Use of oligonucleotides to define the site of interstrand cross-links induced by Adriamycin

摘要

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 TA_4T_4GCA_4A 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(lll) 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-fife of the G-G cross-link was 40 h, consistent with that implied with high molecular weight, heterogeneous sequence DNA. Exonuclease HI digests of adducts formed with 20mer deoxyoligo-nucleotides containing single, central G-G, G—I and l-l potential cross-links revealed that a guanine residue is required at bothends of the cross-link. No cross-llnking was observed with a similar oligonucleotide containing only a single central (G centre dot C) bp.