The ultraviolet endonuclease of bacteriophage T4. Further characterization.

The T4 ultraviolet endonuclease was previously shown to produce strand incisions (nicks) in ultraviolet-irradiated DNA on the 5' side of thymine dimers. The present studies demonstrate that the purified endonuclease creates 3'-OH and 5'-P termini at the sites of nicking. Photoreactivation of ultraviolet-sensitive sites, thereby demonstrating directly endonucleause has a molecular weight of approximately 18,000 and attacks ultraviolet-irradiated single-stranded Escherichia coli and M-13 DNA.

studies demonstrate that the purified endonuclease creates 3'-OH and 5'-P termini at the sites of nicking. Photoreactivation of ultraviolet-irradiated DNA in vitro results in a loss of endonuclease-sensitive sites, thereby demonstrating directly that dimers are required for substrate sites in DNA. The endonuclease has a molecular weight of approximately 18,000 and attacks ultraviolet-irradiated single-stranded Escherichia coli and M-13 DNA.
The mutant bacteriophage T4vl is defective in the v gene and is abnormally sensitive to ultraviolet radiation (1). In addition, no excision of thymine dimers is observed following infection of Eschmichia coli with this phage (2). The v gene has been shown to be the structural gene for an endonuclease that in vitro specifically introduces single strand breaks (nicks) into ultravioletirradiated double-stranded DNA (3-8). Unirradiated DNA is unaffected by incubation with the enzyme (3-8). We and others previously have purified and partially characterized this enzyme. It has been shown that the endonuclease activity has no requirement for divalent cation and is active in 10 mM EDTA. It has a broad pH optimum between 7.0 and 8.0 and is insensitive to inhibition by SH group inhibitors, caffeine, or tRNA (3)(4)(5)(6)(7)(8). In the present study we have determined the chemistry of the termini created by endonucleolytic incision of ultraviolet-irradiated DNA. In addition, we present evidence that pyrimidine dimers are recognized as substrate sites by the enzyme in single-stranded DNA. Finally, a molecular weight for the purified enzyme has been determined.

Materials
Enzymes T4 ultraviolet endonuclease (Fractions IV and V) were purified as previously described (4). The enzyme was stored at 4" in 10 mM Tris-HCl buffer, pH 8.0, with 3y0 polyethylene glycol and 0.1 mM EDTA.1 Purified exonuclease I (9) and bacterial alkaline phosphatase of Escherichia coli (10)  DNA Unlabeled and 8H-labeled T7 DNA were prepared by the procedure of Thomas and Abelson (13)

Gel Electrophoresis
Electrophoresis followed the procedure of Laemmli (16), except that it was performed in flat slabs of polyacrylamide gel approximately 1.8 mm X 10.0 cm X 14.5 cm. The stacking gel consisted of 3% acrylamide and 0.08% N, N'-methylenebisacrylamide in 125 mM Tris-HCl buffer, pH 6.7. The running gel consisted of 10%

RESULTS
Thymine Di.mers in DNA Are Substrate Sites-In an attempt to demonstrate directly that pyrimidine dimers are required to produce substrate sites in ultraviolet-irradiated DNA, the following experiment was carried out. I'hage T7 DNA was ultravioletirradiated at a fluence of 40 J per m2. This DNA was incubated in the presence and absence of Escherichia coli photoreactivating enzyme and the thymine dimer content of an aliquot of each was determined.
