zinostatin and dimethyl-sulfate

Incorporation of NAD into the nuclei was determined autoradiographically in cultured HeLa cells and in cryostat sections of rat organs by incubating them with 3H-NAD after fixation with various agents. Acetone fixation was the best to render the cells permeable to NAD while preserving the cell's enzymatic activity to incorporate NAD into nuclear macromolecules. Various evidence supported that such incorporation of NAD is due mostly to the synthesis of poly(ADP-ribose) on chromatin proteins. In the sections of rat jejunum and esophagus the rate of NAD incorporation was higher in the actively proliferating cell nuclei than in the differentiated cell nuclei within the same epithelia. These results suggested that the capacity of the cells to synthesize poly(ADP-ribose) is associated with cell growth and differentiation.

Topics: Autoradiography; Bleomycin; Cell Nucleus; DNA Damage; HeLa Cells; Humans; Kinetics; Mutagens; NAD; Sulfuric Acid Esters; Tritium; Zinostatin

The alkaline sucrose density gradient centrifugation method was modified to permit detection of 1 lesion/10(9) daltons of DNA. With this technique, the involvements of DNA polymerases in DNA repair of damage by dimethyl sulfate, UV irradiation, neocarzinostatin, and bleomycin were studied in HeLa cells with the aid of the DNA polymerase inhibitors aphidicolin and 2',3'-dideoxythymidine. DNA repair after UV-induced damage seemed to involve only polymerase alpha, while repair of damage by the other three agents involved both polymerase alpha and a non-alpha polymerase, probably polymerase beta. But repair after damage by dimethyl sulfate differed from that after damage by neocarzinostatin or bleomycin with respect to the co-operations of polymerase alpha and polymerase beta: in repair of dimethyl sulfate-induced damage, both polymerases operated on the same lesions, whereas after damage by neocarzinostatin or bleomycin, polymerase alpha and polymerase beta functioned independently on different lesions.

Topics: Aphidicolin; Bleomycin; Dideoxynucleosides; Diterpenes; DNA Polymerase I; DNA Polymerase II; DNA Repair; HeLa Cells; Humans; Hydroxyurea; Kinetics; Sulfuric Acid Esters; Thymidine; Ultraviolet Rays; Zinostatin

We have characterized the binding of lambda phage replication initiation protein O to the phage origin of replication. The minimal DNA segment required for O binding is the single iteron, a 19-bp sequence of hyphenated dyad symmetry that is repeated with variations four times in the origin. The isolated amino terminus of O protein is also sufficient to bind DNA. Electrophoretic studies show that the amino terminus of O protein induces bending of a single iteron. The DNA-protein interaction was characterized by ethylation interference, dimethyl sulfate protection and neocarzinostatin footprinting. Points of DNA-protein contact are largely concentrated in two areas symmetrically disposed with respect to the dyad symmetry of the iteron. This suggests the protein interacts as a dimer with half sites in the DNA. However, a few non-symmetrical contacts are found, indicating that O protein may distort the helix. This may correlate with the bending effects demonstrated electrophoretically. Cylindrical DNA projections were used to model O protein binding to the lambda origin and compare it with the lambda repressor-operator interaction. Whereas bound repressor nearly encircles the DNA in the major groove, O protein leaves the major groove on the opposite side exposed.

Topics: Alkylation; Bacteriophage lambda; Base Sequence; Binding Sites; DNA Replication; DNA-Binding Proteins; DNA, Viral; Nucleic Acid Conformation; Sulfuric Acid Esters; Viral Proteins; Virus Replication; Zinostatin