Single-Stranded Deoxyribonucleic Acid-specific Nuclease from Vaccinia Virus EKDONUCLEOLYTIC An’D EXONUCLEOLYTIC ACTIVITIES

A deoxyribonuclease purified from to have a specificity for single-stranded DNA.

A deoxyribonuclease purified from vaccinia virus was shown to have a specificity for single-stranded DNA. Undenatured HeLa cell DNA was digested at less than 2% of the rate of thermally denatured DNA. The low rate of digestion was probably due to single-stranded regions in the isolated HeLa cell DNA since no nicking of native T7 DNA was detected by alkaline sucrose gradient sedimentation. Evidence of endonucleolytic activity was obtained by sedimentation, gel filtration, and DEAE-cellulose chromatography of the digestion products.
Exonucleolytic activity was also indicated by the high percentage of mononucleotides formed.
The single-strand specificity and the endo-and exonucleolytic activities are similar to the previously described properties of the S1 nuclease from Aspergillus oryzae. The vaccinia deoxyribonuclease was also able to nick closed single-stranded circular DNA as well as closed superhelical double-stranded circles.
The susceptibility of the latter probably resulted from the presence of weakly hydrogenbonded regions in the DNA.
In the preceding paper (1)  HeLa cell I>NA (12.9 nmoles, specific activity 6226 cpm per nmole). At the times indicated, samples were removed and precipitated with perchloric acid. The acid-soluble nucleotides were counted in scintillation fluid. FIG. 2 (celrter). Alkaline sucrose gradient sedimentation of native and denatured T7 I)NA after incubation with deoxyribonuclease. 1Squal amounts of [3H]thymidine-labeled native or denatured T7 L>NA (13.9 nmoles, specific activity 305 cpm per nmole) were added to the standard reaction mixtures containing 0.2 unit of purified deoxyribonuclcase.
As controls native or denatured T7 IINA was added to parallel reaction mixtures in the absence of enzyme. All samples were incubated at 37" for 20 min. The reaction was stopped by the addition of NnCl and NaOH to RESULTS

Specificity
for Single-Stranded DNA--V1 dcosyribonuclease, purified from vaccinia virions as described in the accompanying paper (l), exhibits a specificity for single-stranded DNA.
Initially this was tested with [311]thymidine-labeled HeLa cell DNA. Fig. 1 shows the enzymatic hydrolysis of thermally denatured and native DNA as a function of time. When hydrolysis of thermally denatured DNA plateaued at 45 min, approximately 507; or 6.4 nrnoles of nucleotide were rendered acid-soluble.
In contrast, only 1 To of the "undenatured" DNA or 0.15 nmole of nucleotide was acid-soluble during the same period of time. The plateau obtained with denatured DNA was not entirely due to residual duplex DNA or to a limit digestion of single-stranded DNA but to inactivation of the deoxyribonuclease since further hydrolysis occurred after addition of more enzyme (Fig. 1).
Since undcnatured HeLa cell DNA was likely to contain some single-stranded regions produced during the isolation procedures, the single-stranded specificity of the deoxyribonuclcase was tested more rigorously with a much smaller and uniform size DNA from bacteriophage T7. In addition, the assay was made more sensitive by examining the products of digestion 011 alkaline sucrose gradients.
Under conditions in which all molecules of alkaline-denatured T7 DNA were extensively hydrolyzed, no evidence of nicking of native DNA was detected (Fig. 2).
To determine whether the enzyme could use RNA as a substrate, [3H]uridine-labe1ed ribosomal RNA was incubated in the standard reaction mixture containing 0.1 unit of enzyme at 37" for 1 hour. The sample was then made 507, in dimethylsulfoxide, 1 mM EDTA, and 0.2% sodium dodecyl sulfate, and analyzed on a 5 to 20% sucrose gradient in 99% dimethylsulfoxide containing 10 InM LiCl and 0.5 mu EDTA.
The samples were f--n T T FRACTlON NUMBER final concentrations of 0.9 and 0.2 M, respectively. The samples were then analyzed on alkaline sucrose gradients as described under "Materials and Methods." Samples were collected from the bottom of the gradient.
A, denatured T7 DNA without en- sucrose gradient sedimentation of denatured T7 1)NA after partial digestion.
[3H]Thymidine T7 DNA was denatured and treated as described under "Materials and Methods." li:qual amounts of denatured DNA (60 nmoles, specific activity 3500 cpm per nmole) were added to reaction mixtures. Purified enzyme (0.05 and 0.1 unit) was added to the samples which were incubated for 20 min at 37". The reactions were stopped and analyzed on alkaline gradients as Under these conditions there was 110 significant difference in the sedimentation of the ribosomal RNA incubated in the presence or absence of enzyme (results not shown).

Analysis
of Products of Limited Digestion by Sedimentation and Gel Filtration-Evidence of endonucleasc activity was obtained by examining the products of limited digestion of alkaline denatured T7 DNA.
Digested samples containing 0.4% and 7% acid-soluble nucleotides were analyzed on alkaline sucrose gradients. As a control, DNA incubated in the absence of enzyme was similarly analyzed.
The results shown in Fig. 3 indicate a shift in the sedimentation of the DNA consistent with endonuclease action.
Analysis of the products of limited digestion of thermally denatured HeLa cell DNA by gel filtration also provided evidence of endonuclease activity.
Enzymatic digestion of the DNA was allowed to proceed until 6% of the nucleotides were acid-soluble. The product was then applied to a Sephadex G-100 column. DNA incubated for the same period of time without enzyme was similarly analyzed and eluted in the void volume.
Approximately 18% of the digested DNA was shifted from the void volume of the column and eluted as a broad heterogeneous peak (Fig. 4A). 3Iore material was found in the total column volume in enzymatic digests which had a greater per cent of soluble nucleotides.
However, this material did not coincide entirely with the [V]ATP marker. As a control, the product of an exonuclease, snake venom phosphodiesterase, was analyzed on a similar column (Fig. 4B). III contrast to the previous results, the exonuclease product eluted in two peaks coinciding with the void volume and the [32P]ATI' marker. tensive digestion was determined by chromatography. Thermally denatured 321'-labeled HeLa DNA was incubated with purified vaccinia deoxyribonuclease for varying time intervals and the digestion product was analyzed on DEAE-cellulose colui,ms in the presence of 7 M urea. Under these conditions, oligonucleotides are separated according to chain length.
The products produced by digestion of [3H]thymidine-labeled HeLa DNA with an exonuclease, snake venom phosphodiesterase, or an endonuclease, pancreatic DNase I, were used as markers.
As expected 32P-and 3H-labeled peaks overlapped rather than coincided since at pH 7.5 there is partial separation based on nuclcotide composition within peaks and only thymidine was labeled with tritium (4). A representative elution profile from an experiment in which approximately 80% of the thermally denatured HeLa cell [32P]DNA was rendered acid-soluble is shown in Fig. 5. Of the total a2P-labeled material recovered, 15% was mononucleotides, 5% was dinucleotides, 8% was trinucleotides, 7% was tetranucleotides, approximately 8% was pentanuclcotides, and the remainder of the product was of higher molecular weight.
The formation of thymidine monophosphate during digestion by V, nuclease was analyzed further by thin layer chromatography on PEI-cellulose plates (Fig. 6). A significant peak of TMP was detected when 4% of the DNA was made acidsoluble.
These results demonstrate that the deoxyribonuclease has both endonucleolytic and cxonucleolytic activities. pll Optimum of Endonuclease A&&-Since a pH optimum of 4.4 for the deoxyribonuclease was previously obtained using an acid solubility assay which may favor detection of exonuclease action (l), it was important to check the effect of pH using a specific endonuclease assay. Reaction mixtures containing identical amounts of deoxyribonuclease and denatured [3H]thymidine-labeled HeLa cell DNA were prepared at pI-1 3.7, 4.4, and 5.0. After 5 min of incubation at 37", the reactions were stopped and the samples were applied to Sephadex G-100 col- 7 (lefl). Sedimentation of deoxyribonuclease-treated SV40 Form II DNA in alkaline sucrose gradients.
Purified SV40 Form II DNA was denatured as described under "Materials and Methods." Portions of the denatured DNA (0.3 pg, specific activity 1400 cpm per rg) were added to the standard reaction mixtures. The samples were incubated for 30 min at 37" without (O---0) or with (O---O) 0.3 unit of purified deoxyrihonuclease. The reaction was stopped as previously described, immediately layered on an alkaline sucrose gradient, and analyzed as described under "Materials and Methods." Samples were collected from the bottom and counted in scintillation fluid. FIG.
["H]Thymidinelabeled SV40 Form I DNA (0.9 pg, specific activity 11,778 cpm per pg) was incubat,ed in standard reaction mixtures in the presence Cleavage of Circular DNA Jlolecules-The ability of the vaccinin deoxyribonuclease to cleave circular DNA was tested using SV40 Form I and Form II DNAs.
Form I is a double-stranded closed circular molecule; Form II is also a doublestranclcd circular molecule but contains a nick in one straucl. Dcuaturation of Form II rcsult,s in the production of linear and circular strands which sediment in alkaline sucrose gradients at 16 and 18 S, respcctivcly.
130th strands of denatured Form II DNA were clcavecl by vaccinia virus deosyribonucleasc iudicating that the enzyme dots not rquire the presence of free cncls (Fig. 7).
Covalcntly closed circular molecules (e.g. SV40 Form I DNA) contain supcrhelical twists which result in unpaired or weakly hydrogen-bonded regions (6) in the DNA. These regions are nicked by single-stranded DNA-specific nuclcascs such as Neurospora crassa cndonuclease (7). A preparation of native [WIthymidine-labeled SV40 DNA, consisting predominantly of Form I but containing some Form II DNA, was incubated with vaccinia cleosyribonuclca.sc.
The product was then analyzed on an alkaline SIICI'OSC gradient.
Under these conditions of ccntrifugation Form I DNA remains doublestranded and therefore scdimcnts as a 53 S molecule while both Form II and Form III (a double-stranded linear molecule) sediment much more slowly at 16 to 18 S (8). The results shown in Fig. 8 iuclicatc that SV40 Form I DNA was nickccl by the vaccinia clcosyribonuclcase forming a structure seclimentiug at 16 to 18 S. To dctcrminc whether the latter consisted of Form II or both Fom II and Form III DNAs, the reaction product was analyzed on neutral sucrose gradients.
Uuclcr these conditions Form I sediments at 21 S, Form II at 16 S, and Form III at 14 S (9). The results shown in Fig. 9 inclicatc that both Forms II and III wcrc produced iu the prcscnce of vacciuia dcosyribonuclease. (O--O) and the absence (O---0) of deoxyribonuclease (0.2 unit) for 30 min at 37". The reaction was stopped as previously described.
The samples were then layered on an alkaline sucrose gradient and analyzed as described under "Materials and Methods." The location of SV40 Form I (63 S) and Form II and III (16-18 S) DNAs are indicated by arrows.
3H-labeled SV40 Form I DNA (0.9 rg, specific activity 11,778 cpm per pg) was incubated in standard reaction mixtures in the absence and presence of deoxyribonuclease (0.2 unit) for 30 min at 37". Samples were then layered immediately on neutral sucrose gradients as described under "Materials and Methods." The positions of Form I (21 S) and Form II (16 S) DNAs are indicated by arrows.