Under the incubation conditions described in the legend to Table I, 60% of the thymine dimers were monomerized. The rest of the DNA then was incubated with T4 ultraviolet endonuclease and sedimented in alkaline sucrose density gradients. As shown in Table I

Rate of incorporation of [*H]TMP into ultravioletirradiated
nicked and unnicked DNA: T7 DNA was prepared as described in the text and maintained in 50 mM Tris-HCI, pH 8.0. The DNA was ultraviolet-irradiated at 50 J per m2 and 41.25 nmol (as nucleotide) was incubated at 37" for 60 min with or without the addition of 120 units of T4 ultraviolet endonuclease (Fraction V) in a total volume of 0.5 ml. Both nicked and unnicked DNA (8.25 nmol) were preincubated with or without bacterial alkaline phosphatase (0.7 unite) in a total of 0.2 ml. Incubation was at 45" for 60 min. Each reaction tube then was supplemented with N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer (50 mu, pH 7.4), MgClt (5.0 mM), the four usual deoxynucleoside triphosphates (each at 0.05 mM), and [aH]TTP (0.5 pCi) and purified Eschetichia colz' DNA polymerase I (1.8 units). The final volume of the reaction mixtures was 0.3 ml. Control samples containing no DNA polymerase were included. Incubation was at 37" for the times indicated. Reactions were terminated by the addition to each tube of 1.0 ml of calf thymus DNA (0.5 mg per ml) in 0.1 M sodium pyrophosphate. Tubes were boiled for 2 min, quenched on ice and 1.3 ml of 10% cold trichloroacetic acid were added. The suspension was filtered through 2.4-cm GF/C glass filter discs which were washed with 5yo trichloroacetic acid and then with 95yo ethanol. Radioactivity was determined by counting the dried discs in 5.0 ml of scintillation mixture containing 5.04 g of Omnifluor per liter of toluene, in a liquid scintillation spectrometer. Triangles, ultraviolet-irradiated DNA plus endonuclease; circles, ultravioletirradiated DNA without endonuclease; closed symbols, bacterial alkaline phosphatase added; open symbols, no bacterial alkaline phosphatase.
precipitable DNA is significantly increased compared to unnicked ultraviolet-irradiated DNA. This result is unaffected by preincubation with bacterial alkaline phosphatase. These data indicate that 3'-OH termini are produced at the sites of endonucleolytic incision. Previous studies (26) have shown that incubation of nicked T7 ultraviolet-irradiated DNA with DNA polymerase J results in excision of thymine dimers. Thus the 5' terminus apparently is digested rather than displaced in this reaction. Further confirmation of the 3'-OH terminus is provided by the results shown in Figs. 2, 3, and 4. Fig. 2 demonstrates that the rate of degradation of specifically nicked and then denatured T7 DNA by purified exonuclease I of E. COG is significantly greater than that of mmicked ultraviolet-irradiated denatured DNA. This result, too, is essentially unaffected by preincubation with alkaline phosphatase. Since exonuclease I of E. coli specifically requires 3'-OH termini in single-stranded DNA, these data confirm the presence of 3'-OH sites produced by endonuclease treat- at 200 J per me and treated with T4 ultraviolet endonuclease (Fraction V) ss follows. Incubation mixtures (0.8 ml) contained 5.4 nmol of DNA (as nucleotide), 10 mre Tris-HCl buffer (pH 8.0), 1.0 mM EDTA, and 96 units of endonuclease or an equivalent volume of Tris-HCl buffer. Incubation was at 37" for 90 min following which the reaction tubes were boiled for 10 min and immediately quenched in ice. To 0.1 ml of each mixture containing 0.675 nmol of DNA the following were added: glycine buffer, pH 9.2,50 mr.r; MgClp, 5.0 mM; and 3.5 units of bacterial alkaline phosphatase or an equivalent volume of water. The final volume of this reaction mixture was 