Adenovirus DNA synthesis in vitro in an isolated complex

Abstract

DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.

Role of DNA polymerase .gamma. in adenovirus DNA replication. Mechanism of inhibition by 2',3'-dideoxynucleoside 5'-triphosphates

“…A role of DNA polymerase a has been suggested from the inhibition of adenovirus DNA replication by phosphonoacetic acid (Frenkel, 1978) and aphidicolin, a specific inhibitor of this enzyme (Krokan et al, 1978;Longiaru et al, 1979). However, a 300-400-fold difference exists between the sensitivity of Ad5 and cellular DNA replication to aphidicolin.…”

Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

“…These individual components of chromosome replication, as well as enzymes, structural proteins, and other associated factors, can then be isolated and characterized, and their function can be deduced, through reconstitution experiments. This approach has been encouraged by the development of subnuclear systems that can complete replication of simian virus 40 (SV40) (13,14,26) or adenovirus (5,17,18,31) DNA and by the discovery that chromatin assembly accompanies DNA replication in vitro (25). SV40 as well as polyoma virus provide welldefined genomes that are particularly suited to studying the biochemical mechanisms involved in the replication of mammalian chromosomes.…”

The DNA polymerases associated with the adenovirus type 2 replication complex: effect of 2′-3′-dideoxythymidine-5′-triphosphate on viral DNA synthesis

“…Other in vitro Ad DNA replication systems have been isolated frcm nuclei of infected cells (6,7,8). Each of these systems is able to incorporate deoxynucleotide triphosphates into DNA molecules which had been ¢ Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England initiated in vivo.…”