AN INVESTIGATION INTO THE SEQUENCE SPECIFICITY OF VARIOUS DNA-BINDING DRUGS USING FOOTPRINTING AND RESTRICTION ENDONUCLEASE CLEAVAGE INHIBITION
Abstract
It has been suggested that sequence specificities exhibited by a number of DNA-binding ligands might affect the way genes are expressed and consequently control cellular mechanisms. The pharmacological importance of acridines and their ability to bind to DNA make them very attractive compounds to investigate using both physico-chemical and biochemical techniques. Ultra-violet spectrophotometric measurements show that several 9-aminoacridine derivatives bind to DNA with association constants of 0.74-1.3 x 10('7)M('-1). Restriction endonuclease cleavage inhibition by small intercalators suggest that they bind randomly along the DNA helix. This has been inferred by the observed reduction in the cleavage rate of the endonucleases AluI, DraI, HinfI, NaeI, NCiI and RsaI in the presence of a number of acridine derivatives and the antileukemic xanthone psorospermin. Since, for a particular enzyme, each restriction site is identical, preferential recognition of the site by either the enzyme or the ligand must depend upon the preferential recognition of the sequence around the cleavage site. Actinomycin D, a known GC specific ligand, produces an alteration in the time-course restriction pattern of several endonucleases suggesting preferential recognition of certain sites. Footprinting with DNaseI shows that simple intercalators do not protect the phosphodiester bond from cleavage by the nuclease. Actinomycin D prevents cleavage of certain GC rich areas in both single stranded and duplex DNA. This is the first report that actinomycin D produces footprint patterns in single stranded fragments. Not all GC areas are protected to the same extent; an indication that preferences for particular regions might be dependent on the secondary structure of the DNA imposed by the base sequence. Sequence specificities of DNA-binding ligands stem primarily from an optimization of the thermodynamics involved in the interaction with individual base pairs along a defined sequence.
Degree
Ph.D.
Subject Area
Biochemistry
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