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Title: Length quantization of DNA partially expelled from heads of a bacteriophage T3 mutant

DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNA missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection. - Graphical abstract: Highlights: • We implement directed evolution- and DNA-sequencing-based phage assembly genetics. • We purify stable, mutant phage heads with a partially leaked mature DNA molecule. • Native gels and DNase-protection show leaked DNA segments to have quantized lengths. • Native gels after DNase I-removal of leaked DNA revealmore » the capsids to vary in radius. • Thus, we hypothesize leaked DNA quantization via variably quantized capsid radius.« less
Authors:
 [1] ;  [1] ; ;  [2]
  1. Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (United States)
  2. Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 (United States)
Publication Date:
OSTI Identifier:
22435036
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 456-457; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BACTERIOPHAGES; CONTROL; DNA; DNA SEQUENCING; ELECTRON MICROSCOPY; ELECTROPHORESIS; EVOLUTION; GELS; GENE MUTATIONS; GENETICS; IN VITRO; LEAKS; MOLECULES; MUTANTS; PACKAGING; PHENOTYPE; QUANTIZATION; SAFETY; ULTRACENTRIFUGATION