This research is aimed at providing an understanding of how the anthracycline antitumor drugs, particularly daunomycin, interact with DNA in the state it is found in all nuclei, namely chromatin, its subunits, and possible aggregates. In addition, experiments designed to discover the targets actually responsible for cell toxicity of the drug are planned. Finally, efforts will be made to prepare drug derivatives capable of rebinding to specific DNA sequences, with the long range objective of targeting antitumor drugs to DNA onc gene and related sequences.
Specific aims i nclude study of binding affinities by equilibrium dialysis and optical methods, and examination of drug-induced conformational changes using transient electric dichroism, sedimentation velocity, and light scattering. Drug binding kinetics will be studied using the temperature jump method, with fluorescence detection. Ethidium will serve as a comparison compound for the physical studies. Possible protein targets for drug action in cells and cell nuclei will be examined using a new photoaffinity analog of daunomycin (DNG), which reacts specifically with typical protein nucleophiles. Excess production of drug-protein crosslinked species will be sought in cell lines which are resistant to the drug. The effect of daunomycin on transcription initiation, propagation, termination, and promoter selection will be examined using gel electrophoresis methods, and posssible DNG-RNA polymerase photoadducts will be investigated. DNG-lac repressor and DNG-CAP protein photoadducts will serve as the starting point for isolation of daunomycin-peptide conjugates capable of specific rebinding to target DNA sequences. New methods of derivatizing daunomycin will be sought, and monitored by cell uptake and toxicity studies.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Biophysics and Biophysical Chemistry A Study Section (BBCA)
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Yale University
Schools of Arts and Sciences
New Haven
United States
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Shrader, T E; Crothers, D M (1989) Artificial nucleosome positioning sequences. Proc Natl Acad Sci U S A 86:7418-22
Cera, C; Egbertson, M; Teng, S P et al. (1989) DNA cross-linking by intermediates in the mitomycin activation cascade. Biochemistry 28:5665-9
Cera, C; Crothers, D M (1989) Modulation of mitomycin cross-linking by DNA bending in the Escherichia coli CAP protein-DNA complex. Biochemistry 28:3908-11
Teng, S P; Woodson, S A; Crothers, D M (1989) DNA sequence specificity of mitomycin cross-linking. Biochemistry 28:3901-7
Haran, T E; Crothers, D M (1988) Phased psoralen cross-links do not bend the DNA double helix. Biochemistry 27:6967-71
Rice, J A; Crothers, D M; Pinto, A L et al. (1988) The major adduct of the antitumor drug cis-diamminedichloroplatinum(II) with DNA bends the duplex by approximately equal to 40 degrees toward the major groove. Proc Natl Acad Sci U S A 85:4158-61
Fritzsche, H; Wahnert, U; Chaires, J B et al. (1987) Anthracycline antibiotics. Interaction with DNA and nucleosomes and inhibition of DNA synthesis. Biochemistry 26:1996-2000
Straney, D C; Crothers, D M (1987) Effect of drug-DNA interactions upon transcription initiation at the lac promoter. Biochemistry 26:1987-95
Sen, D; Crothers, D M (1986) Influence of DNA-binding drugs on chromatin condensation. Biochemistry 25:1503-9
Sen, D; Crothers, D M (1986) Condensation of chromatin: role of multivalent cations. Biochemistry 25:1495-503

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