DNA binding properties and processive proofreading of herpes simplex virus type 1 DNA polymerase.
The DNA binding properties of herpes simplex virus type 1 DNA polymerase (HSV pol), an alpha-like DNA polymerase, were investigated using an optimized band-shift assay. With linear double-stranded DNA (dsDNA), HSV pol formed two complexes. The favored DNA template was dsDNA with protruding 5'-phosphoryl termini. Stable binding of HSV pol was observed with a DNA hairpin containing a primer region of 9 bp of dsDNA, a 6-base loop and a 12-base 5'-terminal single-stranded extension. For the polymerization activity of HSV pol on poly(dT) an optimal primer length of 8 to 10 nucleotides was determined. The DNA binding event could be clearly separated from the enzymatic activities by its unique response to divalent cations and salt. Under ionic strength conditions where HSV pol exerts optimal polymerization activity in vitro, novel polymerase-DNA complexes were detected by band-shift analysis. These new complexes were similar while either in DNA polymerase or 3',5' exonuclease mode. Using a polymerase trap method and high-resolution polyacrylamide gel electrophoresis, HSV pol demonstrated internal switching from 3',5' exonuclease to polymerase-active mode during one DNA binding event. These results support the role of HSV pol as a true replicase, which proofreads without dissociating from the DNA template.
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Forschungsschwerpunkt Genomforschung und Bioinformatik H0601, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, D-69120 Heidelberg, Germany.
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D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006493:Heparin / Q000494:pharmacology; D011061:Poly A / Q000378:metabolism; D011071:Poly T / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity / Q000235:genetics; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D012492:Salts; D014764:Viral Proteins; D011061:Poly A; D011071:Poly T; C015465:poly(dA); D006493:Heparin; D004247:DNA; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus