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DNA replication of bacteriophage T5. 2. structure and properties of the slow sedimenting form of intracellular T5 DNA

Everett, R.D., and Lunt, M.R. (1980) DNA replication of bacteriophage T5. 2. structure and properties of the slow sedimenting form of intracellular T5 DNA. Journal of General Virology, 47 (1). pp. 133-150. ISSN 0022-1317 (doi:10.1099/0022-1317-47-1-133)

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Publisher's URL: http://dx.doi.org/10.1099/0022-1317-47-1-133

Abstract

The intracellular DNA of bacteriophage T5 contains two major DNA forms distinguishable by sedimentation rate through neutral sucrose gradients. The slow sedimenting form (ssf) which moves at a rate similar to that of DNA extracted from mature T5 virus particles, was prepared free of the fast sedimenting form and capsid-associated DNA by electrophoresis on agarose gels in which it migrated as peak II DNA. The ssf DNA thus obtained was subjected to extensive structural analysis. The number and location of single-stranded regions was studied using the single-strand-specific S1 nuclease and electron microscopy. The frequency and position of the single-stranded regions, which could occur both at internal and terminal locations, was reproducible in the population of ssf DNA molecules as a whole but individual molecules were not identical. After analysis of these results in conjunction with results of studies on the location of single-strand interruptions in ssf DNA it is suggested that single-stranded regions mainly occur at those sites corresponding to the sites of the major nicks in mature T5 virion DNA. When the ssf was isolated by gel electrophoresis and maintained in low ionic strength solutions it was found to be associated with several phage-specific proteins including some which are constituents of the mature virus particle. The presence of these proteins reduced the electrophoretic mobility of the DNA from that expected on the basis of its mol. wt. alone. This property of reduced mobility could be reproduced in vitro using exogenous ssf DNA and crude extracts from phage-infected, but not from uninfected, cells. By contrast, when DNA purified from phage T7 particles was incubated with crude extracts of T5-infected bacteria, the electrophoretic mobility of the T7 DNA was unaltered; thus the effect was due to T5-specific proteins on T5-specific DNA. The contour lengths of both peak II DNA and ssf were measured by electron microscopy and compared to that of mature T5 virion DNA. Unexpectedly it was found that both were reproducibly about 12% shorter than the mature molecule; therefore it appears unlikely that the ssf is a true intermediate in the pathway of T5 DNA replication. These observations are discussed in relation to the current models of excision from concatemers and encapsulation of mature phage DNA.

Item Type:Article
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Everett, Prof Roger
Authors: Everett, R.D., and Lunt, M.R.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
Journal Name:Journal of General Virology
Journal Abbr.:J. Gen. Virol.
Publisher:Society for General Microbiology
ISSN:0022-1317
ISSN (Online):1465-2099

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