Journal of Molecular Biology
A Defined in Vitro System for DNA Packaging by the Bacteriophage SPP1: Insights into the Headful Packaging Mechanism
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.
References (48)
- et al.
Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4
J. Mol. Biol.
(1999) - et al.
Specific interaction of terminase, the DNA packaging enzyme of bacteriophage λ, with the portal protein of the prohead
J. Mol. Biol.
(1995) - et al.
Sequential headful packaging and fate of the cleaved DNA ends in bacteriophage SPP1
J. Mol. Biol.
(1996) - et al.
Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing gene 1 to 6: the products of gene 1 and gene 2 are required for pac cleavage
J. Mol. Biol.
(1992) - et al.
Characterization of the small subunit of the terminase enzyme of the Bacillus subtilis bacteriophage SPP1
Virology
(1998) - et al.
Analysis of the Bacillus subtilis bacteriophage SPP1 and SF6 gene 1 product: a protein involved in the initiation of headful packaging
Virology
(1994) - et al.
Functional analysis of the terminase large subunit, gp2, of Bacillus subtilis bacteriophage SPP1
J. Biol. Chem.
(2000) In vitro packaging of bacteriophage T4 DNA
Virology
(1981)- et al.
A defined in vitro system for packaging of bacteriophage T3 DNA
Virology
(1986) - et al.
Biochemical characterization of bacteriophage lambda genome packaging in vitro
Virology
(2003)
Bacillus subtilis bacteriophage SPP1 DNA packaging motor requires terminase and portal proteins
J. Biol. Chem.
Early events in DNA packaging in a defined in vitro system of bacteriophage T3
Virology
In vitro packaging of bacteriophage φ29 DNA restriction fragments and the role of the terminal protein gp3
J. Mol. Biol.
Construction of a defined in vitro DNA packaging system using purified terminase proteins
J. Mol. Biol.
In vitro packaging of DNA of the Bacillus subtilis bacteriophage SPP1
J. Mol. Biol.
Characterization of the bacteriophage T3 DNA packaging reaction in vitro in a defined system
J. Mol. Biol.
Structural organisation of the head-to-tail interface of a bacterial virus
J. Mol. Biol.
A discontinuous headful packaging model for packaging of less than headful length DNA molecules by bacteriophage T4
J. Mol. Biol.
Headful packaging revisited: the packaging of more than one DNA molecule into a bacteriophage P1 head
J. Mol. Biol.
The small subunit of the terminase enzyme of Bacillus subtilis bacteriophage SPP1 forms a specialized nucleoprotein complex with the packaging initiation region
J. Mol. Biol.
Bacillus subtilis bacteriophage SPP1 terminase has a dual activity: it is required for the packaging initiation and represses its own synthesis
Gene
In vitro packaging of mature phage DNA by Salmonella phage P22
Virology
In vitro packaging of plasmid DNA oligomers by salmonella phage P22: independence of the pac site, and evidence for the termination cut in vitro
Virology
A phage T4 in vitro packaging system for cloning long DNA molecules
Gene
Cited by (40)
Viral genome packaging machines: Structure and enzymology
2021, EnzymesCitation Excerpt :Terminase enzymes generally function as heterooligomeric complexes consisting of a large catalytic subunit that contains both maturation and packaging activities (TerL) and a small DNA recognition subunit that is required for site-specific assembly at the packaging initiation site (TerS; Fig. 1C); both subunits are essential for virus development in vivo [5,6,14,27]. In some cases, such as phages λ and P22, the holoenzymes can be isolated as stable complexes of both subunits [28,29], while in others, such as phages T4 and SPP1, the two subunits do not strongly interact until they assemble the maturation complex on DNA [30–32]. Although phage ϕ29-like viruses do not encode a DNA recognition subunit (TerS), they must similarly solve the problem of finding and binding their genomic DNA end among the multitude of nucleic acids in the cell.
Conformational changes leading to T7 DNA delivery upon interaction with the bacterial receptor
2015, Journal of Biological ChemistryFunction and horizontal transfer of the small terminase subunit of the tailed bacteriophage Sf6 DNA packaging nanomotor
2013, VirologyCitation Excerpt :In apparent contradiction to this view, removal of twenty C-terminal amino acids, which make up most of the C-terminal tubular β-barrel domain of the P22 TerS, does not impair its oligomerization but does block its DNA binding capability (Nemecek et al., 2008; Roy et al., 2012). T4 TerS is dispensable in a T4 in vitro DNA packaging system (Al-Zahrani et al., 2009; Zhang et al., 2011), while in P22 and SPP1 TerS protein is required but specific recognition of the packaging target site is not required in vitro (Poteete and Botstein, 1979; Schmieger, 1984; Schmieger and Koch, 1987; Oliveira et al., 2005). Thus much remains to be understood regarding small terminase subunit function and its role in DNA packaging motor initiation.
Headful DNA packaging: Bacteriophage SPP1 as a model system
2013, Virus ResearchCitation Excerpt :However, initiation of packaging shall start at different regions of the T4 genome as this phage shows complete circular permutation (Streisinger et al., 1967). The virulent Bacillus subtilis siphovirus SPP1 is a well-established model system for phages that package their DNA by a headful mechanism (Dröge and Tavares, 2000, 2005; Oliveira et al., 2005). Encapsidation is initiated by recognition and cleavage by the terminase of the nucleotide sequence pac followed by unidirectional translocation of a concatemeric DNA to the interior of the procapsid.
"Push Through One-Way Valve" Mechanism of Viral DNA Packaging
2012, Advances in Virus Research