A map of local curvature of the pBR322 DNA has been established by electron microscopy analysis of linearized plasmid molecules. To determine their polarity these molecules are one end labelled with an avidin‐ferritin‐biotin complex and the images are digitized. Local curvature is calculated from two mathematical treatments of the DNA trajectory and expressed in term of a mean dinucleotide wedge angle. Eight regions of curvature are distinguished. The four main regions of curvature have a high content of phased AA runs. The experimental curvature map is compared to theoretical maps of curvature obtained from four available models for DNA curvature.
The removal of inherent curving in Crithidia fasciculata kinetoplast DNA by various small DNA ligands, groove binders and mono- and bisintercalators, has been studied by gel retardation and electron microscopy. The migration of the kinetoplast DNA fragment is highly retarded during gel electrophoresis. We demonstrate that this retardation is suppressed by DNA ligands such as distamycin and ditercalinium, which have different modes of binding and sequence specificities. Observation by electron microscopy confirms that the effect of ditercalinium on gel migration of curved DNA is linked to DNA uncurving. As the drug is progressively added to DNA, a large broadening of the retarded band is observed during gel electrophoresis for distamycin and ditercalinium. In the case of distamycin, the retarded DNA band splits into two broad bands, whereas the noncurved DNA bands remain homogeneous. This indicates that the drug-DNA exchange is extremely slow in the gel and that a limited number of specific sites on DNA are critical for the removal of bending. GC-specific quinomycin, monointercalators, and bisintercalators act in a manner similar to that of AT-specific distamycin. This indicates that direct drug binding at the dAn tracts is not required for DNA uncurving. We propose that the uncurving of kinetoplast DNA by drugs is caused by a global alteration of DNA structure; subsequent increased flexibility leads to the suppression of rigid bending at the AT tract junctions.
The 8-methoxy- and 8-hydroxy-11H-pyrido[2,3-a]-, -[3,4-a]-, -[4,3-a]-, and [3,2-a]carbazoles were synthesized as potential DNA intercalating antitumor drugs. The structure of these compounds was confirmed by 1H NMR study including NOE experiments. The DNA binding properties of substituted and unsubstituted (8-H) heterocycles were determined by using their hydrochlorides or methiodides. These derivatives are able to bind to DNA with an affinity varying from 2.0 X 10(4) to 1.0 X 10(6) M-1, but most of them are unable to intercalate in contrast with the behavior of 6H- and 7H-pyridocarbazole analogues. The cytotoxicity of 11H-pyridocarbazoles, measured on L1210 cells in vitro, is much lower than those of 6H- and 7H-pyridocarbazole analogues.
The bending of kinetoplast DNA from Crithidia fasciculata is thought to be related to the periodic distribution of AA or TT cluster sequences. The sensitivity to DNase I of the two strands of this DNA was analyzed at nucleotide resolution by sequencing gel electrophoresis. The effect on the DNase I cleavage pattern of two drugs, ditercalinium and distamycin, that are able to remove bending was analyzed. The same analysis was done on a pBR 322 DNA fragment of random sequence as a control. The periodic distribution of the AA or TT clusters in the bent DNA fragment was first analyzed by computing the autocorrelation function of the AA or TT clusters in the bent DNA fragment. It is shown that the AT tracts are on average 10.5 base pairs apart. This value is almost identical with that of the B-DNA helix pitch in solution [10.5 (Wang, 1979); 10.6 +/- 0.1 (Rhodes & Klug, 1980)]. To reveal the periodic pattern of DNase I cleavage on this bent DNA, alone or in presence of drugs, the cross correlation between the different bands obtained from DNAse I cleavage and the presence of AA or TT sequences was computed. This shows that GC and mixed sequences are the most sensitive regions. These data also suggest that there is a periodic fluctuation in the width of the minor groove in the bent fragment. Ditercalinium and distamycin alter the DNase I cutting pattern of the bent DNA fragment but in an inverse fashion.(ABSTRACT TRUNCATED AT 250 WORDS)
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