0.255 ml. Incubation was at 37" for 30 min, following which 58.4 units of exonuclease I of E. coli were added for the times indicated. Reactions were terminated by the addition of 0.1 ml of 1% bovine serum albumin and 0.4 ml of cold 10% trichloroacetic acid. The tubes, including controls with no exonuclease addition were centrifuged at 5,000 X g and the acid-soluble fraction saved. Radioactivity was determined by counting 0.4 ml of the acid-soluble fraction with 10.0 ml of the scintillation mixture described in the legend to Table I Fig. 3 shows the results of an experiment designed to demonstrate 5'-P termini in specifically nicked ultraviolet-irradiated DNA. Bovine spleen phosphodiesterase degrades denatured DNA with 5'-OH termini. DNA degradation is, however, inhibited by the presence of pyrimidine dimers (22, 23). As seen in the figure, the rate of degradation of ultraviolet-irradiated, nicked, and then denatured T7 DNA by spleen phosphodiesterase, is increased only when the DNA has been incubated with both photoreactivating enzyme and bacterial alkaline phosphatase to remove pyrimidine dimers and terminal phosphate groups. In control experiments T7 DNA that had been preincubated with micrococcal nuclease (which produces 5'-OH termini) and then denatured was extensively degraded by bovine spleen phosphodiesterase. These experiments indicate the presence of 5'-P termini at the sites of nicking by T4 ultraviolet endonucleaae and confirm that the nicks occur on the 5' side of the dimers. *H-labeled T7 DNA was ultraviolet-irradiated at 106 J per m* and incubated with or without T4 ultraviolet endonuclease (Fraction V). Incubations (0.5 ml) contained T7 DNA (50 nmol), EDTA (10 mM), p-chloromercuriphenyl sulfonic acid (1.0 mM), and endonuclease (120 units). Incubation was at 37" for 60 min, following which the DNA was extracted twice in cold buffered phenol and dialyzed extensively against 29.0 mu potassium phosphate buffer pH 7.2. A second incubation with or without photoreactivating enzyme was carried out in reaction volumes of 0.25 ml containing nicked or unnicked DNA (12.0 nmol). notassium nhosnhate buffer (20.0 mM. DH 7.21. EDTA (1.0 mM);'dithiothreitbl (6.1 mM), and photoreactivating enzyme of Escherichia cola' (918 unite or an equivalent volume of potassium phosphate buffer, pH 7.2). Incubation was for 2 hours at 35" using the photoreactivating conditions described in Table I. Following incubation, 0.04 ml aliquots of DNA treated with and without photoreactivating enzyme were placed into thick walled hvdrolvsis vials together with 0.5 ml 10 mM Tris-HCl buffer CDH 8:0), 0.55 ml of l$!&bovine serum albumin, and 0.6 ml of cold I$" trichloroacetic acid. The tubes were centrifuged at 6,000 X g and the supernatant was discarded. The thymine dimer content of the precipitate was measured as previously described (17), and it was determined that incubation with photoreactivating enzyme caused monomerization of 87% of the thymine dimers. The DNA samples were dialyzed against 10 mM Tris-HCl buffer (pH 8.0) and incubated at 60" for 45 min with or without the addition of 3.5 units of bacterial alkaline phosphatase. The tubes then were boiled for 10 min and rapidly quenched in ice. Each of the four DNA samples (treated with or without photoreactivating enzyme and alkaline phosphatase) was divided into 0.04-ml aliquots to which were added 0.15 ml of 0.2 M sodium acetate buffer pH 5.0 and 0.075 units of bovine spleen phosphodiesterase.
Incubation was at 37" for the times indicated. Reactions were terminated by the addition of 0.05 ml of 1% bovine serum albumin and 0.25 ml of 10% cold trichloroacetic acid. The tubes were centrifuged at 5,000 '2 g and 0.25 ml of the acid-soluble fraction was used for determination of radioactivitv.
