A Small (58-nm) Attached Sphere Perturbs the Sieving of 40-80-Kilobase DNA in 0.2-2.5% Agarose Gels: Analysis of Bacteriophage T7 Capsid-DNA Complexes by Use of Pulsed Field Electrophoresis

Philip Serwer, Shirley J. Hayes, Elena T. Moreno, Christopher Y. Park

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel. The data are explained by the following assumptions: (1) The capsid-DNA complexes are sterically arrested at 2-6 V/cm when DNA passes through pores that are too small to allow passage of the capsid; either Brownian motion (if the electrical potential gradient is low enough) or reverse pulsing prevents arrest. (2) The electrical potential gradient necessary to produce arrest decreases as the distance of the capsid from the most distal DNA end increases. Use of PFGE for isolating capsid-DNA complexes from lysates of T7-infected cells is demonstrated; a complex with dimeric DNA and a complex with trimeric DNA are detected.

Original languageEnglish (US)
Pages (from-to)8397-8405
Number of pages9
JournalBiochemistry
Volume31
Issue number36
DOIs
StatePublished - Feb 1 1992

ASJC Scopus subject areas

  • Biochemistry

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