The effects of the antitumor protein auromomycin on HeLa S3 nuclei. Release of soluble chromatin.
The protein antitumor agent auromomycin releases chromatin from intact HeLa S3 nuclei by introducing strand scissions in DNA linker regions. The ability of auromomycin to solubilize nuclear chromatin was compared with the well-characterized action of micrococcal nuclease. Production of acid-soluble material (DNA 10-20 base pairs or less) did not increase significantly over a range of drug levels or incubation times. Release of chromatin from auromomycin-treated nuclei was not affected by EDTA but required dithiothreitol for optimal activity. The soluble chromatin, when resolved by agarose gel electrophoresis, consisted of discrete nucleosome bands ranging from monomer to hexamer size and unresolved higher molecular weight oligomers. Sufficiently high concentrations of drug convert most of the higher molecular weight oligonucleosomes to dimer and monomer material. These nucleosome bands were quite broad and could indicate strand scission occurring at random throughout the linker region. The average size of the drug-generated mononucleosomes from multiple experiments was 175 +/- 4 base pairs. In contrast to micrococcal nuclease, higher concentrations of drug did not degrade mononucleosomes to core particles. Solubilized chromatin is detectable following drug treatments as short as 5 min. Chromatin produced at early time points was mostly small and very heterogeneous. Chromatin from drug-digested nuclei analyzed under denaturing conditions appeared as discrete bands similar to those observed on native gels. The ratio of single- to double-stranded breaks may be close to 2:1. When chromophore isolated from the holantibiotic was incubated with nuclei, a similar pattern of chromatin degradation was observed.
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D004229:Dithiothreitol; D004492:Edetic Acid; D006367:HeLa Cells; D006801:Humans; D008836:Micrococcal Nuclease; D010455:Peptides / Q000494:pharmacology; D012995:Solubility
D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D002843:Chromatin; D010455:Peptides; C001666:auromomycin; D004492:Edetic Acid; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; D008836:Micrococcal Nuclease; D004229:Dithiothreitol