The pattern of gamma-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.

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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...

MRE11 and RAD50 are known to be required for nonhomologous joining of DNA ends in vivo. We have investigated the enzymatic activities of the purified proteins and found that Mre11 by itself has 3' to 5' exonuclease activity that is increased when Mre11 is in a complex with Rad50. Mre11 also exhibits endonuclease activity, as shown by the asymmetric opening of DNA hairpin loops. In conjunction with a DNA ligase, Mre11 promotes the joining of noncomplementary ends in vitro by utilizing short homologies near the ends of the DNA fragments. Sequence identities of 1-5 base pairs are present at all of these junctions, and their diversity is consistent with the products of nonhomologous end-joining observed in vivo.