Abstract
The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.
Highlights
I1 DNA topoisomerase action on double-stranded DNA cient mutants display a “DNA-delay’’ phenotype, characterhas been explored by studying the sites where DNA ized by a delayed production of DNA
In the reaction catalyzed by type I1 topoisomerases, the tion siteswere observed on native T4 DNA, which enzyme creates a transient break in a double-stranded DNA
Studies of a similar isomerase is that the addition of the cofactor ATP intermediate in the DNA gyrase reaction were greatly facilistrongly stimulates the topoisomerase-induced cleav- tated by the finding that nalidixic acid and oxolinic acid, two age of native T4 DNA, but has only a slight effect on closely related antibacterial agents, trap this intermediate and cleavage of cytosine-containing T4 DNA
Summary
Oxolinic AcidInduces DNA Cleavage by the T4TopoisornermM dithiothreitol, 0.5 mM Na3EDTA, 0.5 mM ATP, human serum use-As discussed above, an intermediate in the reaction of albumin a t 30 pg/ml and oxolinic acid a t 500 pg/ml. Topoisomerase reactions contained 0.3 pg of linear 4x174 larger GF/A filter, and 150 p1 of binding buffer was delivered slowly duplex DNA, the indicated amount of topoisomerase, and either no to the top filter (the buffer soaks through ftihlter pair by capillary oxolinicacid (0)or 500 pg/mloxolinic acid ( 0 ) .After 10 min a t action) After this prewash, the filter pair was moved to a dry areaof 30 “C, the reactionws ere terminated by adding SDS, and treated the GF/A filter, and the spin dialysis eluate was applied, followed with proteinase K as described under“Experimental Procedures.”.
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