Vaccinia virus DNA polymerase. In vitro analysis of parameters affecting processivity.
The polymerization and proofreading activities of the vaccinia virus DNA polymerase reside within a 116-kDa catalytic polypeptide. We report here an investigation of the intrinsic processivity of this enzyme on both natural and homopolymeric DNA templates. Inclusion of the Escherichia coli helix destabilizing protein allowed the viral enzyme, which lacks strand displacement activity, to utilize a singly primed M13 DNA template. In the presence of either 10 mM MgCl2 or 1 mM MgCl2 + 40 mM NaCl, synthesis was achieved in a highly distributive manner. RFII formation required a significant excess of enzyme, and < or = 10 nucleotides (nt) were added per primer-template binding event. The apparent rate of primer elongation varied with the enzyme/template ratio and reached a maximum of 8 nt/s. A similar lack of processivity was observed on a poly(dA390)-oligo(dT12-18) template. In contrast, highly processive synthesis was achieved on both templates in the presence of 1 mM MgCl2 and the absence of NaCl. A primer extension rate of 30 nt/s was observed, and > or = 2000 nt were added per binding event. These studies suggest that the catalytic polypeptide of the vaccinia virus DNA polymerase will require accessory protein(s) to form a stable enzyme-template interaction and direct processive DNA synthesis under isotonic conditions in vivo.
McDonald
W F
WF
Department of Microbiology, Cornell University Medical College, New York, New York 10021.
Traktman
P
P
D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D011089:Polydeoxyribonucleotides / Q000378:metabolism; D011108:Polymers; D012965:Sodium Chloride / Q000494:pharmacology
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011089:Polydeoxyribonucleotides; D011108:Polymers; D015636:Magnesium Chloride; D012965:Sodium Chloride; C090592:DNA polymerase, vaccinia virus; D004259:DNA-Directed DNA Polymerase