Visualization of the unwinding of long DNA chains by the herpes simplex virus type 1 UL9 protein and ICP8

Alexander M. Makhov, Paul E. Boehmer, I. Robert Lehman, Jack D. Griffith

Research output: Contribution to journalArticle

37 Scopus citations

Abstract

UL9 protein and ICP8 encoded by the herpes simplex virus type 1 (HSV-1) were shown to catalyze a highly active, non-origin-dependent unwinding of DNA. UL9 protein, the HSV-1 origin binding protein, as a modest helicase activity that is greatly stimulated by the HSV-1 single strand (ss) binding protein, ICP8. Here, electron microscopy has been applied to examine the mechanics of this reaction. Negative staining of the proteins revealed particles consisting primarily of ICP8 monomers and UL9 protein dimers. When the binding of UL9 protein to double strand (ds) DNA containing ss tails was examined by shadowcasting methods, UL9 protein was seen bound to the ss tails or ss/ds junctions; addition of ATP led to its appearance internally along the ds segment. When UL9 protein and ICP8 were incubated together with the tailed dsDNA in the presence of ATP, a highly ordered unwinding of the DNA was observed by negative staining that appeared to progress through four distinct stages: (1) binding of ICP8 to the ss tail and progressive coverage of the ds portion by UL9 protein; (2) formation of highly condensed regular filaments; (3) relaxation of the condensed structures into coiled-coils; and (4) unwinding of the coils and release of ICP8-covered linear ssDNAs. This process represents a mechanism of unwinding that is very different from ones that proceed by a progressive unwinding at Y-shaped forks that move along the DNA.

Original languageEnglish (US)
Pages (from-to)789-799
Number of pages11
JournalJournal of Molecular Biology
Volume258
Issue number5
DOIs
StatePublished - May 24 1996

Keywords

  • Electron microscopy
  • Helicase
  • Herpes simplex
  • ICP8
  • UL9

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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