The herpes simplex virus type 1 helicase-primase. Analysis of helicase activity.
The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' single-stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding protein, infected cell protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.
Falkenberg
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Department of Biochemistry, Beckman Center, Stanford University, Stanford, California 94305-5307, USA.
Elias
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P
Lehman
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IR
D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D019915:DNA Primase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases; D008274:Magnesium