1) Energetics and mechanism of ATP-driven DNA translocation into the prohead; 2) Structure of packaged phage DNA; 3) Terminase and headful cutting of DNA in packaging; 4) Interactions ofd DNA packaging with other DNA processes in vivo; 5) DNA structural requirements for packaging; 6) In vitro packaging in recombinant DNA

DNA Packaging in dsDNA Bacteriophages

Phage lambda, like a number of other large DNA bacteriophages and the herpesviruses, produces concatemeric DNA during DNA replication. The concatemeric DNA is processed to produce unit-length, virion DNA by cutting at specific sites along the concatemer. DNA cutting is co-ordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the lambda DNA packaging process is the phage-encoded enzyme terminase, which is involved in (i) recognition of the concatemeric lambda DNA; (ii) initiation of packaging, which includes the introduction of staggered nicks at cosN to generate the cohesive ends of virion DNA and the binding of the prohead; (iii) DNA packaging, possibly including the ATP-driven DNA translocation; and (iv) following translocation, the cutting of the terminal cosN to complete DNA packaging. To one side of cosN is the site cosB, which plays a role in the initiation of packaging; along with ATP, cosB stimulates the efficiency and adds fidelity to the endonuclease activity of terminase in cutting cosN. cosB is essential for the formation of a post-cleavage complex with terminase, complex I, that binds the prohead, forming a ternary assembly, complex II. Terminase interacts with cosN through its large subunit, gpA, and the small terminase subunit, gpNu1, interacts with cosB. Packaging follows complex II formation. cosN is flanked on the other side by the site cosQ, which is needed for termination, but not initiation, of DNA packaging. cosQ is required for cutting of the second cosN, i.e. the cosN at which termination occurs. DNA packaging in lambda has aspects that differ from other lambda DNA transactions. Unlike the site-specific recombination system of lambda, for DNA packaging the initial site-specific protein assemblage gives way to a mobile, translocating complex, and unlike the DNA replication system of lambda, the same protein machinery is used for both initiation and translocation during lambda DNA packaging.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1365-2958.1995.tb02333.x

Virus DNA packaging: the strategy used by phage lambda.

This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage λ genome packaging. The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a preformed capsid. Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging ‘machine.’ Models for the assembly and interconversion of these complexes are presented. Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed. This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription. Terminase is thus part of a growing number of examples of biological ‘machines’ or molecular ‘motors.’

The terminase enzyme from bacteriophage lambda: a DNA-packaging machine.

The mature chromosome of bacteriophage X is a linear duplex of a unique sequence 48,502 nucleotide pairs long (34). The 5'-terminal ends of this molecule protrude as self-complementary single-stranded chains of 12 nucleotides which can anneal to generate circles. When a X DNA molecule is injected into the host, it is circularized and closed covalently by DNA ligase action. Early during lytic infection, X DNA undergoes several rounds of replication in which circles generate circles. At later times of infection, rolling-circle replication takes over and individual rings give rise to linear concatemers (3, 8, 9, 28) which may be 10 chromosomal units long (in the absence of packaging). During DNA packaging, monomeric chromosomal units are cut from these replicative oligomers by the enzyme X DNA terminase, which regenerates the unique single-stranded ends by introducing two specific nicks staggered 12 nucleotides apart on opposite strands of the duplex, followed by melting of the joint. The DNA site of action of terminase is called cos. By its interaction with cos, terminase not only catalyzes the formation of ends but, in coupling proheads to DNA, establishes the origin and direction of packaging. The overview model for packaging and cutting of X DNA which is currently accepted is as follows: (i) random selection of a cos site from among those present on a concatemer for packaging initiation, (ii) polarity of chromosome entry into the prohead from the Nul end to the R end, (iii) dependence of cos cutting on the packaging of a minimal length ofDNA, and (iv) processive packaging oftwo or three chromosomes in series from a concatemer (8, 9, 28). For previous reviews on this subject, see references 8, 9, and 28. With a few exceptions, references included in these reviews will not be cited specifically; instead, the reader will be directed to one or more of these three reviews, as the case may be.

K Matsubara

Papers

Lambda phage DNA sequences affecting the packaging process.