Closed Finally, Fig. 4 demonstrates that phosphodiester bond breaks created in ultraviolet-irradiated double-stranded DNA by incubation with T4 ultraviolet endonuclease can be rejoined by incubation with T4 polynucleotide ligase. The data shown in the figure are from an experiment in which the nicked DNA was pretreated with photoreactivating enzyme in order to monomerize dimers. Calculation of the weight average molecular weight of the DNA from the sedimentation profiles indicates that approxi- was ultravioletirradiated at 50 J per ml, and then incubated in a reaction volume of 0.50 ml with T4 ultraviolet endonuclease (Fraction V,, 120 units), EDTA (10 mM), and p-chloromercuriphenyl sulfonic acid (1.0 mM). Incubation was at 37" for 1 hour following which the DNA was extracted in cold buffered phenol at pH 7.0 and extensively dialyzed against 20 mM potassium phosphate buffer (pH 7.2). The DNA was incubated with or without photoreactivating enzyme of Escherichia coli. The incubation mixture (0.5 ml) contained nicked DNA (22.5 nmoles), potassium phosphate buffer (pH 7.2, 20 mM), EDTA (1.0 mM), dithiothreitol (0.1 mM), and either photoreactivating enzyme (918 units) or an equivalent volume of potassium phosphate buffer. Incubation was at 37" for 2 hours. The DNA was again phenol extracted and dialyzed against 10 mM Tris-HCl buffer (pH 8.0). Incubation with T4 DNA ligase was carried out in a reaction volume of 0.2 ml and contained nicked DNA (preincubated with or without photoreactivating enzyme, 4.5 nmoles), Tris-HCl buffer (pH 8.0, 50 mM), MgClz (4.0 mM), ATP (0.2 mru), dithiothreitol (10.0 mM), bovine serum albumin (5Opg per ml), and DNA ligase (2 units). Incubation was at 20" for 1 hour. Reactions were terminated by the addition of 0.02 ml of 0.1 M EDTA. A total of approximately 10,000 cpm of radioactivity contained in 0.05 ml of each incubation mixture was layered onto a 5 to 20y0 alkaline sucrose gradient containing 0.8 M NaCl and 0.2 N NaOH. The gradients were sedimented in a SW 56 rotor at 40,000 rpm for 210 min at 20". The radioactivity in 8-drop fractions was determined as described in the legend to Table I mately 50% of the endonucleolytic incisions were rejoined in the presence of DNA ligase. Qualitatively similar results (data not shown) have been obtained without photoreactivation, indicating that T4 polynucleotide ligase can seal nicks adjacent to pyrimidine dimers at 20". These results confirm the presence of 3'-OH and 5'-P termini. Future studies are aimed at detailing the kinetics of the joining reaction in the presence and absence of dimers in the DNA. Degradation of Single-stranded DNA by T4 Ultraviolet Endonuclease-Evidence is presented that purified T4 ultraviolet endonuclease attacks ultraviolet-irradiated but not unirradiated single-stranded DNA. Fig. 5 shows the sedimentation profiles of denatured E. coli DNA and demonstrates that ultravioletirradiated DNA incubated with T4 ultraviolet endonuclease sediments at a significantly slower rate than such DNA without endonuclease incubation. Further experiments (data not shown) demonstrate a linear relationship between total ultraviolet Escherichia coli DNA by T4 ultraviolet endonuclease. 3H labeled E. coli DNA (120 nmol per ml) was diluted 1:5 into 0.2 N NaOH and allowed to stand at room temperature for 15 min. The DNA then was dialyzed in the cold against 1.0 mM Tris-HCl buffer (pH 8.0) and ultraviolet-irradiated at 21.0 J per m2. The DNA was incubated with T4 ultraviolet endonuclease as follows. Reaction mixtures (0.5 ml) contained denatured DNA (unirradiated or ultravioletirradiated at the fluence indicated, 1.2 nmol), Tris-HCl buffer (pH 8.0,lO mM), EDTA (1.0 mM), and T4 ultraviolet endonuclease (Fraction V), 60 unite. Incubation was at 37" for 15 min following which tubes were placed on ice. An aliquot (0.1 ml) of each tube containing about 14,000 cpm of radioactivity was placed onto a 5 to 26Q/, alkaline sucrose gradient prepared as indicated in the legend to Table I. Sedimentation was at 38,000 rpm for 3 hours in a SW 56 rotor at 20". Fractionation of the gradients and measurement of radioactivity were carried out as described in the legend to fluence to the DNA and the extent of endonucleolytic degradation. Although E. coli DNA was denatured in alkali and then neutralized in low ionic strength in order to facilitate incubation with enzyme, we were concerned about the possibility that sufficient renaturation may have occurred so that pyrimidine dimers attacked by the endonuclease were actually in regions of DNA with a duplex conformation. In order to minimize this potential problem, we carried out experiments using M-13 DNA from aH-labeled purified phage. The DNA was maintained and incubated at a maximal ionic strength of 50 mM, under which conditions it is believed to exist primarily in the single-stranded form (24). Fig. 6 shows that unirradiated M-13 DNA incubated with T4 ultraviolet endonuclease does not undergo degradation detectable by sedimentation in cesium chloride velocity gradients, while ultraviolet-irradiated DNA does.
