Adenovirus DNA replication in vitro: Duplication of single-stranded DNA containing a panhandle structure

Abstract

Adenovirus DNA replicates by displacement of one of the parental strands followed by duplication of the displaced parental single strand (complementary strand synthesis). Displacement synthesis has been performed in a reconstituted system composed of viral and cellular proteins, employing either the viral DNA-terminal protein complex as template or linearized plasmids containing the origin. Previously, evidence was obtained that in vivo complementary strand synthesis requires formation of a panhandle structure originating from hybridization of the inverted terminal repeats. To study the conditions for complementary strand synthesis in vitro, we have constructed an artificial panhandle molecule that contains a double-stranded inverted terminal repetition (ITR) region and a single-stranded loop derived from the left and right terminal Xma I fragments of Ad2. Such a molecule appeared to be an efficient template and could initiate by the same protein-priming mechanism as double-stranded DNA, employing the precursor terminal protein. The efficiency of both types of template was comparable. Like for replication of the duplex molecule initiation of panhandle replication was stimulated by nuclear factors I and III, proteins that bind to specific double-stranded regions of the ITR. The Ad DNA-binding protein is essential and the 39 kDa C-terminal domain of this protein that harbors the DNA-binding properties is sufficient for its function. These results support the hypothesis that panhandle formation is required for duplication of the displaced strand.

The adenovirus DNA binding protein enhances intermolecular DNA renaturation but inhibits intramolecular DNA renaturation

“…Single-stranded M13 phage mprL9, containing both Ad2 inverted terminal repeats, flanked by EcoRl sites and separated by 1.3 kb of adenovirus sequences, cloned in the EcoRl site of Ml3mp9, was isolated and digested with EcoRI for 4 hrs at 370C (24). The product was Klenow end-labelled to give the panhandle probe, containing at either end an adenovirus inverted terminal repeat, separated by adenovirus sequences and an unlabelled linear Ml3mp9 molecule (see Fig.…”

“…We investigated whether DBP could also promote intramolecular renaturation To this end we used a single sftaded Ml13 molecule in which both adenovirus qwp 2 inverted terminal repeats have been cloned in reverse orientation, separated by approximately 1300 nucleotides of adenovirus sequences (24). After appropriate renaturation this molecule can be cleaved by EcoRI, resulting in a panhandle molecule, containing the adenovirus inverted terminal repeat (Fig.…”

“…During the elongation phase DBP performs a dual role. It binds the displaced single strand and protects it from nuclease attack (21)(22)(23)(24). Furthermore it binds the double-stranded template and enhances the rate of synthesis and the processivity of the polymerase (22,23).…”

Clinical features and treatment of adenovirus infections

“…The pTP remains attached to the ITRs but becomes cleaved by the Ad proteinase into the smaller TP during the late stage of the infection. Once one of the parental strands has been duplicated entirely, the displaced single-stranded molecule can in turn serve as a template for DNA synthesis via the formation of a panhandle structure through its self-complementary ITRs [16].…”

Generation of Adenovirus Vectors Devoid of All Viral Genes by Recombination between Inverted Repeats

“…Three nonexclusive mechanisms are proposed for the second step, the replication of the displaced parental strand. (i) Displaced single strands can form partial duplexes by base pairing of the ITRs on which a second round of DNA synthesis may be initiated (22,36). (ii) When two oppositely moving displace-ment forks meet, the two parental strands can no longer be held together and therefore separate, resulting in partially duplex and partially single-stranded molecules; the synthesis is then completed on the displaced parental strand (21).…”