DISCUSSION
A deosyribonuclease purified from vaccinia virus was shown to have a specificity for single-stranded DNA.
Undenatured HeLa cell DNA was digested at 2y0 or less of the rate of thermally denatured DNA.
The low rate of digestion was probably due to single-stranded regions in the isolated HeLa cell DNA, since no nicking of native 'I'7 DNA was detected by alkaline sucrose gradient sedimentation.
Evidence of endonucleolytic activity was obtained by sedimentation, gel filtration, and DEAE-cellulose chromatography of the digestion products. Exonuclease activity was also indicated by the high percentage of mononucleotides formed.
Although we cannot rule out the possibility that the cndo-and exonucleolytic activities result from two separate enzymes, the pI-I optima determined by acid solubility and gel filtration were identical.
The deosyribonuclease activity of intact vaccinia virus cores at low pII as well as of a purified deoxyribonuclease from infected cell extracts were previously thought to be exclusively esonucleolytic (10-13).
However, the only documented evidence of this was based on nitrocellulose filter assays. Nevertheless, because of the possibility that the low pH endonuclease activity may be masked while in cores, we examined the products produced by limited digestion of denatured [3H]thymidine-labeled HeLa cell DNA at pH 4.4. Filtration of the products on Sephadex G-100 columns clearly indicated the production by intact cores of large oligonucleotidc fragments.2 Thus, at 111-I 4.4 endonuclease activity can be demon&rated by both intact cores and purified VI nuclcasc.
The sensitivity of Form I DNA to the vaccinia deoxyribonu-3296 clcase probably results from the presence of unpaired or weakly hydrogenbonded regions in the superhelical DNA (6). Both the N. crassa endonuclease (7) and the Si nuclease (14) cleave superhelical DNAs.
The latter enzyme also has a low but significant activity on linear double-stranded DNA (14). Further studies will be needed to determine whether the conversion of some Form II and Form III DNA by vaccinia deoxyribonuclease resulted from a low ability of the enzyme to nick double-stranded DNA or to rccognizc an interruption in the polynucleotide chain resulting from the cleavage of a single phosphotlicster bond. It is possible that the esonucleasc activity of the enzyme could recognize a single cleaved phosphodiester bond in the double-stranded DNA and thus make t,he gap wider.
The endonuclease activity of the enzyme would then attack the opposite strand of the molecule, thus converting Form II to Form III.
The biological function of the vaccinia deoxyribonuclease and the reason for its presence in the viral core are unknown.
The two strands of vaccinia DNA behave as if they are cross-linked, possibly at the terminal ends (15). Bcrns and Silverman (15) suggested that a single-stranded DNA-specific endonuclease would be needed for strand separation if replication were semiconservative.
Strand separation may then be a possible role of the deoxyribonuclease found in the core of the virus. I'ogo and Dales (12) have suggested the possibility that deosyribonucleases might be released from the virion after infection and inhibit host DNA4 synthesis.
Of the nucleases studied so far, the vaccinia Vi nuclease described here most closely resembles the Si nuclease of Aspergillus oryzae (16). Both show specificity for single-stranded DNA, e~ldo-and exonucleolytic activities, and pH optima of approximately 4.5 (1,14,16,17). Although some ribonuclease activity nlay be associated with preparations of Si nuclcase, it is thought to be a contaminant (16). The molecular weight of the Sr en-molecular weights of 50,000. The S1 nuclease has been used to discriminate between single-and double-stranded DNA for analytical and preparative purposes.
Whether the Vi nuclease offers any practical advantages because of the absence of ribonuclease activity remains to be seen.