Two Nucleic Acid-dependent Nucleoside Triphosphate Phosphohydrolases from Vaccinia Virus NUCLEOTIDE SUBSTRATE AND POLYNUCLEOTIDE COFACTOR SPECIFICITIES

SUMMARY The two purified nucleoside triphosphate phosphohydrolases from vaccinia virus are distinct enzymes as judged by their nucleotide substrate and polynucleotide cofactor specificities.

The range of K, values for ATP and dATP hydrolysis by phosphohydrolase I (1.5 to 1.6 X lop4 M) was lower than the range of K, values for ATP, dATP, TTP, GTP, CTP, and UTP hydrolysis by phosphohydrolase II (6.4 to 11.2 X 1o-4 M). Phosphohydrolase I had little or no detectable activity in the absence of added nucleic acids. Phosphohydrolase II frequently had some endogenous activity, probably due to trace amounts of nucleic acid present in the eluate from the DNA-cellulose column.
This endogenous activity could be reduced by passage through a DEAE-cellulose column.
Neither phosphohydrolase I nor II was stimulated by completely double-stranded DNA unless the DNA was first denatured.
Only phosphohydrolase II was stimulated by RNA but again only single-stranded forms were usable. Phosphohydrolase II was also stimulated by all tested homopolynucleotides of either the ribose or deoxyribose series and by all possible hybrid forms.
Phosphohydrolase I exhibited a greater specificity and was incapable of using single-stranded homopolynucleotides. However, homopolydeoxyribonucleotide duplexes and some homopolyribonucleotide: homopolydeoxyribonucleotide duplexes were effective. These results suggest that phosphohydrolase I requires some secondary structure such as adjacent single-stranded and hydrogen-bonded regions. This interpretation was supported by the preferential inhibition of phosphohydrolase I with actinomycin D and low concentrations of proflavine. The ability of both enzymes to form stable complexes with nucleic acids was shown by glycerol gradient sedimentation. It was concluded that both nucleoside triphosphate phosphohydrolases have unique properties which make them distinct from any previously described enzymes.
Their biological role is unknown, although it is tempting to speculate an involvement in replication and packing of DNA or transcription and extrusion of nascent RNA from viral cores.  (Table I). Since phosphohydrolase I and phosphohydrolase II are separable by DNA-cellulose chromatography (l), the column fractions were assayed with each of the four radioactively labeled ribonucleoside triphosphates.
The results indicated that phosphohydrolase I hydrolyzed only ATP while phosphohydrolase II hydrolyzed all four ribonucleoside triphosphates tested ( Fig. 1). Further evidence that phosphohydrolase II contained all four hydrolytic activities was provided by gel filtration. A single peak of activity capable of hydrolyzing ATP, GTP, UTP, and CTP was eluted from a Sephadex G-200 column after application of a sample of phosphohydrolase II (data not presented). The hydrolysis of deoxyribonucleoside triphosphates by purified phosphohydrolase I and phosphohydrolase II was tested using dATP and TTP as representative purine and pyrimidine deoxyribonucleoside triphosphates. The data in Table II indicated that the purified phosphohydrolase I used dATP as efficiently as ATP but did not hydrolyze TTP to any appreciable extent.
Purified phosphohydrolase II, on the other hand, hydrolyzed both dATP and TTP as efficiently as ATP.
The K, values for each of the ribo-and deoxyribonucleoside triphosphate substrates tested were determined from Lineweaver-Burk plots of the saturation data and are presented in Table  III. Several important points may be derived. First, the K, of phospho-hydrolase I for ATP and dATP are virtually identical. Second, the K, of phosphohydrolase I for either substrate is lower than the corresponding values for phosphohydrolase II. Third, the K, of phosphohydrolase II for each substrate lies within a narrow range (6.4 to 11.2 x 10e4).
EJect of Native and Denatured DNA on Activity of Phospho-. hydrolase I and Phosphohydrolase II-The effects of native and denatured DNA on the hydrolysis of ATP by phosphohydrolase I and phosphohydrolase II are shown in Fig. 2. In this experiment adenovirus DNA was used. Both phosphohydrolase I and phosphohydrolase II were stimulated by denatured DNA but not to any appreciable extent by native DNA.
An appreciable rate of ATP hydrolysis in the absence of added DNA was found with some purified phosphohydrolase II preparations,  while in other preparations endogenous activity was quite low. Filtrat.ion of the DNA-cellulose-purified phosphohydrolase II enzyme through a' DEAE-cellulose column served to reduce the endogenous activity and suggested that residual nucleic acid may sometimes by present in the enzyme preparations after DNAcellulose chromatography.
It is also evident from Fig. 2  breaks. Only when the DNA was sheared and then denatured did it function as a cofactor for both phosphohydrolase I and phosphohydrolase II, again indicating the requirement by both enzymes for single-stranded DNA. The ability of both phosphohydrolase I and phosphohydrolase II to use the closed circular single-stranded form of 4X174 DNA as efficiently as when it had been sheared suggested that ends are not required for the nucleic acid-enzyme interreaction resulting in ATP hydrolysis.
This point must be explored further since some linear molecules were present in the unsheared preparation.
