Autographa californica nuclear polyhedrosis virus DNA polymerase: measurements of processivity and strand displacement

Abstract

The DNA polymerase (DNApol) of Autographa californica nuclear polyhedrosis virus was purified to homogeneity from recombinant baculovirus-infected cells. DNApol was active in polymerase assays on singly primed M13 template, and full-length replicative form II product was synthesized at equimolar ratios of enzyme to template. The purified recombinant DNApol was shown to be processive by template challenge assay. Furthermore, DNApol was able to incorporate hundreds of nucleotides on an oligo(dT)-primed poly(dA) template with limiting amounts of polymerase. DNApol has moderate strand displacement activity, as it was active on nicked and gapped templates, and displaced a primer in a replication-dependent manner. Addition of saturating amounts of LEF-3, the viral single-stranded DNA-binding protein (SSB), increased the innate strand displacement ability of DNApol. However, when LEF-3 was added prior to the polymerase, it failed to stimulate DNApol replication on a singly primed M13 template because the helix-destabilizing activity of LEF-3 caused the primer to dissociate from the template. Escherichia coli SSB efficiently substituted for LEF-3 in the replication of a nicked template, suggesting that specific protein-protein interactions were not required for strand displacement in this assay.

FIG. 1

Purification of DNApol. Nuclear extracts (NE) prepared from AcDNApol-infected cells (lane 3) were subjected to chromatography on DE52 (lane 4), heparin (lane 5), MonoQ (lane 6), MonoS (lane 7), and ssDNA agarose (lane 8). Lanes 4 to 8 contain 10 μg of protein from the peak fractions of each column. Lane 2 contains 10 μg of crude nuclear extracts prepared from RP6-S/C-infected cells. The positions of protein molecular markers electrophoresed in lane 1 are shown in kilodaltons on the left; the position of DNApol is indicated on the right. Samples were separated on SDS–8% polyacrylamide gels and stained with Coomassie blue.

FIG. 2

Replication of singly primed M13 DNA by AcNPV DNApol. Purified DNApol (20, 50, or 100 fmol; lanes 2 to 4) was incubated with 20 fmol of singly primed M13 DNA. Reaction products were denatured and separated on a 0.8% alkaline agarose gel. Lane 1 contains ³⁵S-labeled HindIII-digested λ DNA. Sizes of the molecular markers are shown in kilobases on the left; the position of full-length M13 DNA (7.2 kb) is indicated on the right.

FIG. 3

Processivity of DNA polymerases on poly(dA)-oligo(dT). Reaction mixtures contained 336 fmol of poly(dA)-oligo(dT) template and 5.25, 10.5, 21, 42, 84, 168, 336 fmol of each polymerase. Klenow fragment was used in the reaction mixtures in lanes 1 to 7; those in lanes 8 to 14 contain T4 DNA polymerase; those in lanes 15 to 21 contain AcNPV DNApol. A control reaction with no enzyme is shown in lane 22. Reactions were electrophoresed on a 7 M urea–12% polyacrylamide gel. Sizes are indicated in nucleotides.

FIG. 4

Template challenge assay: demonstration of the processive nature of AcNPV DNApol on singly primed M13 and φX174 templates. Reaction mixtures of 50 μl contained 20 fmol of purified recombinant AcNPV DNApol and 20 fmol of each template. Lane 2 contains singly primed M13 template alone without φX174 challenge template; lanes 3 to 7 contain reactions in which the polymerase was allowed to assemble on the M13 template before addition of the φX174 challenge template and dCTP. Aliquots were removed at the times (minutes) indicated at the top. Lane 8 contains φX174 template alone; lanes 9 to 13 contain reaction mixtures in which the polymerase was allowed to assemble on the φX174 template before addition of excess M13 challenge template and dCTP. Aliquots were removed at the times indicated at the top. DNA products were fractionated on a 0.8% alkaline agarose gel. Sizes are indicated in kilobases.

FIG. 5

Mung bean nuclease sensitivity of AcDNApol replication products. Standard singly primed M13 assays were extracted with phenol and precipitated with ethanol. DNA was resuspended in mung bean nuclease buffer and incubated in the presence (lane 3) or absence (lane 2) of mung bean nuclease. Reaction products were analyzed by 0.8% alkaline agarose gel electrophoresis. The migration of molecular markers is shown in lane 1, and the relevant sizes are indicated in kilobases on the left. The migration of full-length M13 DNA is shown on the right.

FIG. 6

Strand displacement assay on a gapped DNA template. (A) Schematic of gapped DNA substrate; (B) strand displacement activity of AcNPV DNApol with and without LEF-3 on a gapped DNA substrate. Reaction mixtures contained 20 fmol of DNA template and 50 fmol of the indicated DNA polymerase. Lane 1, AcNPV DNApol incubated with singly primed M13 template; lane 2, gapped M13 template with T4 DNA polymerase; lane 3, gapped M13 template incubated with Klenow fragment; lane 4, gapped M13 template incubated with AcNPV DNApol; lane 5, gapped M13 template incubated with AcNPV DNApol and 10 pmol of LEF-3. 290, 290-nt product.

FIG. 7

Replication-dependent displacement of a radiolabeled primer. (A) Schematic of the gapped DNA substrate and assay. dNTPs, deoxynucleoside triphosphates. (B) Gapped template was incubated with indicated amounts of AcNPV DNApol. Samples were electrophoresed on a 12% polyacrylamide gel to separate the displaced oligonucleotide from the template. The amount of displacement primer was quantitated by PhosphorImager analysis. Control reactions were run in the absence of dCTP, and the values shown for synthetic displacement were adjusted for background radioactivity in the absence of dCTP.

FIG. 8

LEF-3 stimulates replication in a manner that depends on order of addition. The products fractionated in lanes 1, 4, 7, 10, 13, and 16 were taken from a reaction mixture in which 45 pmol of LEF-3 was added first, followed by incubation for 5 min on ice, addition of 300 fmol of DNApol, and incubation at 37°C; 300 fmol of DNApol was added first to the reaction mixture distributed in lanes 2, 5, 8, 11, 14, and 17; after 5 min on ice, 45 pmol of LEF-3 was added; 300 fmol DNApol only was added to the reaction mixture allocated to lanes 3, 6, 9, 12, 15, and 18. Aliquots of 25 ml from each of the three reaction mixtures were removed 1, 3, 5, 10, 20, and 30 min after incubation at 37°C. Sizes are indicated in kilobases.

FIG. 9

Strand displacement on nicked template. (A) Strand displacement in the presence of LEF-3. DNApol (3.8 pmol) or Klenow fragment (0.5 U) was incubated with 1 pmol of nicked DNA template, and then the indicated amount of LEF-3 was added. (B) DNApol (3.8 pmol) or Klenow fragment (0.5 U) was added to each reaction, and then the indicated amount of E. coli SSB was added. Each point represents the average of three separate experiments.