration and quantified. The ROME assay was then used to characterize DNA helicase activities in ECB. Optimum parameters (metal, nucleotide, and pH requirements) for the measurement of ECB DNA helicase activities were determined.; The effect of cis-platin treatment on pol-alpha and helicase was tested. Treatment of human BRCC (SKBR3, MCF-7, and MDA-468) with cis-platin (anti-cancer drug) proved to induce apoptosis in each cell line. The progress of apoptosis was determined by phosphatidyl serine flopping with a novel dye (PSS-380) and DNA laddering. Activation of caspase-3 was observed within 24 hours of treatment. A dose dependent decrease of activities of pol-alpha and helicases was observed after 48 hours. Treatment with low concentration of cis-platin was able to break up the complex while high concentration treatment caused proteolysis of the proteins in the complex. |