Native vaccinia DNA exhibited a limited capacity to serve as cofactor for phosphohydrolase II but not for phosphohydrolase I. Since vaccinia DNA is rather large, with a molecular weight of 160 x lo6 (6), and difficult to isolate intact, this limited capacity of vaccinia DNA to serve as cofactor for phosphohydrolase II may be due to some single-stranded DNA present in the preparation.
The fact that phosphohydrolase II is saturated at much lower concentrations of denatured DNA than is phosphohydrolase I may account for the preferential utilization of the DNA by the former enzyme. Alternatively, a specific association of phosphohydrolase II with a specific region on the vaccinia DNA is possible.
The ability of native XDNA to serve to some extent as cofactor for both phosphohydrolase I and phosphohydrolase II may be due to the single-stranded regions at the ends of the XDNA molecule (7).
Commercial preparations of native salmon sperm DNA and calf thymus DNA as well as native HeLa cell DNA all served to varying degrees as cofactors for both phosphohydrolase I and phosphohydrolase II. This was probably due to single-stranded DNA present in these preparations.
Yeast ribosomal RNA and reovirus RNA and to a lesser extent E. co&soluble RNA functioned as cofactors for phosphohydrolase II but not for phosphohydrolase I. As seen with native and denatured reovirus RNA, phosphohydrolase II could use only the denatured form as cofactor.
This result was similar to the requirement for denatured DNA.
The deoxysugar polynucleotide specificity of phosphohydrolase I provided another means of distinguishing the two enzymes in addition to the nucleotide substrate specificity.  II responded quite differently to synthetic polynucleotides (Fig. 3). Phosphohydrolase II was stimulated by the single-stranded homopolymers or by the synthetic duplex poly(dA) :poly(dT).
In contrast, phosphohydrolase I was stimulated only by the duplex (Fig. 3). This was a totally unexpected result in view of the previous demonstration that phosphohydrolase I had shown a complete dependence on thermally denatured DNA and was not stimulated by double-stranded DNA.
These findings led us to investigate the ability of a number of synthetic ribo-and deoxyribohomopolymers as well as their respective hybrids to serve as cofactors for phosphohydrolase I and phosphohydrolase II. Single-Stranded Synthetic Homopolymers as Cofactors for Phosphohydrolase I and Phosphohydrolase II-The effects of various synthetic homopolymers on the hydrolysis of ATP, UTP, CTP, and GTP by phosphohydrolase I and phosphohydrolase II are presented in Table V. Phosphohydrolase II could use any of the synthetic homopolymers tested as cofactor for the hydrolysis of any of the four ribonucleoside triphosphates.
There was no clear preference by phosphohydrolose II for any synthetic homopolymer as cofactor, nor did phosphohydrolase II indicate any preference for a ribonucleoside triphosphate substrate. Phosphohydrolase I, on the other hand, did not use to an appreciable extent any of the tested synthetic homopolymers as cofactor for the hydrolysis of ATP with exception of poly(dC). The latter was used by phosphohydrolase I only about one-fifth as well as commercial salmon sperm DNA.
Except Eflect of Actinomycin D, ProJEavine, and Rifampicin on Phosphohydrolase I and Phosphohydrolase II Activity-When commercial salmon sperm DNA was used as a cofactor, actinomycin D preferentially inhibited phosphohydrolase I activity (Table  VIII). Actinomycin D had little or no effect on phosphohydrolase II. Similarly, proflavine, which binds more readily to duplex than to single-stranded polynucleotide structures (8)) inhibited phosphohydrolase I more strongly than it inhibited phosphohydrolase II activity (Table VIII). This specificity was especially evident at lower concentrations of the drug.
Rifampi- tin was not found to inhibit either phosphohydrolase I or phosphohydrolase II activity (Table VIII).
DNA Binding of Phosphohydrolase I and Phosphohydrolase II -WC took advantage of the ability of phosphohydrolase I and phosphohydrolase II to bind to DNA-cellulose for their purification (1). Additional studies showed that under standard assay conditions both enzymes bind strongly to DNA in solution and form stable DNA-enzyme complexes that can be isolated by glycerol gradient sedimentation (Fig. 4). Control experiments indicated that phosphohydrolase I and phosphohydrolase II incubated in a standard enzyme reaction without nucleic acid remained on top of glycerol gradients (Fig. 4). The conditions needed to form the DNA-enzyme complex, such as requirements for divalent cation, ATP, single-or double-stranded nucleic acid, will be the subject of future studies.

Phosphohydrolase
I and phosphohydrolase II appear to be distinct enzymes as judged by their elution from DNA-cellulose, divalent cation requirements, nucleotide substrate specificities, polynucleotide cofactor requirements, and inhibition by intercalating agents. A summary of the major differences is presented in Table IX.
The most striking difference between the two enzymes is their specificity for hydrolysis of nucleoside triphosphates.
Phosphohydrolase II hydrolyzed any of the four 7. Inhibition by actinomycin D + TTP, while phosphohydrolase I hydrolyzed only ATP and dATP to any appreciable estent.
The second major difference is the polynucleotide cofactor requirement. Phosphohydrolase II utilized denatured DNA or RNA, while phosphohydrolase I common ribonucleoside triphosphates as well as dATP and utilized only denatured DNA. Furthermore, phosphohydrolase