A defined in vitro system for DNA packaging by the bacteriophage SPP1: insights into the headful packaging mechanism.
Tailed icosahedral bacteriophages and other viruses package their double-stranded DNA inside a preformed procapsid. In a large number of phages packaging is initiated by recognition and cleavage by a viral packaging ATPase (terminase) of the specific pac sequence (pac cleavage), which generates the first DNA end to be encapsidated. A sequence-independent cleavage (headful cleavage) terminates packaging, generating a new starting point for another round of packaging. The molecular mechanisms underlying headful packaging and its processivity remain poorly understood. A defined in vitro DNA packaging system for the headful double-stranded DNA bacteriophage SPP1 is reported. The in vitro system consists of DNA packaging reactions with highly purified terminase and SPP1 procapsids, coupled to a DNase protection assay. The high yield obtained enabled us to quantify directly the efficiency of DNA entry into the procapsids. We show that in vitro DNA packaging requires the presence of both terminase subunits. The SPP1 in vitro system is able to efficiently package mature SPP1 DNA as well as linear plasmid DNAs. In contrast, no DNA packaging could be detected with circular DNA, signifying that in vitro packaging requires free DNA extremities. Finally, we demonstrate that SPP1 in vitro DNA packaging is independent of the pac signal. These findings suggest that the formation of free DNA ends that are generated by pac cleavage in vivo is the rate-limiting step in processive headful DNA packaging.
Oliveira
Leonor
L
Unité de Virologie Moléculaire et Structurale, UMR CNRS 2472, UMR INRA 1157 and IFR 115, Bat. 14B, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France. oliveira@vms.cnrs-gif.fr
Alonso
Juan C
JC
Tavares
Paulo
P
D001435:Bacteriophages / Q000235:genetics / Q000502:physiology*; D004279:DNA, Viral / Q000235:genetics*; D004720:Endonucleases / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D015398:Signal Transduction; D013379:Substrate Specificity
D004279:DNA, Viral; D004720:Endonucleases