Intercalator and minor groove binding drug effects on intrinsic DNA curvature
Abstract
DNA displays a large variety of conformations beyond the commonly accepted notion of it being just a monotonously linear molecule. A property inherent to some DNA molecules is a stable deflection of the helix caused by a compressed minor groove of certain sequences of nucleotides called bending elements. When the position of each bending element matches the helical repeat of the DNA, the summation of the individual deflections results in a macromolecular curvature of the DNA helix. This intrinsically curved DNA plays an essential role in a variety of biological systems including replication and transcription. A computer program designed to predict DNA curvature from a given sequence of nucleotides identified a significant number of circular structures in retroviral and cellular genomes. The integrase gene of the human immunodeficiency virus type I (HIV-I) contains an 830 base-pair circular structure. A comparison of predicted curvature to the electrophoretic mobility of 57 mutated sequences resulted in a correlation coefficient of 0.93. The relative effect of three minor groove binding compounds on DNA curvature was determined. DAPI $(4\sp\prime$-6-diamidino-2-phenylindole) was more effective than either Hoechst 33258, or distamycin A at reducing the curvature of a 198 base-pair circular DNA fragment from Caenorhabditis elegans. Decreased curvature was demonstrated by the reduction of anomalous electrophoretic mobility in polyacrylamide gels, or by the reduced formation of closed circular products in a T4 ligase mediated cyclization reaction. Cytofluorimetry indicated nearly identical binding affinities of the C. elegans DNA for DAPI and Hoechst 33258. An intercalating drug that unwinds the DNA helix would be expected to reduce the curvature of the C. elegans circular DNA by disrupting its unilateral positioning of bending elements. Neither actinomycin D, chloroquine, nor ethidium bromide reduced the intrinsic curvature of the C. elegans DNA circle as determined by electrophoretic and cyclization studies. Studies of these DNA-drug complexes have dual objectives: to add to our understanding of DNA intrinsic curvature, and to analyze the relative binding properties of the drugs with the goal of designing target specific compounds.
Degree
Ph.D.
Advisors
Anderson, Purdue University.
Subject Area
Molecular biology|Pharmacology
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.