SEQUENCE-DEPENDENT PRIMER SYNTHESIS BY THE HERPES-SIMPLEX VIRUS HELICASE-PRIMASE COMPLEX

The herpes simplex virus helicase-primase complex, a heterotrimer of t he 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 deoxycytos ine-rich, directing the synthesis of a primer initiating with several guanine residues, The size and sequence requirements for primer synthe sis were determined using oligonucleotides containing variations of th e predominant template, Although the efficiency of primer synthesis on oligonucleotides was influenced by template size, it was absolutely d ependent on nucleotide sequence, Conversely, the ATPase activity on ol igonucleotide templates was dependent on template size rather than nuc leotide sequence, Furthermore, only oligonucleotides containing primas e templates were inhibitory in a coupled primase-polymerase assay usin g phi X174 DNA as template, suggesting that primer synthesis or primas e turnover is rate-limiting, Additionally, stimulation of helicase-pri mase by the UL8 component and that by the ICP8 protein were shown to d iffer mechanistically using different templates: the UL8 component sti mulated the rate of primer synthesis on phi X174 virion DNA and oligon ucleotide templates, while ICP8 stimulation occurred only on phi X174 virion DNA.