The Enzymatic Repair of Dna, I. Formation of Circular Xdna* by Malcolm L

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 recombination 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 termini 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 alkaline 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 sedimented 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 increasing time, structural changes in the superhelix became evident with the appearance of a DNA 240 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 conversion 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% "alkaline 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 concentration 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 extract (160 ml) was diluted to 200 ml with the above buffer and treated with 67 ml of 5% streptomycin 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 centrifugation 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- 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 phosphate, pH 7.5, and 100 ml of 0.5 M ammonium phosphate, pH 7.5. Ten-ml fractions were collected. The active fractions were eluted with 0.5 M phosphate buffer, pooled (67 ml), and adjusted to 70% with ammonium sulfate. The precipitate was collected by centrifugation and dissolved 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 followed by rapid cooling. Denatured protein was removed by centrifugation (ASIJI heated), Bio-Rad 70 chromatography: The heated ASIII fraction was dialyzed against a total of 1500 ml of 0.02 M sodium succinate buffer, pH 6.5, over a 3-hr period. This was added to a Bio-Rad 70 column (3 X 15-cm) previously equilibrated with 0.02 M sodium succinate buffer, pH 6.5. The column was washed with 50-ml vol of 0.05 M sodium succinate buffer, pH 6.5, 0.05 M sodium succinate buffer, pH 6.5, + 0.05 M NaCl, 0.05 M sodium succinate buffer, pH 6.5, + 0.1 M NaCl, and 0.05 M sodium succinate buffer, pH 6.5, + 0.2 M NaCl. The enzyme activity was recovered in the latter part of the 0.05 M sodium succinate buffer, pH 6.5, + 0.1 M NaCl eluate. The active fractions were pooled and dialyzed for 12 hr against 50 vol of 50% glycerol, which concen- trated the enzyme solution 5- to 7-fold and stored at -10°. At the stage of ASIII, DNA polymerase activity was detected with dAT copolymer as primer (e.g., 1 mumole of dTMP incorporation per mg protein in 30 min at 380). The ASIII-heated fraction contained no demonstrable DNA polymerase activity using 20 jig of protein. Results.-Properties of the product formed: (a) Sedimentation in "alkaline sucrose": The sedimentation profile of reaction products using "Hershey circle" XDNA as a substrate is shown in Figure 1. Increasing concentrations of enzyme yielded more of the rapidly sedimenting DNA with a concomitant decrease in DNA sedimenting as linear molecules. The product sediments four times faster than linear XDNA as reported for a collapsed circular species of XDNA10 11 and by Gellert4 for the circular product formed in vitro. (b) Sedimentation in "neutral sucrose": The sedimentation profile of reaction products was examined in neutral sucrose gradients. Reactions were terminated by heating at 750 for 3 minutes followed by rapid cooling. P32-labeled, twisted and untwisted, circular XDNA was introduced as a marker (Fig. 2). The twisted circle isolated in vivo and the untwisted circle sediment 1.9 and 1.19 times faster than linear XDNA, respectively. The twisted circle sediments 1.7 times faster than the untwisted form. Reaction mixtures lacking enzyme resulted in the pattern shown in Figure 2A. Incubation of XDNA with sealase resulted in the conversion of part of the C14-XDNA substrate to a form which cosedimented with the circular untwisted P32-DNA marker (Fig. 2B). Reaction mixtures which were subjected to shear by several passages through a no. 26-gauge needle, prior to layering on the gradient, resulted in a preferential breakage of linear molecules, thereby permitting a clearer demonstration of a circular form of XDNA (Fig. 2C). The sedimentation profile of the starting substrate, product, and P32-linear XDNA were also compared in neutral sucrose (Fig. 3). A portion of the C14-XDNA substrate (--30%) cosediments with linear DNA (Fig. 3A), while the majority of the C14-XDNA sediments 1.13 times faster than the linear marker.3 In addition to these species, other hydrogen-bonded forms of XDNA are also evident. The results described below indicate