Investigations of DNA adducts of adriamycin and molecular interactions between DNA and xUBF Box 1

Abstract

Duplex DNA incubated with adriamycin, dithiothreitol (DTT) and Fe 3+ under aerobic, aqueous conditions yields double stranded (DS) DNA bands by denaturing polyacrylamide gel electrophoresis (DPAGE) analysis, characteristic of DNAs which are interstrand cross-linked. Another laboratory provided evidence that formaldehyde produced under these conditions promotes the covalent linkage of adriamycin to one strand of DNA and suggested that this monoadduct results in this appearance of interstrand cross-linking upon DPAGE analysis. This dissertation provides strong support for this interpretation. We show that mixtures of DNA and adriamycin incubated with DTT/Fe3+, H 2O2, or formaldehyde all show DS DNA bands on DPAGE analysis. We have also developed an indirect, GC-MS analytical technique that quantitatively detects formation of this lesion. Using this technique, we show that the DS DNA bands and the formaldehyde-mediated lesion form with similar time courses, and in similar amounts. We also show that the DNA in the DS DNA bands contains approximately one such lesion per DNA, whereas the single stranded DNA is devoid of it. Finally, we cursorily examine the effect of sequence context on lesion formation. All of our results further support the interpretation that adriamycin does not create interstrand cross-links in DNA, and that the DS DNA observed in DPAGE experiments derives from the formaldehyde-mediated monoadduct.The N-terminal HMG box of xUBF (xUBF Box 1) is investigated for binding to synthetic oligonucleotides. A library of oligonucleotides with cisplatin intrastrand crosslinks was prepared and their relative affinities for xUBF Box 1 were determined using the electrophoretic mobility shift assay (EMSA). Due to the extremely poor binding of these oligonucleotides, an electrophoretic binding assay based on the ability to compete with the binding of four-way junction DNA to xUBF Box 1 was developed. Although the efficacy of this competition assay is demonstrated, very little difference is seen in the affinity of the oligonucleotide library, either platinated or native, to xUBF Box 1. Despite this, an NMR-size sample of one of these platinated sequences has been prepared and its binding to the protein is currently being studied.