Journal of Molecular Biology
Volume 353, Issue 3, 28 October 2005, Pages 529-539
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A Defined in Vitro System for DNA Packaging by the Bacteriophage SPP1: Insights into the Headful Packaging Mechanism

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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.

Introduction

One of the most fascinating and intriguing issues in the field of viral morphogenesis is the mechanism by which DNA is packaged into a preformed protein shell (prohead or procapsid). DNA packaging into procapsids is a general strategy for chromosome condensation followed by many tailed double-stranded DNA (dsDNA) bacteriophages and also by some animal viruses such as herpes virus.1, 2, 3 DNA packaging in these viruses requires assembly of a complex molecular machine at a specific vertex of the icosahedral procapsid. This vertex is characterized by the presence of the portal protein, a turbine-like oligomer possessing a central channel through which DNA translocation occurs.4, 5, 6, 7, 8 Initiation of packaging involves the specific interaction of the viral DNA concatemer with the viral-encoded DNA recognition and cleavage protein complex, named terminase. This complex is normally composed of two subunits, a small terminase subunit involved in DNA recognition (termed gp1 in phage SPP1), and a large terminase subunit (termed gp2 in SPP1) possessing endonucleolytic and ATPase activities. The DNA-terminase nucleoprotein complex then interacts with the portal vertex and DNA translocation begins, fuelled by the ATPase activity of the terminase.9, 10, 11

Two general modes for packaging in dsDNA virus have been described. In the first one, packaging and cutting occur specifically at a unique and precise sequence (termed cos in phage λ), thus generating unit-length encapsidated molecules. This packaging process is well characterized in phages like λ, T3 and T7.12, 13 In the second mode (phages like P22, T4, P1, T1 and SPP1), headful sized DNA fragments are sequentially packaged from the concatemeric DNA precursor.3, 14 Packaging starts with the recognition of a specific sequence, called pac in SPP1,15 leading to an initial endonucleolytic cut (pac cleavage). When a threshold amount of DNA, representing ∼103% of the genome, has been packaged, the terminase introduces a sequence-independent cut (headful cleavage). This termination cut separates the first headful from the concatemer, which serves now as a substrate to fill a second prohead. Subsequent encapsidation begins at the end created by the previous event, regardless of the position in the genome, and proceeds unidirectionally (Figure 1). This processive headful mechanism leads to the generation of terminally redundant and partially circularly permuted DNA molecules.3 The DNA packaging machinery thus uses two substrates for packaging: a pac sequence, in the first packaging cycle, and a DNA end generated by the headful cleavage in the following encapsidation cycles. The control of substrate specificity by the packaging apparatus is essential to ensure the processivity of the reaction that can lead to more than 12 sequential packaging cycles along a single substrate concatemer.14, 16 The study of this and other key questions was hampered to a significant extent by the lack of an in vitro DNA encapsidation system with purified components whose yield is enough high to assay directly the DNA encapsidated by a headful packaging motor. Here we describe the first system of this type using purified terminase subunits and procapsids of bacteriophage SPP1. Our results provide insights into the substrate specificity of the DNA packaging machinery and enable us to propose a model to explain the processivity of the headful packaging reaction.

Section snippets

Purification of the SPP1 DNA packaging components

For studying DNA packaging in vitro it is vital to maximize control of the compounds present during the packaging reaction. This requires the production of sufficient amounts of purified and biologically active subunits of the terminase and procapsids. In view of that we attempted to optimize the production and purification of the small and large SPP1 terminase subunits, gp1 and gp2, respectively, as well as SPP1 procapsids.

Gp1 and gp2 were produced and purified from Escherichia coli cells as

Discussion

Viral DNA packaging is a complex process requiring intricate interactions between the substrate DNA, the two terminase subunits and the procapsid portal vertex.1, 2, 3, 4 The key questions that remain to be addressed in molecular detail include the assembly of the packaging machinery, the dynamics of DNA translocation, termination of the DNA encapsidation reaction, and the basis of processive headful DNA packaging. Their study requires the use of performing in vitro defined DNA packaging

Bacterial strains, plasmids and phages

Escherichia coli strains BL21(DE3) and BL21(DE3)(pLysS) were as described.44 SPP1sus7015 and plasmids pCB191,17 pBT11515 and pREP4 (QIAGEN) have been described. Plasmid pSK (pBluescript SK+) was from Stratagene.

Enzymes and reagents

Ultrapure acrylamide and isopropyl-1-thio-β-d-galactopyranoside (IPTG) were purchased from Euromedex; agarose was from BioRad. Syber Gold was from Molecular Probes. Protein molecular mass markers and DNA restriction enzymes were from Biolabs. Proteinase K was from Roche. Lysozyme, DNase,

Acknowledgements

We thank Dr Jean-Lepault, Unité de Virologie Moléculaire et Structurale, for electron microscopy observations of SPP1 procapsids. This work was supported by grants from the CNRS (ATIP) and from the Fondation pour la Recherche Médicale, France (to P.T.). L.O. was supported by grants from the European Molecular Biology Organization and from the Fundação para a Ciência e a Tecnologia, Ministério da Ciência e Ensino Superior, Portugal.

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