Sequence-dependent primer synthesis by the herpes simplex virus helicase-primase complex.
The herpes simplex virus helicase-primase complex, a heterotrimer of the UL5, UL8, and UL52 proteins, displays a single predominant site of primer synthesis on phi X174 virion DNA (Tenney, D. J., Hurlburt, W. W., Micheletti, P. M., Bifano, M., and Hamatake, R. K. (1994) J. Biol. Chem. 269, 5030-5035). This site was mapped and found to be deoxycytosine-rich, directing the synthesis of a primer initiating with several guanine residues. The size and sequence requirements for primer synthesis were determined using oligonucleotides containing variations of the predominant template. Although the efficiency of primer synthesis on oligonucleotides was influenced by template size, it was absolutely dependent on nucleotide sequence. Conversely, the ATPase activity on oligonucleotide templates was dependent on template size rather than nucleotide sequence. Furthermore, only oligonucleotides containing primase templates were inhibitory in a coupled primase-polymerase assay using phi X174 DNA as template, suggesting that primer synthesis or primase turnover is rate-limiting. Additionally, stimulation of helicase-primase by the UL8 component and that by the ICP8 protein were shown to differ mechanistically using different templates: the UL8 component stimulated the rate of primer synthesis on phi X174 virion DNA and oligonucleotide templates, while ICP8 stimulation occurred only on phi X174 virion DNA.
Tenney
D J
DJ
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492, USA.
Sheaffer
A K
AK
Hurlburt
W W
WW
Bifano
M
M
Hamatake
R K
RK
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism* / Q000502:physiology; D019915:DNA Primase; D017931:DNA Primers / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D008969:Molecular Sequence Data; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology*; D014764:Viral Proteins / Q000502:physiology
D017931:DNA Primers; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; C062021:helicase-primase, Human herpesvirus 1; D000251:Adenosine Triphosphatases; D004265:DNA Helicases