Further evidence supporting the ability of the T4 ultraviolet endonuclease to recognize pyrimidine dimers in single-stranded DNA is provided by the data shown in Table II. The table shows a correlation between the number of endonucleolytic incisions calculated from the fraction of "P label in M-13 DNA rendered acid-soluble during incubation with endonuclease, and the cal- The DNA was incubated with T4 ultraviolet endonuclease as follows. Reaction mixtures (0.068 ml) contained 0.57 nmol of DNA (as nucleotide, either unirradiated or ultraviolet-irradiated at 400 J per m2), Tris-HCl buffer (pH 8.0, 44 mM), EDTA (7.4 mM), T4 ultraviolet endonuclease (Fraction V, 24 units). Incubation was at 37" for 30 min following which 0.005 ml of 1% sodium dodecyl sulfate was added to each tube and the tubes were kept on ice. An aliquot of each reaction mixture (0.02 ml) was layered onto an alkaline cesium chloride gradient with a density from 1.3 to 1.4 containing 0.1 N NaOH and 1.0 mM EDTA. Sedimentation was in a SW 56 rotor at 40,006 rpm for 3.5 hours. Fractionation of gradients and measurement of radioactivity was performed as described in the legend to function of increasing ultraviolet fluence. In order to be sure that reactions were carried to completion, M-13 DNA irradiated at the highest fluence used in Table II was incubated under identical conditions for periods between 10 to 120 min. The release of a2P following incubation with bacterial alkaline phosphatase reached a maximum at 30 min of incubation. These results indicate that all pyrimidine sites in ultraviolet-irradiated M-13 DNA are attacked by the T4 ult.raviolet endonuclease. Thus, even if some degree of double-stranded structure exists in M-13 DNA under our experimental conditions, it is unlikely that all dimers are located in these regions.

Molecular Weight Determination-Electrophoresis
of a lyophilized preparation of Fraction V of the T4 ultraviolet endonuclease was carried out in polyacrylamide gel containing sodium dodecyl sulfate. The gel showed a single major band at a molecular weight calculated at 17,700 relative to the standard proteins used (Fig. 7). A single extremely faintly staining band at a Relationship between pyrimidine dimers and endonucleolytic incisions in M-13 DNA **P-labeled M-13 DNA was prepared as described under "Experimental Procedures." The DNA was ultraviolet-irradiated at fluences between 0 to 1,300 J per ma. Incubation mixtures (0.15 ml) contained: unirradiated or irradiated M-13 DNA, 0.13 nmol; Tris-HCl buffer, pH 8.0, 17.0 maa; EDTA, 16.6 maa; T4 ultraviolet endonuclease (Fraction V), 43 units. Incubation was at 37" for 60 min. Reactions were terminated by placing the incubation mixture in a boiling water bath for 10 min. To each tube was added 3.5 units of bacterial alkaline phosphatase and incubation was continued for 60 min at 37". To each tube were added 0.2 ml of 1% bovine serum albumin and 0.6 ml of 10% cold trichloroacetic acid. The tubes were centrifuged at 6,006 X g and the supernatant fractions were saved. Radioactivity was measured by counting 0.5 ml of each supernatant in 10.0 ml of a mixture of Omnifluor in toluene with Triton X-100 as described in the legend to Table I DNA molecule 3.9 11.9 22.2 40.0 molecular weight of 51,000 was visible with the naked eye; however, this band was not recordable on a densitometer tracing of the gel. Sephadex gel filtration of Fraction IV of the enzyme revealed a single peak of ultraviolet endonuclease activity with a molecular weight of approximately 18,000 relative to the standards used (Fig. 7, inset).

