Conformation of DNA packaged in bacteriophage T7. Analysis by use of ultraviolet light-induced DNA-capsid cross-linking

Philip Serwer, Shirley J. Hayes, Robert H. Watson

Research output: Contribution to journalArticlepeer-review

20 Scopus citations

Abstract

The conformation of the linear, double-stranded, 39,936 kilobase-pair DNA packaged in the protein capsid of bacteriophage T7 is investigated here by use of short wavelength ultraviolet light-induced DNA-capsid cross-linking. To detect both DNA-capsid and DNA-DNA cross-links, DNA is expelled from the T7 capsid and the products of expulsion are analyzed by use of Nycodenz buoyant density centrifugation, followed by either pulsed field gel electrophoresis or invariant field gel electrophoresis. Short wavelength ultraviolet light is found to progressively induce both DNA-DNA and DNA-protein cross-links in intact bacteriophage T7, but not in T7 from which DNA had been expelled before exposure to ultraviolet light. Protein-protein cross-links are not induced. When DNA expelled from previously cross-linked T7 is cleaved with restriction endonuclease (1 to 3 sites cleaved), analysis of the resulting fragments reveals no regions on T7 DNA that are excluded from cross-linking to the capsid. However, the efficiency of cross-linking decreases as the distance from the left end (last end packaged) of the packaged DNA increases. Electron microscopy of negatively stained capsid-DNA complexes reveals no DNA-retaining structure other than the outer shell of the capsid. Together with previously reported data that indicate lack of protein-based specificity for ultraviolet light-induced cross-linking, these observations are interpreted by the assumptions that, within the limits of resolution of these experiments: (1) no region of packaged T7 DNA is excluded from contact with the outer shell of the T7 capsid; (2) the probability of contacting the outer shell decreases as the distance from the left end of packaged T7 DNA increases. Thus, T7 DNA packaging concentrates the last end packaged near the inner surface of the outer shell of the T7 capsid.

Original languageEnglish (US)
Pages (from-to)999-1011
Number of pages13
JournalJournal of Molecular Biology
Volume223
Issue number4
DOIs
StatePublished - Feb 20 1992
Externally publishedYes

Keywords

  • DNA
  • agarose gel
  • bacteriophage
  • buoyant density
  • centrifugation
  • conformation of
  • cross-linking
  • electrophoresis
  • structure of
  • ultraviolet light-induced

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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