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.

Polymerases:

Topics:

Accessory Proteins/Complexes, Source / Purification, Historical Protein Properties (MW, pI, ...), Nucleotide Incorporation

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
AcNPV McDougal VV1999 Nick Extension Yes, via strand displacement
AcNPV McDougal VV1999 Molecular Weight 1.1E+05 Dalton
AcNPV McDougal VV1999 Cloned or native Insect cells
AcNPV McDougal VV1999 Tagged No
AcNPV McDougal VV1999 Full length or truncated Full length
AcNPV McDougal VV1999 Processivity 4kb
AcNPV McDougal VV1999 Strand Displacement Yes
AcNPV McDougal VV1999 Other accessory protein(s) P143, IE-1, LEF-1, LEF-2, and LEF-3
AcNPV McDougal VV1999 Gap Filling Yes

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