The development of a general method for studying the sequence specificity of small ligands which bind to DNA is proposed. The method involves partial digest of a drug ligated, singly end-labeled (32P), DNA restriction fragment using a nonspecific endonuclease. The DNA fragments produced in the digest along with the base specific reaction products of the Maxam-Gilbert sequencing method, are separated using electrophoresis in a polyacrylamide sequencing gel. Autoradiography and microdensitometry allows quantitative comparison of the digest patterns obtained in the presence of the drug with those obtained in its absence. The regions of protection from nuclease attack, allow direct visualization of the binding sequence of the drug as well as the determination of thermodynamic properties associated with the drug DNA contact. In addition to examining the relationship between a drug's structure and its sequence specificity for linear DNA's, the method will be adapted to the study of drug-DNA interactions using supercoiled DNA and chromatin as binding substrates.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM031895-03
Application #
3280311
Study Section
Biophysics and Biophysical Chemistry A Study Section (BBCA)
Project Start
1983-04-01
Project End
1986-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
3
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Syracuse University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Fish, E L; Lane, M J; Vournakis, J N (1988) Determination of equilibrium binding affinity of distamycin and netropsin to the synthetic deoxyoligonucleotide sequence d(GGTATACC)2 by quantitative DNase I footprinting. Biochemistry 27:6026-32
Kissinger, K; Krowicki, K; Dabrowiak, J C et al. (1987) Molecular recognition between oligopeptides and nucleic acids. Monocationic imidazole lexitropsins that display enhanced GC sequence dependent DNA binding. Biochemistry 26:5590-5
Dabrowiak, J C; Skorobogaty, A; Rich, N et al. (1986) Computer assisted microdensitometric analysis of footprinting autoradiographic DATA. Nucleic Acids Res 14:489-99
Lown, J W; Sondhi, S M; Ong, C W et al. (1986) Deoxyribonucleic acid cleavage specificity of a series of acridine- and acodazole-iron porphyrins as functional bleomycin models. Biochemistry 25:5111-7
Ward, B; Skorobogaty, A; Dabrowiak, J C (1986) DNA cleavage specificity of a group of cationic metalloporphyrins. Biochemistry 25:6875-83
Ward, B; Skorobogaty, A; Dabrowiak, J C (1986) DNA binding specificity of a series of cationic metalloporphyrin complexes. Biochemistry 25:7827-33
Lown, J W; Krowicki, K; Bhat, U G et al. (1986) Molecular recognition between oligopeptides and nucleic acids: novel imidazole-containing oligopeptides related to netropsin that exhibit altered DNA sequence specificity. Biochemistry 25:7408-16
Bromley, S D; Ward, B W; Dabrowiak, J C (1986) Cationic porphyrins as probes of DNA structure. Nucleic Acids Res 14:9133-48
Goodisman, J; Dabrowiak, J C (1985) Theoretical analysis of the footprinting experiment. J Biomol Struct Dyn 2:967-79
Lane, M J; Vournakis, J N; Dabrowiak, J C (1985) Footprinting analysis as a means of quantitating antitumor drug-DNA interactions. Prog Clin Biol Res 172B:145-53