DISCUSSION
A role for the T4 ultraviolet endonuclease in the excision repair of ultraviolet-irradiated phage T4 would appear to be clearly established. Mutants defective in this activity (u gene mutants) are both abnormally ultraviolet sensitive and are unable to effect excision of thymine dimers either in tiuo (2) or in vilro (25). Thus a detailed study of the physicochemical properties of this enzyme may contribute significantly to an understanding of the molecular mechanism of pyrimidine dimer excision in general.
Previously published properties of the enzyme have been referred to in the introductory section (3-8). In addition, recent studies have investigated the kinetics of thymine dimer excision by purified E. coli DNA polymerase I and the 36,066 molecular weight fragment of that enzyme (which retains only 5' + 3' exonuclease activity) from ultraviolet-irradiated T7 DNA specifically incised with T4 ultraviolet endonuclease (26). These results showed that endonucleolytic incisions occur on the 5' side of the dimer. This conclusion is supported by the present studies which show that degradation of prenicked and denatured DNA by bovine spleen phosphodiesterase (an enzyme that degrades denatured DNA in the 5' --* 3' direction) is precluded by the DNA in t&o without further modification of the termini. Indeed, our results show that in vitro, E. coli DNA polymerase I will utilize nicks for DNA synthesis in the 5' + 3' direction. These studies also demonstrate that the T4 ultraviolet endonuclease has a molecular weight of approximately 18,000, with no evidence for subunit structure. A question of considerable interest with respect to this endonuclease is its substrate specificity. Previous studies have demonstrated a stoichiometric relationship between the calculated number of dimers and the number of endonucleolytic incisions in DNA, thereby providing indirect evidence that pyrimidine dimers provide substrate sites in DNA (4, 7). The present studies have approached this question more directly. Thus, if the concentration of thymine dimers in DNA is reduced by incubation with photoreactivating enzyme, the number of endonucleolytic incisions in the DNA is reduced. The only known catalytic activity of E. coli photoreactivating enzyme is the splitting of the cyclobutane ring covalently linking two pyrimidines in ultravioletirradiated DNA (27). Similar results have been obtained by introducing purified T4 endonuclease into Brij-treated ultravioletirradiated E. coli (28). In this case all endonuclease sensitive sites measurable by sedimentation velocity of the DNA are removed by exposing the cells to photoreactivating light.
Given that pyrimidine dimers are necessary to produce substrate sites for the T4 ultraviolet endonuclease, the possibility exists that the enzyme recognizes a localized conformational distortion in the secondary structure of the DNA rather than the dimer directly. It has been previously demonstrated with Form I SV 40 DNA containing an average of 1 pyrimidine dimer per molecule, that the endonuclease makes only single strand breaks in ultraviolet-irradiated double-stranded DNA (7). Thus, one needs to consider the possibility that either the dimer-containing strand or the opposite strand at a dimer site is attacked. Evidence available at this time indicates that only the dimer-containing strand is attacked. Fig. 4, photoreactivation of nicked DNA is required to facilitate degradation at 5' termini by bovine spleen phosphodiesterase. This suggests that all substrate sites created for the phosphodiesterase are close to pyrimidine dimers rather than opposite them. In a simila? vein it has previously been shown that when ultraviolet-irradiated T7 DNA incised with T4 ultraviolet endonuclease is incubated with a partially purified extract of T2-infected E. coli that contains a phagecoded dimer excision activity, 80 to 90% of the thymine dimers can be excised with a loss of only 10 to 20 nucleotides per dimer into the acid-soluble phase, suggesting that at least 80 to 90% of the endonucleolytic incisions are close to pyrimidine dimers (29).

As shown by the results presented in
We believe that these experiments rule out the possibility of an equally probable attack at either strand at a dimer site in duplex DNA; however, attack at the strand opposite a dimer may occur at a low frequency. We are currently investigating this question by constructing DNA duplexes in which only one strand contains pyrimidine dimers. Further studies also are being carried out to determine the relative efficiency with which the enzyme attacks ultraviolet-irradiated single-and double-stranded DNA. WEISS,