that the product formed has the same sedimentation VOL., 58, 1967 BIOCHEMISTRY: GEFTER, BECKER, AND HURWITZ 243 profile as "Hershey circle" XDNA. Both the C14-XDNA substrate and P32-linear 1200 _ No enzyme XDNA sediment congruently after heat 800So treatment at 750 (Fig. 3B). In contrast, the enzymatic product, after heat treat- 400_ ment, sediments 1.13 times faster than linear DNA. In addition, the aggregated forms of XDNA also resist the heat treat- E 200 tEnzyme(.57unit) ment and probably represent forms which , have also been covalently sealed. > 800 These results suggest that the product of 0 the sealase reaction has the sedimentation D400 characteristics of an untwisted circular form of XDNA; products formed with crude + Enzyme(1.42units) extracts are similarly untwisted. 1200 _ Requirements of the enzyme: At the ASIJI 800S - stage, the sealing reaction is dependent on "Hershey circle" XDNA and Mg++ (Table 400_ 2). Linear XDNA is not converted to covalent circles. Variable stimulation due to 5 10 15 20 25 30 the addition of ATP and the four deoxy- FRACTION NUMBER nucleoside triphosphates was observed using FIG. 1.-Effect of enzyme concentration less purified fractions.16 In contrast to the on the formation of C'4-labeled xDNA circles. Three separate reaction mix- ASIJI fraction, the most highly purified tures, each containing 2.5 m4moles of enzyme fraction was dependent on the ad- "Hershey circle" XDNA were incubated in the presence of and 1.42 units dition of heated crude extract. The latter of sealase, respectively.0, 0.57, Conditions for was not replaced by ribo- or deoxynucleoside reaction, and "alkaline sucrose" sedimentation were as described in Materials and triphosphates alone or in combination. Methods. Sedimentation is from right to Extent of XDNA closure: With rela- left. tively high concentrations of enzyme, the reaction proceeded until 30 per cent of the total DNA was converted to a circular form. The addition of more enzyme at this point did not cause a significant in- crease in covalent circle formation; the addition of more DNA, however, caused an immediate further appearance of product. The amount of covalent circle formed after the second addition of DNA was twice that observed in the first stage of the incubation. Sealing of "nicked DNA": The requirement of "Hershey circles" as substrate for alkali-resistant circle formation indicated the need for a double-stranded duplex in the reaction. Similar observations have been made with linear XDNA. DNA was pretreated with pancreatic DNase (described above) in a manner which did not alter the sedimentation profile of the DNA in "neutral sucrose." However, as revealed in alkaline sucrose gradients, there was marked evidence of breakage (Fig. 4). Such substrates. when treated with sealase (ASIII), were partially repaired; and addition of heated crude extract (100° for 10 min) resulted in repair of nearly all single-strand breaks present in the XDNA molecules. Similar studies with heat- denatured "nicked XDNA" gave no evidence of repair, indicating a requirement for a duplex structure. When linear XDNA (300 mjumoles) was treated with exonuclease III (sufficient G OL 4i

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3 5 10 IS 20 25 30 35 FRACTIsFRACTION20A5WRNUMBER ~~~~~~~FRACTION NUMKR (LReft) FIG. 2.-Sedimentation of various species of XDNA in "neutral sucrose." The dashed line represents P3 radioactivity of superhelical DNA and its decay products. (A) The sedimentation pattern of Ct4linear XDNA or DNA isolated from a standard reaction mixture lacking enzyme (see "enzyme assay") which were heated as described in the text. (B) The sedimentation pattern of DNA treated with sealase as described in the text; (C) is the same as (B), except that the reaction mixture was subjected to mechanical shear prior to sedimentation. Centrifugation was for 105 min at 150at 37,000 rpm. (Right) FIG. 3.-Sedimentation of XDNA substrate and product in "neutral sucrose." In (A), (B), and (C), the dashed line represents P32 radioactivity of linear XDNA. (A) The sedimentation pattern of the DNA substrate prepared as described in Materials and Methods, and incubated in a standard reaction mixture without enzyme. (B) The same as (A), except the reaction mixture was heated prior to centrifugation. (C) The same as (A), except enzyme was included and following incubation the reaction mixture was heated. Centrifugation was for 105 min at 250 at 50,000 rpm. VOL. .58, 1967 BIOCHEMISTRY: GEFTER, BECKER, AND HURWITZ 245

