Studies on the Mechanism of Replication of Adenovirus DNA II. The Nature of Single-Stranded DNA in Replicative Intermediates

Abstract

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the singlestranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in AT base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.

Adenovirus type 2 DNA replication. II. Termini of DNA replication

“…3, panel A) co-sediments with marker DNA, isolated from intact virions (18), whereas the radioactivity eluted in the presence of caffeine sediments nonhomogeneously ( Fig. 3, panel C) with S values ranging from 34S, the marker position, up to 50 to 60S (13,16). In alkaline gradients, the radioactivity from the 1 M sodium chloride eluate (Fig.…”

“…Pulse-labeled viral DNA consists of a mixture of mature, completed DNA molecules and a spectrum of replicating DNA molecules (9,18). These have previously been shown to contain extensive single-stranded regions (9,13,16). Equilibrium centrifugation in neutral cesium chloride of viral DNA, synthesized in 5-, 10-, and 30-min pulses (Fig.…”

“…In this system, the kinetics of appearance of newly synthesized adenovirusand simian virus 40-specific DNA appear to be consistent with initiation at both molecular ends of the viral DNA. Moreover, replicating Ad2 DNA has been shown to contain extended single-stranded regions (9,13,16,18) whereas detailed electron microscopy analyses (3) never appeared to reveal the presence of either circular or linear concatomeric intermediates, typically observed in replication of other circular and linear DNA molecules.…”

“…Double-stranded, mature adenovirus type 2 (Ad2) DNA can be isolated from intact virions in a linear (5) or circular form (11) of molecular weight 23 x 106 (5). Its replication in nuclei from infected human cells in tissue culture proceeds discontinuously (18) through replicative intermediates which are characterized by their increased buoyant density (13,16,18) and their sensitivity to single-stranded DNAspecific nucleases as compared to mature DNA molecules (9). Electron microscopy analysis (3) of newly synthesized viral DNA revealed the presence of various types of branched and unbranched linear intermediates containing extended regions of single-stranded DNA.…”

Mappings of adenovirus type 7 cytoplasmic RNA species synthesized early in lytically infected cells and synthesized in transformed cells

“…Ad7 early mRNA saturates 20% of the r strand and 35% of the 1 ,ug) were prepared from in vivo-labeled Ad7 HindIII fragments by gel electrophoresis as described by Hayward (9) and purified by self-annealing at 68°C for a time equivalent to about 5OX CotI/2 of Ad7 DNA to remove any contamination of complementary strands. The H and L strands of whole Ad7 DNA were prepared as described by Sussenbach et al (16). Ad7 DNA in 0.1 M Tris-hydrochloride (pH 8.1) containing 5 mM EDTA was alkaline denatured and adjusted to the density of 1.77 g/ml by adding CsCl.…”