| Abstract
| - We investigated the effects of ssDNA template sequence on both primer synthesis and NTPhydrolysis by herpes simplex virus 1 helicase−primase. Primer synthesis was found to be profoundlydependent upon template sequence. Although not absolutely required, an important sequence feature forsignificant production of longer primers (beyond four nucleotides in length) is a deoxyguanylate−pyrimidine−pyrimidine (3‘-G-pyr-pyr-5‘) triplet in the template. The deoxyguanylate serves both to directprimase to initiate synthesis opposite the adjacent pyrimidine and to dramatically increase primer length.While primase synthesized significantly more long primers on those templates containing a G-pyr-pyrtriplet, the enzyme still synthesized massive quantities of di- and trinucleotides on many templates containingthis sequence. Varying the sequences around the G-pyr-pyr recognition sequence dramatically alteredboth the rate of primer synthesis and the fraction of primers longer than four nucleotides, indicating thatprimase must interact with both the G-pyr-pyr and flanking sequences in the template. In contrast to thelarge effects that varying the template sequence had on primase activity, ssDNA-dependent NTPase activitywas essentially unaffected by changes in template sequence, including the presence or absence of theG-pyr-pyr trinucleotide. In addition to hydrolyzing NTPs the NTPase could also hydrolyze the 5‘-terminalphosphate from newly synthesized primers.
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