Use of oligonucleotides to define the site of interstrand cross-links induced by Adriamycin.
It has been known for several years that Adriamycin forms adducts and interstrand cross-links when reacted for long periods of time with bacterial and mammalian DNA in vitro, with the cross-link being restricted to 2 bp elements containing GpC sequences. The self-complementary 20mer deoxyoligonucleotide TA4T4GCA4T4A has been used in this study as a model of the apparent G-G cross-linking site at GpC sequences. The rate of formation of cross-links, as well as the dependence on both Adriamycin and Fe(III) concentration, were similar with this oligonucleotide as compared with calf thymus DNA. The cross-linking was demonstrated on both denaturing and non-denaturing sequencing gels. The half-life of the G-G cross-link was 40 h, consistent with that implied with high molecular weight, heterogeneous sequence DNA. Exonuclease III digests of adducts formed with 20mer deoxyoligonucleotides containing single, central G-G, G-I and I-I potential cross-links revealed that a guanine residue is required at both ends of the cross-link. No cross-linking was observed with a similar oligonucleotide containing only a single central (G.C) bp.
Cutts
S M
SM
School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Phillips
D R
DR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry; D004317:Doxorubicin / Q000494:pharmacology*; D004355:Drug Stability; D005090:Exodeoxyribonucleases / Q000378:metabolism; D006147:Guanine / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*
D003432:Cross-Linking Reagents; D009841:Oligonucleotides; D006147:Guanine; D004317:Doxorubicin; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III