Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro
| Autor: | Donna Louie, Mao Sun, Philip Serwer |
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| Rok vydání: | 1999 |
| Předmět: |
Models
Molecular Concatemer Biophysics Genome Viral Biology Cleavage (embryo) medicine.disease_cause Genome Bacteriophage chemistry.chemical_compound Capsid Bacteriophage T7 Fluorescence microscope medicine Escherichia coli Viscosity biology.organism_classification Molecular biology Kinetics chemistry Microscopy Fluorescence DNA Viral Thermodynamics DNA Research Article |
| Zdroj: | Biophysical journal. 77(3) |
| ISSN: | 0006-3495 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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