Two nucleic acid-dependent nucleoside triphosphate phosphohydrolases from vaccinia virus. Nucleotide substrate and polynucleotide cofactor specificities.

The two purified nucleoside triphosphate phosphohydrolases from vaccinia virus are distinct enzymes as judged by their nucleotide substrate and polynucleotide cofactor specificities. Phosphohydrolase I hydrolyzes only ATP and dATP, whereas phosphohydrolase II hydrolyzes the four common ribonucleoside triphosphates as well as dATP and TTP. 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. The preceding paper (1) described the purification and properties of two nucleic acid-dependent enzymes that hydrolyze