Michael Feiss

The Bacteriophage DNA Packaging Motor

An ATP-powered DNA translocation machine encapsidates the viral genome in the large dsDNA bacteriophages. The essential components include the empty shell, prohead, and the packaging enzyme, terminase. During translocation, terminase is docked on the proheads portal protein. The translocation ATPase and the concatemer-cutting endonuclease reside in terminase. Remarkably, terminases, portal proteins, and shells of tailed bacteriophages and herpes viruses show conserved features. These DNA viruses may have descended from a common ancestor. Terminases ATPase consists of a classic nucleotide binding fold, most closely resembling that of monomeric helicases. Intriguing models have been proposed for the mechanism of dsDNA translocation, invoking ATP hydrolysis-driven conformational changes of portal or terminase powering DNA motion. Single-molecule studies show that the packaging motor is fast and powerful. Recent advances permit experiments that can critically test the packaging models. The viral genome translocation mechanism is of general interest, given the parallels between terminases, helicases, and other motor proteins.

VIRUS DNA PACKAGING : THE STRATEGY USED BY PHAGE LAMBDA

Phage λ, like a number of other large DNA bacterio‐phages 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 coordinated with DNA packaging, the process of translocation of the cut DNA into the preformed capsid precursor, the prohead. A key player in the λ DNA packaging process is the phage‐encoded enzyme terminase, which is involved in (i) recognition of the concatemeric λ 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 lo 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 endo‐nuclease 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, gpNul, 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 λ has aspects that differ from other λ DNA transactions. Unlike the site‐specific recombination system of λ, for DNA packaging the initial site‐specific protein assemblage gives way to a mobile, translocating complete, and unlike the DNA replication system of λ, the same protein machinery is used for both initiation and translocation during λ DNA packaging.

Packaging of the bacteriophage λ chromosome: Effect of chromosome length

We performed helper-packaging experiments to investigate the relation between chromosome length and efficiency of packaging of the bacteriophage λ chromosome. In these experiments, repressed tandem prophages were packaged during lytic growth of a heteroimmune helper phage. The prophage chromosome from which packaging was initiated contained a cohesive end site (cos) duplication so that depending on which of the two sites is attacked, either a duplication or haploid chromosome was packaged. With random initiation at cos sites, the yield of duplication and haploid chromosomes is a measure of the relative efficiency of packaging of the two lengths. A number of such comparisons resulted in several conclusions. In the length range from 1.05 to 0.80, where the λ+ length is 1.00, chromosomes were packaged with constant efficiency. There was an upper limit to efficient packaging: Chromosomes with a length of 1.088 were packaged with decreased efficiency (ca. 20%). Physical studies showed that the phage particles with oversized chromosomes were fully infectious. In the vicinity of length = 0.75, there was a lower limit below which efficiency of packaging also decreased. A fraction of the particles bearing short chromosomes was not infectious, perhaps due to an injection defect. The relevance of these observations to λ DNA packaging and evolution is discussed.

Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication

SummaryWe show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr-dnaK-dnaJ-leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

The Bacteriophage DNA Packaging Machine

Large dsDNA bacteriophages and herpesviruses encode a powerful ATP-driven DNA-translocating machine that encapsidates a viral genome into a preformed capsid shell or prohead. The key components of the packaging machine are the packaging enzyme (terminase, motor) and the portal protein that forms the unique DNA entrance vertex of prohead. The terminase complex, comprised of a recognition subunit (small terminase) and an endonuclease/translocase subunit (large terminase), cuts viral genome concatemers. The terminase-viral DNA complex docks on the portal vertex, assembling a motor complex containing five large terminase subunits. The pentameric motor processively translocates DNA until the head shell is full with one viral genome. The motor cuts the DNA again and dissociates from the full head, allowing head-finishing proteins to assemble on the portal, sealing the portal, and constructing a platform for tail attachment. A body of evidence from molecular genetics and biochemical, structural, and biophysical approaches suggests that ATP hydrolysis-driven conformational changes in the packaging motor (large terminase) power DNA motion. Various parts of the motor subunit, such as the ATPase, arginine finger, transmission domain, hinge, and DNA groove, work in concert to translocate about 2 bp of DNA per ATP hydrolyzed. Powerful single-molecule approaches are providing precise delineation of steps during each translocation event in a motor that has a speed as high as a millisecond/step. The phage packaging machine has emerged as an excellent model for understanding the molecular machines, given the mechanistic parallels between terminases, helicases, and numerous motor proteins.

Terminase and the recognition, cutting and packaging of λ chromosomes

Abstract Terminase is a phage λ enzyme that has multiple functions in λ DNA packaging: it binds to λ DNA and proheads, thereby insuring packaging of the proper DNA; it introduces the staggered nicks that generate the cohesive ends of virion DNA; and it acts processively during sequential packaging of λ chromosomes. Recent studies identify the terminase binding and nicking sites on λ DNA, and show that terminase consists of a linear array of functional domains.

The terminase of bacteriophage λ: Functional domains for cosB binding and multimer assembly☆

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.

DNA Packaging and Cutting

INTRODUCTION The head of bacteriophage λ is assembled when the products of two separate synthetic pathways come together. One, the DNA replication pathway, produces a linear polymer of λ chromosomes called a concatemer. In the other pathway, λ head proteins assemble to produce the empty capsid shell known as the prohead (Georgopoulos et al., this volume). DNA and proheads interact, and a chromosome from the concatemer is inserted into the cavity of the prohead and is separated from the unencapsidated remainder of the DNA by endonucleolytic cutting. Knowledge of λ head assembly was presented by Yarmolinsky (1971) and by Kellenberger and Edgar (1971) in The Bacteriophage Lambda . The review of Hohn and Katsura (1977) offers a more recent summary of the subject. Casjens and King (1975), Murialdo and Becker (1978b), and Wood and King (1979) have written comparative reviews on head morphogenesis and DNA packaging. Most recently, the problem of DNA packaging in the double-stranded DNA phages has been updated in an elegant synthesis by Earnshaw and Casjens (1980). Because of space limitations in this paper, we restrict discussion of other viruses to results not emphasized in other reviews. CHROMOSOME STRUCTURE AND DNA PACKAGING The linear λ chromosome is injected into the cell, and annealing and ligation of the cohesive ends rapidly cyclize the molecule. Early in the growth cycle, bidirectional replication results in progeny rings. Later, rolling-circle replication produces the concatemers that are the substrates for DNA packaging (Skalka 1977; Furth and Wickner, this volume). DNA packaging generates mature...

A new host gene (groPC) necessary for lambda DNA replication

SummaryThe isolation of a bacterial mutation in a gene, designated groPC, which affects the growth of phages lambda and P2 is described. Lambda replication is severely limited in the strain, and some lambda π mutations, which map in (or near) the P gene, allow growth. The gro mutation, groPC259, is recessive to wild type and maps between threonine (thr) and diaminopimelate (dapB) on the E. coli chromosome. The possibility that the groPC gene is concerned with host DNA replication is discussed.

A functional domain of bacteriophage λ terminase for prohead binding

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage λ assembly. Terminase is made of gpNu1 and gpA, the products of the phage λ Nu1 and A genes. Early during DNA packaging terminase binds to λ DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA : terminase : prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of λ . The head genes of λ and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of λ and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, λ -21 hybrid 33, which is the product of a crossover between λ and 21 within the terminase genes. λ -21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of λ -21 hybrid 33 requires λ proheads for packaging. We have determined the position at which the crossover between λ DNA and 21 DNA occurred to produce the hybrid phage. λ -21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of λ -21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of λ gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.