TABLE 2 REQUIREMENTS FOR CIRCULAR XDNA FORMATION Circular XDNA formed Enzyme fraction Additions (mpmoles/10 min) ASIII 1. Complete 0.30 2. Complete; omit Mg++ 0.023 3. "Hershey circle" XDNA heated at 750, for 3 mm 0.027 4. Complete + RNase (1 ;g) 0.41 5. Complete + DNase (0.1 pg) <0.02 6. Complete + ATP + deoxynucleoside triphosphates 0.38 7. Complete + boiled crude extract 0.25 Bio-Rad 70 1. Complete + boiled crude extract 0.67 2. Complete + ATP + deoxynucleoside triphosphates 0.031 3. Complete 0.033 4. Omit sealase + boiled crude extract <0. 02 The complete system (0.3 ml) contained: 20 pmoles Tris buffer, pH 7.5, 2 pmoles MgCI2, 1.7 mpmoles C14 "Hershey circle" XDNA, 2 pmoles dithiothreitol, and 2.8 pg of ASIII. Reactions were incubated at 250 for 10 min. Where indicated, 50 mpmoles of ATP and 2.5 mpmoles of each of the 4 deoxynucleoside triphosphates were added. In the experiment with the Bio-Rad 70 enzyme fraction, 0.15 #g of protein was added with 3.2 mpmoles of C14 "Hershey circle" XDNA; boiled crude extract was prepared by heating crude extracts for 5 min at 1000. After cooling, the mixture was centrifuged and 0.005 ml of the supernatant solution was added as boiled crude extract. to remove 20 /4tmoles of nucleotide residues) and then converted to "Hershey circles," it was inactive in the sealase reaction. However, when the four deoxynucleoside triphosphates and DNA polymerase were present with sealase, covalent circular XDNA was formed. The nucleotide requirement was almost completely satisfied by dATP + deoxythymidine triphosphate (dTTP) (70%) suggesting that AT-rich regions are clustered near the 3'-hydroxyl termini in contrast to the GC cluster near the 5'-phosphate end.17 These results suggest that DNA polymerase can repair internal single-stranded regions of a DNA duplex and, in conjunction with sealase, can lead to a complete repair and joining of DNA fragments. Evidence of phosphodiester bond formation during sealing: Polynucleotide kinase products (see Methods) were exposed to the action of sealase, and following treatment with alkaline phosphatase, the material was degraded as summarized in Table 3. Aliquots of the nuclease digest were separated by electrophoresis and the distribution of p32 was determined. The labeled 5'-mononucleotides are predom- inantly pA and pT ;3, 4however, the sealase reaction has resulted in a distribution of

TABLE 3 DISTRIBUTION OF p32 AMONG DEOXYMONONUCLEOTIDES P32 in Deoxymononucleotides C A G T Degradatiion of DNA Distribution (%) DNase + ven(om diesterase 6 45 2 47 Micrococcal nuclease + spleen diesterase 31 23 24 22 Reaction mixtures (1 ml) containing 208 mpmoles of 5'-P3t-labeled calf thymus DNA (5.07 X 106 cpm; spec. act. of 5 phosphate termini, 2400 cpm per ~ppmole), 30 pmoles Tris buffer, pH 7.5, 8 pmoles MgCl2, 0.5 Emole of dithiothreitol, 0.1 ml boiled crude extract, and 0.35 pig of sealase (Bio- Rad 70 fraction) were incubated for 45 min at 250; the reaction was halted by the addition of 3 pmoles of p-hydroxymercuribenzoate (which quantitatively inhibited sealase activity) neutralized to pH 9.2 with NaOH and treated with 500 pg of alkaline phosphatase for 1.5 hr at 650. This procedure yielded 4.7 ppIumoles of alkaline phosphatase-resistant p32. The reaction mixture was then treated with 20 pmoles of 2-mercaptoethanol and heated at 100° for 15 min to inactive alkaline phosphatase. Approximately 2 ppsmoles of P32-labeled product was used for each of the above enzymatic degradation studies as previously described.7 The mononucleotides were separated by high- voltage electrophoresis (6000 v) for 2 hr at pH 3.5 in 0.05 M citrate buffer. No detectable radioactivity was noted past the Tp marker in electrophoretic separations carried out with digests formed after the action of micrococcal nuclease and spleen diesterase. This indicated the absence of nucleoside diphosphates. 246 BIOCHEMISTRY: GEFTER, BECKER, AND HURWITZ PROC. N. A. S.

Position of A TABLE 4 0 ntoct A DNA ! J. DISTRIBUTION OF SEALASE IN STRAINS OF E. coli _ | Activity 'l_ ~~~~~~~~~~~~(mumoles/10min Crude extract from E. coli strain mg protein) 800_ B 0.074 1000 ^ B,,_1 0.072 Bs2 0.030 600 _ B/r 0.95 AB1157 (uvr+ rec+) 1.08 400-_ _ AB2463 (uvr+ rec- 13) 0.70 AB2470 (uvr+ rec- 21) 0.20 200 _1 AB1186 (uvr A-rec+) 0.47 I ,Reaction] mixtures were as described in Materials and I |* * I & |Methodst and included sRNA.

