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Related Experiment Videos

Sequence-dependent primer synthesis by the herpes simplex virus helicase-primase complex

D J Tenney1, A K Sheaffer, W W Hurlburt

  • 1Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Connecticut 06492, USA.

The Journal of Biological Chemistry
|April 21, 1995
PubMed
Summary

The herpes simplex virus helicase-primase complex synthesizes primers on specific DNA sequences. Primer synthesis efficiency depends on template sequence, while ATPase activity depends on template size.

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Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • The herpes simplex virus helicase-primase complex is essential for viral DNA replication.
  • This complex is a heterotrimer composed of UL5, UL8, and UL52 proteins.

Purpose of the Study:

  • To investigate the primer synthesis site and requirements of the herpes simplex virus helicase-primase complex.
  • To determine the influence of template size and sequence on primer synthesis and ATPase activity.
  • To compare the stimulatory mechanisms of UL8 and ICP8 on helicase-primase activity.

Main Methods:

  • Mapping of the primer synthesis site on phi X174 virion DNA.
  • Oligonucleotide-based assays to determine primer synthesis and ATPase activity requirements.
  • Coupled primase-polymerase assays to assess rate-limiting steps.

Related Experiment Videos

  • Comparative analysis of UL8 and ICP8 stimulation using different DNA templates.

    • Main Results:

    • A predominant primer synthesis site was identified on deoxycytosine-rich regions of phi X174 DNA, directing guanine-initiated primer synthesis.
    • Primer synthesis efficiency was highly dependent on template nucleotide sequence, whereas ATPase activity was influenced by template size.
    • Oligonucleotides with primase templates inhibited coupled primase-polymerase assays, indicating primer synthesis or turnover is rate-limiting.
    • UL8 stimulated primer synthesis on both viral DNA and oligonucleotide templates, while ICP8 stimulation was specific to viral DNA.

    Conclusions:

    • The herpes simplex virus helicase-primase complex exhibits specific sequence recognition for primer synthesis.
    • Template sequence and size differentially affect primer synthesis and ATPase activity.
    • UL8 and ICP8 proteins play distinct roles in modulating helicase-primase function.