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...

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ATP-dependent DNA supercoiling catalyzed by Escherichia coli DNA gyrase was inhibited by oxolinic acid, a compound similar to but more potent than nalidixic acid and a known inhibitor of DNA replication in E. coil. The supercoiling activity of DNA gyrase purified from nalidixic acidresistant mutant (naL4U) bacteria was resistant to oxolinic acid. Thus, the nalA locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (cou). Supercoiling of X DNA in E. coli cells was likewise inhibited by oxolinic acid, but was resistant in the nalAR mutant. The inhibition by oxolinic acid of colicin El plasmid DNA synthesis in a cell-free system was largely relieved by adding resistant DNA gyrase.In the absence of ATP, DNA gyrase preparations relaxed supercoiled DNA; this activity was also inhibited by oxolinic acid, but not by novobiocin. It appears that the oxolinic acid-sensitive component of DNA gyrase is involved in the nicking-closing activity required in the supercoiling reaction. In the presence of oxolinic acid, DNA gyrase forms a complex with DNA, which can be activated by later treatment with sodium dodecyl sulfate and a protease to produce double-strand breaks in the DNA. This process has some similarities to the known properties of relaxation complexes. Previous work (1-3) has described an enzyme activity, DNA gyrase, that is responsible for the supercoiling of DNA in Escherichia colh. As isolated from extracts of E. colh, the enzyme introduces negative superhelical turns into covalently closed circular DNA in an ATP-dependent reaction; the hydrolysis of ATP presumably provides the free energy needed to accumulate mechanical strain energy in the DNA.One genetic locus (cou), which determines resistance to coumermycin A1 and novobiocin, has been identified as controlling the activity of DNA gyrase (2). The enzyme isolated from wild-type cells is inhibited by both these antibiotics, while DNA gyrase from a coumermycin-resistant mutant strain is unaffected. Intracellular DNA supercoiling is similarly blocked by coumermycin.In this paper we report the involvement of a second genetic locus (nalA), which determines resistance to nalidixic acid and oxolinic acid (4, 5), in controlling DNA gyrase activity. These two drugs are inhibitors of DNA replication in E. coli (4, 5). They also inhibit replication in cell-free systems of colicin El plasmid (ColEl) DNA (6, 7) and of phage qX174 replicative form DNA (8), but they do not inhibit the synthesis of the complementary strand of /X174 single-stranded DNA (8). These properties are parallel to those described for coumermycin A1 and novobiocin.Nalidixic acid-resistant mutants of two classes have been identified and mapped (9). Mutations at one locus (naIB, 57 min on the standard E. coli map) are responsible for low-level resistance and have been characterized as interfering with the permeability of the cells to nalidixic acid. Mutations ...

We find the heptamer sequence to be the most important; specifically, the three bases closest to the recombination crossover site are critical. The nonamer is not as rigidly defined, and it is not important to maintain the five consecutive As that distinguish the consensus nonamer sequence. Both types of signals display very similar sequence requirements and have in common an intolerance for changes in spacer length > 1 bp. Although the two signal types share sequence motifs, we find no evidence of a role in recombination for homology between the signals, suggesting that they serve primarily as protein recognition and binding sites. Mature genes encoding the component chains of immunoglobulin (Ig) and T-cell receptor (TCR) proteins are assembled early in lymphoid development from germ-line arrays of variable (V), diversity (D), and joining (J) gene segments (for recent reviews, see Blackwell and Alt 1988;Davis 1988). By allowing for a large variety of coding sequences, this combinatorial process is the key to diversity of antigen-binding proteins. Seven loci subject to rearrangement by V(D)J recombination have now been identified, and a great deal is known about the highly complex and distinctive organization of the endogenous genes. Gene assembly occurs within a restricted period of early B-and T-cell development and in a preferred temporal order within each lineage. [Key Words: V(D)JDNA sequence motifs, found adjacent to all V-, D-, and J-coding elements at every locus known to rearrange, were implicated in recombination because of their strategic locations

P1 ParA is a member of the Walker-type family of partition ATPases involved in the segregation of plasmids and bacterial chromosomes. ATPases of this class interact with DNA non-specifically in vitro and colocalize with the bacterial nucleoid to generate a variety of reported patterns in vivo. Here, we directly visualize ParA binding to DNA using total internal reflection fluorescence microscopy. This activity depends on, and is highly specific for ATP. DNA-binding activity is not coupled to ATP hydrolysis. Rather, ParA undergoes a slow multi-step conformational transition upon ATP binding, which licenses ParA to bind non-specific DNA. The kinetics provide a time-delay switch to allow slow cycling between the DNA binding and non-binding forms of ParA. We propose that this time delay, combined with stimulation of ParA's ATPase activity by ParB bound to the plasmid DNA, generates an uneven distribution of the nucleoid-associated ParA, and provides the motive force for plasmid segregation prior to cell division.

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