Mechanism and base specificity of DNA Breakage in intact cells by neocarzinostatin.
When electrophoresed on an agarose gel, the DNA isolated from neocarzinostatin- (NCS-) treated HeLa cells migrates in a ladder of discrete bands indicative of preferential breakage in the linker region of the nucleosomes. The 5'-termini of the drug-induced DNA strand breaks were characterized by reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of 32P from [gamma-32P]ATP by polynucleotide kinase and treatment of the DNA with hot alkali and alkaline phosphatase prior to the kinase assay to give the total 5'-termini. In DNA isolated from NCS-treated cells, nucleoside aldehyde accounts for 30-45% of the drug-generated 5' ends; the remainder have PO4 termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the 32P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP much greater than CMP greater than GMP, a distribution in excellent agreement with that obtained for in vitro drug-treated DNA. DNA sequencing experiments, using the 340 base pair alphoid DNA fragment isolated from drug-treated cells, show that the pattern of breakage produced by NCS within a defined sequence of DNA in intact cells is similar to that in the in vitro reaction, with a preferential attack at thymidylate residues, but a much higher concentration of the drug was required to cause comparable breakage in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001483:Base Sequence; D004249:DNA Damage; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000302:isolation & purification; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D009353:Zinostatin / Q000494:pharmacology*
D000903:Antibiotics, Antineoplastic; D004273:DNA, Neoplasm; D009353:Zinostatin; D005978:Glutathione