Products and substrate/template usage of vaccinia virus DNA primase

Abstract

Vaccinia virus encodes a 90-kDa protein conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded varphiX174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which (32)P was transferred from [alpha-(32)P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.

Fig.1

Effects of enzyme concentration and divalent and monovalent cations on primase activity. (A) Primase reactions were carried out under standard conditions with a ϕX174 DNA template and 6 mM Mg²⁺. The amount of recombinant D5 primase varied from 0.07 to 2.2 pmol. Products were digested with calf intestinal alkaline phosphatase, denatured and resolved by electrophoresis on a 20% polyacrylamide/urea gel. A phosphoimager scan is shown. Origin indicates the position of the well. The arrow points to the position of the band migrating at the position of a phosphorylated 14-nucleotide marker. (B) The data in panel A were quantified by making a standard curve with known amounts of [α-³²P]CTP. Symbols: ■, “14-nucleotide product”; ●, extended products; ▲, total. (C) Effect of Mg²⁺ and Mn²⁺ concentrations. Primase reactions were carried out under standard conditions with 1.1 pmol of primase except that the divalent cation concentrations were varied as shown. Positions of ³²P-end labeled oligonucleotide markers are indicated on the left. (D) Effect of K⁺ concentration. Primase reactions were carried under standard conditions except that the monovalent cation concentraton was varied.

Fig. 2

Analysis of primase products. (A) The discrete primase product comigrates with dinucleotides GpC and ApC as well as with the phosphorylated 14-nucleotide marker. Primase assays were performed and the products analyzed alongside ³²P-end labeled oligonucleotide markers. In addition, 2 μg of synthetic dinucleotides GpC and ApC were co-electrophoresed and detected by UV shadowing. Positions of GpC and ApC are marked on the left of the autoradiogram. The radioactive dinucleotide product is labeled DN. (B) Agarose gel electrophoresis. The products of reactions with primase (+) and without primase (-) were resolved on a formaldehyde/1% agarose gel. The positions of RNA size markers are on the right.

Fig. 3

Nearest neighbor analysis. Primase reactions were carried out in the presence of [α-³²P]CTP and 1 mM Mn²⁺. (A) Samples were resolved on a denaturing 20% polyacrylamide gel (not shown) and the dinucleotide band was excised, eluted into nuclease free water and digested with piperidine. Chemically treated or untreated products were resolved on a denaturing 20% polyacrylamide gel. Np and DN represent the positions of mononucleotides and dinucleotides, respectively. (B) Chemically treated samples and 20 μg of a mixture of pA, pC, pG and pU were spotted at the bottom left edge of cellulose plates (intersection of arrows A and C). After two-dimensional chromatography (solvent A, 1^st dimension; solvent C, 2^nd dimension), plates were exposed to a 254 nm UV light source to detect nucleotide markers and to X-ray film to detect radioactive products. The migrations of the 5’ nucleotide markers are indicated by full circles in the first dimension and dashed circles in the second. Positions of Ap and Gp radioactive products relative to markers are indicated. Note that 2’, 3’ nucleotides move slightly faster than 5’ nucleotides in the first and second dimensions (Grosjean et al., 2007).

Fig. 4

Incorporation of ribo- and deoxyribonucleotides. Primase assays were performed using [α-³²P]CTP (lanes 1-4) or [α-³²P]dCTP (lanes 5-8) supplemented with combinations of 1.5 mM rNTPs and/or 1.5 mM dNTPs. Positions of ³²P-end labeled oligonucleotide markers are indicated on the left. Origin indicates the top of the well; filled arrow represents dinucleotide product formed with [α-³²P]CTP; open arrow represents the presumed dinucleotide product formed with [α-³²P]dCTP.

Fig. 5

Template recognition of homooligomer templates. Primase reactions were carried out using 100 ng of dT, dC, dG, and dA 18mer homooligomers in the presence of the complementary [α-³²P]NTP and 1 mM Mn²⁺. Products were treated with calf intestinal alkaline phosphatase and then with (+) or without (-) nuclease P1. An autoradiograph of a 20% polyacrylamide gel is shown with the positions of ³²P-end labeled oligonucleotide markers on the left.