The UL8 component of the herpes simplex virus helicase-primase complex stimulates primer synthesis by a subassembly of the UL5 and UL52 components.
The herpes simplex virus type 1 (HSV) UL5, UL8, and UL52 proteins form a helicase-primase complex in infected cells. Several laboratories have demonstrated that helicase and nucleoside triphosphatase activities of the heterotrimer (UL5/8/52) are indistinguishable from that of a subassembly of UL5 and UL52 (UL5/52). Although the UL5/52 subassembly functions in coupled primase-polymerase assays on homopolymeric templates, its activity on natural DNA templates has been reported to require UL8. To determine the role of UL8 in primase assays, the activity of the UL5/52 subassembly was compared to that of the heterotrimer reconstituted by adding UL8 to UL5/52. We detected significant activity of the UL5/52 subassembly in coupled primase-polymerase and oligoribonucleotide primer synthesis assays on phi X174 and M13 virion DNAs. However the addition of UL8 to UL5/52 stimulated this activity in a dose-dependent manner. We demonstrate that stimulation occurred at the level of primer synthesis. UL8 did not affect the amount or size of primers annealed to template, their utilization by DNA polymerase, or the use of specific initiation sites within the template. In kinetic studies, the rate of primer synthesis was increased by UL8 but the Km for phi X174 DNA template was unchanged. These results suggest that a function of the UL8 component of the HSV helicase-primase complex is to increase the efficiency of primer synthesis by UL5/52.
Tenney
D J
DJ
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Hurlburt
W W
WW
Micheletti
P A
PA
Bifano
M
M
Hamatake
R K
RK
D000818:Animals; D016367:Baculoviridae; D002477:Cells; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D019915:DNA Primase; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008322:Mammals; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D014764:Viral Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases