THE ENZYMATIC REPAIR OF DNA, I. FORMATION OF CIRCULAR XDNA* BY MALCOLM L. GEFTER, ANDREW BECKER, AND JERARD HURWITZ DEPARTMENT OF DEVELOPMENTAL BIOLOGY AND CANCER, ALBERT EINSTEIN COLLEGE OF MEDICINE, BRONX, NEW YORK Communicated by Alfred Gilman, April 19, 1967 Physical and genetic studies on bacteriophages indicate that genetic recombina- tion involves the breakage and reunion of DNA molecules." 2 It has been postu- lated that the process of reunion occurs first by the formation of a joint structure containing portions of the parental DNA's linked together by noncovalent bonds, and second by the formation of a phosphodiester "seal" between the internal ter- mini of DNA chains held together in this manner.2 In our search for the enzyme(s) responsible for the covalent joining of polynucleotides, we anticipated that phage XDNA, in the hydrogen-bonded circular form ("Hershey circle"),3 might provide a model system for studying the enzymatic fate of DNA termini held in apposition by hydrogen bonds. In agreement with the results reported by Gellert,4 it was found that extracts of Escherichia coli catalyzed the formation of covalently closed circles from hydrogen-bonded circles of XDNA. We have purified this enzyme activity from E. coli K12 approximately 2000-fold. The enzyme also catalyzes the conversion of XDNA containing single-strand breaks to a form which, in al- kaline sucrose gradients, sediments in a manner indistinguishable from intact XDNA. The enzyme requires double-stranded DNA as substrate and is dependent on a heat-stable factor. Similar results have been observed by Olivera and Leh- man.5 Materials and Methods.-Bacterial strains and bacteriophages: E. coli K12 strain 1100 (endonu- clease I-deficient) was obtained from Dr. S. E. Luria. E. coli CR34 thymine- was obtained from Dr. M. Yarmolinsky. E. coli strains AB2463 (recombination-deficient) and other rec- and excision-negative strains were obtained from Dr. P. Howard-Flanders. E. coli strains B, B/r (radiation-resistant), B,,-, and B,,2 (radiation-sensitive) were obtained from Dr. E. Witkin. Preparation of C'4-labeled XDNA: C"4-thymidine-labeled XDNA was prepared6 from Xvir grown in E. coli CR34 thy-. The DNA had a specific activity of 3.2 X 103 cpm per mAmole of DNA nucleotide. For assay of covalent circle formation, "Hershey circles" were prepared according to Hershey et al.3 (contaminated with 20-40% linear molecules). Preparation of DNA containing single-strand breaks ("nicked DNA"): Reaction mixtures (0.3 ml) containing 10 miumoles C14-XDNA, 33 .umoles NaCl, 3.3 jtmoles Tris-HCl buffer, pH 7.5, 0.2 jAmoles MgC12, and 5 mlg pancreatic DNase (Worthington Biochemical Corp.) were incubated at 250C for 25 min. The reaction was stopped by heating the reaction mixture at 550 for 5 min. Preparation of P32-labeled 6'-phosphate-terminated DNA: Calf thymus DNA was phosphorylated at 5'-hydroxyl termini by treatment with 5'-hydroxyl polynucleotide kinase and -y-P32-adenosine 5'-triphosphate (ATP) (3100 cpm per uAumole) as previously described,7 with the exception that reaction mixtures were incubated with 0.5 mg of alkaline phosphatase at 650 for 60 min8 for each .5 Mmoles of DNA. Preparation of circular xDNA in vivo: The procedure used for the isolation of P32-twisted circular XDNA (superhelix) was that of Meselson.9 This shear-resistant superhelix species of DNA was isolated by preparative sedimentation in "neutral sucrose." The DNA isolated sedi- mented 3.8 times faster than linear XDNA and 1.9 times faster than linear XDNA in "alkaline" and "neutral" sucrose gradients, respectively, as reported by Bode and Kaiser.'0 With increas- ing time, structural changes in the superhelix became evident with the appearance of a DNA 240 Downloaded by guest on October 2, 2021 VOL. 58, 1967 BIOCHEMISTRY: GEFTER, BECKER, AND HURWITZ 241 species that sedimented in "neutral" sucrose gradients 1.13 times faster than linear XDNA.1' The new species presumably represented untwisted circular XDNA, generated by single-strand breaks in the superhelix due to decay of p32 as reported by Ogawa and Tomizawa."1 5'-Hydroxyl polynucleotide kinase was purified from T4 am 82-infected E. coli B.7 12 Zone centrifugation in sucrose density gradients: Sedimentation of XDNA was carried out in a 5-20% sucrose gradient. For sedimentation under alkaline conditions, the sucrose was dissolved in 0.7 M NaCl, 0.3 M NaOH, and 10-3 M ethylenediaminetetraacetate (EDTA) ("alkaline sucrose"). For detection of double-stranded covalent XDNA circles, gradients were centrifuged at 38,000 rpm for 70 min in the SW39 Spinco rotor at 50C. When sedimentation studies were carried out under neutral conditions, the sucrose was dissolved in 0.01 AI Tris-HCl buffer, pH 7.5, 0.1% sodium dodecyl sulfate (SDS), and 10-3 M EDTA ("neutral sucrose"). Enzyme assays: Three methods were used to measure the sealing reaction. These included (a) the conversion of 5'-p32 termini of DNA to an alkaline phosphatase-resistant form;"' (b) the con- version of "nicked" DNA to intact DNA measured in "alkaline sucrose" gradients; and (c) the conversion of "Hershey circle" XDNA to a form sedimenting 4 times faster than linear XDNA in "alkaline sucrose." In the latter assay, reaction mixtures (0.3 ml) contained 20 Jhmoles Tris buffer, pH 7.5, 2,Amoles MgCl2, 2,umoles dithiothreitol, 50 mymoles of ATP, 2.5 m/umoles of each of the 4 deoxynucleoside triphosphates, 80 mjumoles of sRNA (with crude preparation), 1.7 mjumoles of "Hershey circle" C14-labeled XDNA, and enzyme. Reactions were incubated at 250 for 10 min and then halted by the addition of 0.065 ml of a mixture containing 1.15 M NaOH, 0.31 M EDTA, and 1.85 M NaCl. The mixture was layered on top of 5.2 ml of a 5-20% "alka- line sucrose" gradient and centrifuged as described. Fractions were collected into Bray's solution"4 from a hole pierced in the bottom of the centrifuge tube and counted in a Packard scintillation counter. The radioactivity in a discrete peak, sedimenting 3.8-4 times faster than the starting material, was considered the circular DNA product. One unit of enzyme converted 1 mnjmole of "Hershey circle" XDNA nucleotide to a rapidly sedimenting form under the above conditions. The amount of the rapidly sedimenting product formed was proportional to the enzyme concen- tration up to 65-70%" conversion of the input DNA. TABLE 1 PURIFICATION OF SEALASE FROM E. coli K12 Fraction Total activity Spec. act. Crude extract 6250 2.2 Streptomycin sulfate supernatant (ASI) 5900 7.3 AlCy gel (ASII) 4560 60 DEAE-cellulose (ASIII) 2800 184 ASIII heated 2450 490 Bio-Rad 70 1175 4600 Purification of sealase (crude extract): All buffers, unless otherwise mentioned, contained 0.01 M 2-mercaptoethanol and 10-3 M EDTA, and all operations were carried out at 0-40C. E. coli K12 strain 1100 (45 gm) was ground with 90 gm of alumina A-301. The paste was then extracted with 180 ml of 0.05 M Tris buffer, pH 7.5, and 0.01 M MgCl2, and centrifuged at 100,000 X g for 40 min. The highly viscous supernatant solution was decanted. Streptomycin sulfate precipitation followed by ammonium sulfate concentration: The crude ex- tract (160 ml) was diluted to 200 ml with the above buffer and treated with 67 ml of 5% strepto- mycin sulfate; after 15 min at 00, the mixture was centrifuged. The supernatant (250 ml) was treated with 56.5 gm of ammonium sulfate (40%), and the precipitate removed by centrifuga- tion at 10,000 X g. The supernatant (265 ml) was treated with 40.5 gm of ammonium sulfate 65%, and the precipitate collected and dissolved in 16 ml of 0.02 M Tris buffer, pH 7.5 (ASI). Alumina Cy gel treatment: The ASI fraction was passed through a G-25 Sephadex column (3 X 60-cm) previously equilibrated with 0.02 M Tris-HCl buffer, pH 7.5. The protein fractions were pooled, diluted to 133 ml (3 mg of protein/ml), and treated with 50 ml of alumina CG gel suspension (17.7 mg of solids per ml). After 30 min, the gel was collected by centrifugation and washed successively with 65 ml of 0.1 M ammonium sulfate, pH 7.5, and 65 ml of 0.35 M am- Downloaded by guest on October 2, 2021 242 BIOCHEMISTRY: GEFTER, BECKER, AND HURWITZ PROC. N. A. S. monium phosphate buffer, pH 7.5. The latter fraction was adjusted to 70% with solid ammonium sulfate and the precipitate dissolved in 0.02 M Tris buffer, pH 7.5 (ASH). O-(Diethylaminoethyl) cellulose (DEAE-cellulose) chromatography: The ASH fraction (8 ml) was passed through a G-25 Sephadex column (3 X 40-cm) previously equilibrated with 0.02 M Tris buffer, pH 8.0. The desalted fraction was applied to a DEAE-cellulose column (3 X 18-cm) previously equilibrated with the above buffer, and the column was washed with 50 ml of 0.02 M Tris, pH 8.0, 100 ml of 0.08 M ammonium phosphate pH 7.5, 100 ml of 0.2 M ammonium phos- phate, pH 7.5, and 100 ml of 0.5 M ammonium phosphate, pH 7.5. Ten-ml fractions were col- lected. The active fractions were eluted with 0.5 M phosphate buffer, pooled (67 ml), and ad- justed to 70% with ammonium sulfate. The precipitate was collected by centrifugation and dis- solved in 4 ml of 0.05 M Tris buffer, pH 7.5 (ASIII). The sealase activity was further purified by heating the ASIJI fraction at 600 for 5 min fol- lowed by rapid cooling.