Integration host factor (IHF) is a small heterodimeric protein that specifically binds to DNA and functions as an architectural factor in many cellular processes in prokaryotes. Here, we report the crystal structure of IHF complexed with 35 bp of DNA. The DNA is wrapped around the protein and bent by >160 degrees, thus reversing the direction of the helix axis within a very short distance. Much of the bending occurs at two large kinks where the base stacking is interrupted by intercalation of a proline residue. IHF contacts the DNA exclusively via the phosphodiester backbone and the minor groove and relies heavily on indirect readout to recognize its binding sequence. One such readout involves a six-base A tract, providing evidence for the importance of a narrow minor groove.
Relaxed closed-circular DNA is converted to negatively supercoiled DNA by DNA gyrase. This enzyme has been purified from Escherichia coli cells. The reaction requires ATP and Mg++ and is stimulated by spermidine. The enzyme acts equally well on relaxed closed-circular colicin El, phage X, and simian virus 40 DNA. The final superhelix density of the DNA can be considerably greater than that found in intracellularly supercoiled DNA. In the course of studies on integrative recombination of phage X DNA in a cell-free system from Escherichia coli (1, 2) we became aware that the process required a negatively supercoiled DNA substrate. This substrate could be replaced by relaxed closed-circular DNA only if the latter was incubated with an E. cali cell fraction and ATP (K. Mizuuchi, M. Gellert, and H. Nash, manuscript in preparation). The simplest interpretation of these results was that the E. coli extract contains an ATP-dependent activity capable of converting relaxed closed-circular DNA to the supercoiled form. We have obtained a considerable purification of this enzyme, which we call DNA gyrase, and report here the purification procedure and preliminary characterization of the enzyme. was prepared by sealing hydrogen-bonded circular DNA with DNA ligase (2). These DNA samples were deproteinized by shaking with chloroform-isoamyl alcohol (24:1 vol/vol) and repurified by centrifugation in cesium chloride-ethidium bromide density gradients. All DNA samples were dialyzed and stored in 0.01 M Tris.HCl at pH 8.0, 1 mM Na3EDTA, at 4°. MATERIALS AND METHODSAssay of DNA Supercoiling. The assay measures the conversion of relaxed closed-circular Col El DNA to the supercoiled form, as demonstrated by agarose gel electrophoresis. The standard reaction mixture (70,ul) contained 35 mM TrisHCl at pH 7.5, 1.6 mM MgCl2, 18 mM potassium phosphate at pH 7.5, 5 mM spermidine-HCl, 1.4 mM ATP, 90 jig/ml of E. coli tRNA (Calbiochem), 3.6 mg/ml of bovine serum albumin (Armour, crystalline), and 0.4 ,g of relaxed covalently circular Col El DNA. Enzyme (1-5 ,l) was diluted when necessary into 0.2 M potassium phosphate at pH 6.8, 1 mM Na3EDTA, 1 mM dithiothreitol, 10% glycerol (wt/vol), and 3.6 mg/ml of bovine serum albumin.The solution was incubated at 250 for 60 min, and then extracted with an equal volume of chloroform-isoamyl alcohol (24:1 vol/vol). After brief centrifugation, 50 ,l of the aqueous phase were added to 12.5 gl of a mixture of 5% sodium dodecyl sulfate, 25% glycerol, and 0.25 mg/ml of bromphenol blue, and the sample was loaded onto an agarose gel. Up to 30 samples at a time were electrophoresed in a 6 X 230 X 160 mm slab (E-C Apparatus Corp.) of 0.8% agarose (Type II, Sigma Chemical Co.) with Tris-borate-EDTA buffer (10.8 g of Tris base, 5.5 g of boric acid, and 0.93 g of Na2EDTA per liter). After 16 hr of electrophoresis at 40 V, the slab was stained with 1 gg/ml of ethidium bromide in the electrophoresis buffer for 1 hr, and destained in electrophoresis buffer for at least an hour. The slab was photographed using t...
The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.Topoisomerases are ubiquitous enzymes that play important roles in virtually all cells. The defining activity of these enzymes is their capacity to break DNA and, after an interval in which topological changes may occur, to reseal the break without the intervention of a high energy cofactor. Reversible breakage and reunion by topoisomerases is achieved through an intermediate in which a specific tyrosine residue of the enzyme is covalently joined to the DNA at the site of the break. The tyrosine-phosphodiester linkage is formed with one of two distinct polarities. Some enzymes join to the 5' end of the break, liberating a fragment terminated by a 3' hydroxyl; representatives include bacterial topoisomerase I as well as DNA gyrase and its relatives, the topoisomerase TI family. Other topoisomerase enzymes join to the 3' end of the break, liberating a fragment with a free 5' hydroxyl; representatives include the eukaryotic type I topoisomerases and the Int family of site-specific recombinases (for reviews, see refs. 1 and 2).The covalent intermediate between a topoisomerase and DNA is normally quite transient, i.e., strand rejoining is fast relative to strand cleavage. However, when a topoisomerase acts on DNA that contains imperfections (3, 4) or when specific inhibitors are present (5, 6), the rejoining step is slowed or blocked. This shifts the covalent complex from a transient intermediate toward a dead-end product; the associated long-lived break in the DNA backbone can have dire consequences for chromosome stability and cell survival (5,6). In this work, we describe an activity that hydrolyzes the covalent bond that joins topoisomerases to the 3' end of DNA. The specificity, robustness, and widespread distribution of this enzyme suggest that it could be an important element in the repair of DNA that has become stuck in covalent linkage with eukaryotic type I topoisomerases. MATERIALS AND METHODSDNA Substrates. Conventional oligonucleotides were constructed using an Applied Biosystems model 391 DNA synthesizer (Perkin-Elmer) or were purchased from Midland Certified Reagent (Midla...
Covalent intermediates between topoisomerase I and DNA can become dead-end complexes that lead to cell death. Here, the isolation of the gene for an enzyme that can hydrolyze the bond between this protein and DNA is described. Enzyme-defective mutants of yeast are hypersensitive to treatments that increase the amount of covalent complexes, indicative of enzyme involvement in repair. The gene is conserved in eukaryotes and identifies a family of enzymes that has not been previously recognized. The presence of this gene in humans may have implications for the effectiveness of topoisomerase I poisons, such as the camptothecins, in chemotherapy.
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