Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro

Mao Sun, Donna Louie, Philip Serwer

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Abstract

Bacteriophage T7 packages its double-stranded DNA genome in a preformed protein capsid (procapsid). The DNA substrate for packaging is a head-to-tail multimer (concatemer) of the mature 40-kilobase pair genome. Mature genomes are cleaved from the concatemer during packaging. In the present study, fluorescence microscopy is used to observe T7 concatemeric DNA packaging at the level of a single (microscopic) event. Metabolism-dependent cleavage to form several fragments is observed when T7 concatemers are incubated in an extract of T7-infected Escherichia coli (in vitro). The following observations indicate that the fragment-producing metabolic event is DNA packaging: 1) most fragments have the hydrodynamic radius (R(H)) of bacteriophage particles (±3%) when R(H) is determined by analysis of Brownian motion; 2) the fragments also have the fluorescence intensity (I) of bacteriophage particles (±6%); 3) as a fragment forms, a progressive decrease occurs in both R(H) and I. The decrease in I follows a pattern expected for intracapsid steric restriction of 4',6-diamidino-2-phenylindole (DAPI) binding to packaged DNA. The observed in vitro packaging of a concatemer's genomes always occurs in a synchronized cluster. Therefore, the following hypothesis is proposed: the observed packaging of concatemer- associated T7 genomes is cooperative.

Original languageEnglish (US)
Pages (from-to)1627-1637
Number of pages11
JournalBiophysical Journal
Volume77
Issue number3
DOIs
Publication statusPublished - Sep 1999

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ASJC Scopus subject areas

  • Biophysics

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