Studies on the replication of adenovirus-2 DNA

Abstract

Monolayer cultures of KB cells infected with adenovirus type 2 (Ad2) were subjected to short pulses of tritiated thymidine, at the time of maximal viral DNA synthesis. Nascent viral DNA was selectively extracted from the cells, purified, and fractionated into partly single-stranded (or replicating) molecules and double-stranded (or completed) molecules. Completed molecules were cleaved by the restriction endonucleases EcoRI, HindIII, Hpal, or by the latter two enzymes in succession, and the resulting fragments were separated by electrophoresis through agarose-acrylamide slab gels. In the case of replicating molecules, the double-stranded fragments generated after treatment with the same enzymes were first separated from the partly single-stranded fragments by chromatography on benzoylated-naphthoylated DEAE (BND) cellulose before being subjected to electrophoresis. The relative yields of tritium of the various fragments resolved by the gels were then determined. The analysis of the completed molecules obtained after the shorter pulses revealed a strong preferential labelling of the fragments derived from the molecular ends, as expected if both of these ends contained termini for replication. The analysis of double-stranded fragments from replicating molecules revealed a predominant labelling of the central portion of the genome which could be reconciled with initiation occurring either centrally or at both molecular ends. In addition, two gradients of labelling, ascending toward each molecular end and encompassing the outer quarters of the molecule, were noted which slowly receded when the pulse length was increased. The latter finding could reflect an overrepresentation of molecules in the late stages of replication in the pool of nascent DNA, such as one would expect if replication slows down when it nears the termination site(s). This interpretation is consistent with the kinetics of labelling of the various portions of the genome observed in the pool of completed molecules. Our present work deals also with the characterization of three different fractions of replicating Ad2 DNA, separated by MAP chromatography. Two of these fractions, eluted with 140 or 180 mM phosphate respectively, display properties distinct from the third fraction, eluted with 240 mM phosphate or the mature DNA. The progressive disappearance of all three fractions upon chases of sufficient durations, suggest that they are all eventually converted into mature DNA. Based on the density in CsC1 or CS2SO4, before or after treatment by various enzymes (N. crassa nuclease, RNAase, DNA polymerases, alone or in various combinations), it is concluded that the first two fractions contain a substantial amount of ss DNA, or RNA in the form of RNA-DNA hybrid. These fractions may thus be composed of molecules at rather late stages of displacement of incipient stages of complementary strand synthesis. The presence of RNA in these molecules accounts for the high buoyant density difference found in several laboratories (up to 10 mg/mL) between replicating and completed Adenovirus DNA molecules, which cannot be explained on the sole basis of partial single-strandedness