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 (1) reduction of the nucleoside 5'-aldehyde ends to 5'-hydroxyls followed by incorporation of /sup 32/P from (..gamma..-/sup 32/P)ATP by polynucleotide kinase and (2) 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 PO/sub 4/ termini. By contrast, 5'-terminal nucleoside aldehyde in DNA cut with the drug in vitro exceeds 80% of the total 5' ends. Of the /sup 32/P representing nucleoside aldehyde in DNA from NCS-exposed cells, 77% is in TMP; the rest is in AMP >> CMP > 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. Furthermore, in cells depleted of glutathione, DNA strand breakage was greatly reduced, confirming thiol involvement in the activation of the drug in cells, as in the vitro reaction.
- Research Organization:
- Harvard Medical School, Boston, MA
- OSTI ID:
- 6620117
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:2; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ANTI-INFECTIVE AGENTS
ANTIBIOTICS
ANTINEOPLASTIC DRUGS
ATP
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
DAYS LIVING RADIOISOTOPES
DNA
DNA SEQUENCING
DRUGS
ELECTROPHORESIS
ENZYMES
HELA CELLS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
RADIOISOTOPES
STRAND BREAKS
STRUCTURAL CHEMICAL ANALYSIS
TRACER TECHNIQUES
TRANSFERASES