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Enzyme (0 87 unit) C 1000 _Boiled extract i2 15 FIG. 4.-Sedimentation of "nicked" XDNA substrate and product in alkaline Soo _ _ sucrose. (A) The sedimentation pattern of "nicked" XDNA substrate prepared as 600 _ described in Materials and Methods; (B) the sedimentation pattern of "nicked". with as de- 4000-rL,XDNA pretreated enzyme L scribed in the text; (C) the same as (B), except heated crude extract (0.005 ml) 200 - _J ot _ was included in the reaction mixture. Centrifugation was for 100 min at 25° at .1 rpm. 5 loaIs 20 25 30I ' 50,000E sL FRACTION NUMBER p32 to all four 3'-deoxymononucleotides, indicating internal phosphodiester bond formation. Distribution of sealase activity in strains of E. coli: All strains of E. coli examined contained sealase activity. The sealase activity is relatively low in strains of E. coli B and more abundant in E. coli K12 strains. The enzyme activity is present in the recombination-deficient strains (Table 4) as already observed by Gellert.4 Strain B/r contains a level of enzyme similar to the K(12 strains, while strains Bs-1 and Bs-2 do not have appreciably lower levels of enzyme than strain B. Discussion and Summary.-An enzyme which catalyzes the formation of covalent circular DNA from hydrogen-bonded circular DNA has been purified approximately 2000-fold from extracts of E. coli K12. The product formed after action of the enzyme on hydrogen-bonded circular XDNA has the following properties: (1) It sedimented 3.8-4 times faster than linear XDNA in "alkaline sucrose" gradients. (2) In "neutral sucrose" gradients, it VOL. 58, 1967 BIOCHEMISTRY: GEFTER, BECKER, AND HURWITZ 247 sedimented 1.13 times faster than linear XDNA. This sedimentation property indicates circularity is achieved in vitro in the absence of twisting in contrast to the product formed in vivo. (3) The product of sealase action on "Hershey circle" DNA renders the DNA resistant to the action of exonuclease I after heat treatment at 1000 for 4 minutes. (4) The product shows a higher degree of resistance to mechanical shear than linear XDNA. The sealase reaction is not limited to the formation of covalent circular DNA but also catalyzes the repair of "nicked" DNA. A condition necessary for repair is that the DNA exist as a duplex structure. Denaturation of "nicked" DNA or the conversion of "Hershey circles" to a linear form inactivates these compounds as substrates in the reaction. The reaction has a requirement for a heat-stable cofactor for all of the joining reactions studied. In contrast to the sealase activity from E. coli K12, a T4 phage- induced sealase activity, extensively purified, is satisfied specifically by ATP.18' 19 This enzyme, which has been purified approximately 500-fold, catalyzes all of the reactions described here.'9 The distribution of the enzyme activity in various strains suggests that the enzyme may play an important role in the repair of radiation-damaged DNA. The enzyme activity is present in relatively high concentrations in radiation-resistant strains tested. Note added in proof: In agreement with Olivera and Lehman,5 the requirement for the boiled extract is satisfied by DPN in all reactions studied. This to be contrasted with the absolute requirement for ATP when these reactions are catalyzed by the T-even phage-directed sealase. * This work was supported by grants from the National Institutes of Health, the National Science Foundation, the New York City Research Council, and the American Cancer Society. 1 Meselson, M., J. Mol. Biol., 9, 734 (1964). 2'Aknraku, N., and J. Tomizawa, J. Mol. Biol., 11, 501 (1965). 3 Hershey, A. D., E. Burgi, and L. Ingraham, these PROCEEDINGS, 49, 748 (1963). 4 Gellert, M., these PROCEEDINGS, 57, 158 (1967). 6 Olivera, B. M., and I. R. Lehman, these PROCEEDINGS, 57, 1426 (1967). We are indebted to Dr. Lehman for communicating his results to us prior to publication. 6 Kaiser, A. D., and D. S. Hogness, J. Mol. Biol., 2, 392 (1960). 7 Novogrodsky, A., and J. Hurwitz, J. Biol. Chem., 241, 2923 (1966). 8 Dr. C. C. Richardson informed us that treatment with alkaline phosphatase at elevated temperatures results in a dephosphorylation of the 5'-termini present in internal breaks in DNA. 9 Mesels6n, M., personal communication. 10 Bode, V. C., and A. D. Kaiser, J. Mol. Biol., 14, 399 (1965). "Ogawa, H., and J. Tomizawa, J. Mol. Biol., 23, 265 (1967). 12 Becker, A., and J. Hurwitz, J. Biol. Chem., 242, 963 (1967). 13 Becker, A., and J. Hurwitz, Federation Proc., 25, 276 (1966). 14 Bray, G. A., Anal. Biochem., 1, 279 (1960). 15 The extent of conversion of "Hershey circle" XDNA to covalent circular DNA decreased with purification of enzyme. The latter may be due to the removal of DNA polymerase, suggesting that some of the DNA may have "gaps" which can be repaired by the action of DNA polymerase. 16 Becker, A., M. L. Gefter, and J. Hurwitz, Federation Proc., 26, 395 (1967). 17 Strack, H., and A. D. Kaiser, J. Mol. Biol., 12, 36 (1965). "8 Weiss, B., and C. C. Richardson, Federation Proc., 26, 395 (1967). 19 Hurwitz, J., M. L. Gefter, G. Lyn, and A. Becker, unpublished observations.