Adenovirus DNA replication in vitro.

Abstract

Much of the recent dramatic progress in understanding prokaryotic DNA replication is due to the development of soluble in vitro systems that carry out the replication of exogenously added DNA templates of defined structure (1-4). These systems have made it possible to study in detail the biochemical mechanisms involved in the initiation and elongation of DNA chains and to purify and characterize the proteins required for these processes. It is clear that the development of analogous in vitro systems would be extremely valuable for dissecting the mechanisms of eukaryotic DNA replication.The human adenoviruses have several advantages as model systems for studying eukaryotic DNA replication. The adenovirus genome is easily isolated in intact form from purified virions, and its structure and genetic organization are well defined (5, 6). In addition, a good deal is known about the general mechanism of adenovirus DNA replication in vivo (7). This makes it possible to assess the biological relevance of any synthetic reactions observed in vitro. The adenovirus genome is a linear, duplex DNA molecule with a molecular weight of 20-25 X 106 (8). It possesses two novel structural features of possible significance for DNA replication. First, the two ends of the genome have identical nucleotide sequences (refs. 9-11; J. R. Arrand and R. J. Roberts, personal communication), and second, the 5' ends of both DNA strands are covalently linked to a polypeptide of molecular weight 55,000 (12)(13)(14)(15)(16). In vivo studies (reviewed in ref. 7) have shown that initiation of adenovirus DNA replication takes place at or near either end of the viral genome. Daughter strand synthesis then proceeds in the 5' to 3' direction with concomitant displacement of the parental strand with the same polarity. Synthesis of the complementary strand is initiated at or near the 3' end of the displaced parental strand and also proceeds in the 5' to 3' direction. Thus, the overall pattern of adenovirus DNA replication appears ratherThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance' with 18 U. S. C. §1734 solely to indicate this fact. 655 simple. At each growing point only one of the two parental strands is replicated, and, at least in principle, there is no requirement for a discontinuous mechanism of DNA synthesis. This simplicity makes the adenovirus system especially attractive for biochemical studies.In this paper we describe a soluble enzyme system from adenovirus-infected cells that is capable of replicating exogenously added adenovirus DNA molecules. We have shown by several criteria that the DNA synthesis that occurs in this in vitro system closely resembles adenovirus DNA replication in vivo. METHODSPreparation of Nuclei from Adenovirus-Infected Cells. HeLa cells were maintained at 370C in suspension culture in Eagle's minimal essential medium supplemented with horse serum (5%). One liter of cells at a density of 4-5 X 105 cells/ml was ...

ADP-ribosylation in in vitro Systems Synthesizing Adenovirus DNA

“…The $200 system of Shaw et al (1979) is capable of elongating adenovirus DNA from an endogenous template previously initiated in vivo. In contrast, the system described by Challberg & Kelly (1979) (the CK system) appears to be capable of both initiation and elongation from an exogenously added template of adenovirus DNA with the 55K terminal protein attached, with little endogenous DNA synthesis. The latter system very closely mimics the replication pattern seen in vivo.…”

“…The cells were harvested 19 to 21 h after infection, the nuclei isolated by Dounce homogenization and, after freezing in liquid nitrogen and thawing, the nuclei were extracted with 100 mM-NaC1 for 1 h at 4 °C before being removed by centrifugation. The supernatant (the CK extract) was capable of the initiation of adenovirus replication in vitro (Challberg & Kelly, 1979).…”

Expression of rTSβ as a 5-fluorouracil resistance marker in patients with primary breast cancer

“…The procedure for preparing nuclear extracts was described previously (24). Briefly, monolayer cells (2-5x10 7 ) grown to 70% confluence were detached with trypsin-EDTA.…”

Possible Mechanism of Adenovirus Generation from a Cloned Viral Genome Tagged with Nucleotides at Its Ends

“…The synthesized fourth to sixth CAT trinucleotides linked with pTP "jump back" onto the 3'-most GTA trinucleotides of the template strand and elongation of the synthesized strand is then initiated (14,15). In in vitro replication systems of Ad5 (3,29), formation of the pTP-dC complex occur when using linearized plasmid DNA with an internal Ad5 replication origin or supercoiled DNA as templates, but dose occur when the plasmid is linearized in such a way that the origin is located at the end of the molecule (32). This observation is consistent with the in vivo results described above.…”