Cloning, Expression, and Biochemical Characterization of Hexahistidine-tagged Terminase Proteins*

  title={Cloning, Expression, and Biochemical Characterization of Hexahistidine-tagged Terminase Proteins*},
  author={Q Hang and L. Woods and Michael Feiss and Carlos Enrique Catalano},
  journal={The Journal of Biological Chemistry},
  pages={15305 - 15314}
The terminase enzyme from bacteriophage λ is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage λ and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the… 

Biophysical Characterization of the DNA Binding Domain of gpNu1, a Viral DNA Packaging Protein*

The data suggest that gpNU1ΔE68 represents the minimal DNA binding domain of gpNu1, a protein consisting of the N-terminal 68 residues of gpnu1 that is a dimer with no evidence of dissociation or further aggregation.

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

  • C. Catalano
  • Biology, Chemistry
    Cellular and Molecular Life Sciences CMLS
  • 2000
Abstract. 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

Biochemical characterization of bacteriophage lambda genome packaging in vitro.

The construction of a vector that expresses lambda procapsids with a yield that is 40-fold greater than existing systems is described, and evidence for a packaging-stimulated ATPase activity is provided, quantifying the energetic cost of DNA packaging in bacteriophage lambda.

Nucleotides regulate the conformational state of the small terminase subunit from bacteriophage lambda: implications for the assembly of a viral genome-packaging motor.

In this work, nucleotide binding to the gpNu1-DBD was probed using acrylamide fluorescence quenching and fluorescence-monitored ligand binding studies, and the data indicate that the minimal DBD dimer binds both ATP and ADP at two equivalent but highly cooperative binding sites.

Functional identification of the DNA packaging terminase from Pseudomonas aeruginosa phage PaP3

The results presented here suggest that PaP3 utilizes a typical cos site mechanism for DNA packaging and pro- vide a first step towards understanding the molecular mechanism of the PaP 3 DNA packaging reaction.

ATPase center of bacteriophage lambda terminase involved in post-cleavage stages of DNA packaging: identification of ATP-interactive amino acids.

Results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging.

Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor.

Experimental support is provided for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay and circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates.

Energy-independent helicase activity of a viral genome packaging motor.

The assembly of complex double-stranded DNA viruses includes a genome packaging step where viral DNA is translocated into the confines of a preformed procapsid shell. In most cases, the preferred



Cloning, expression, and characterization of a DNA binding domain of gpNu1, a phage lambda DNA packaging protein.

It is proposed that the hydrophobic amino acids found between Lys100 and Pro141 define a self-association domain that is required for the assembly of stable nucleoprotein packaging complexes and that the C-terminal tail of the protein defines a distinct gpA-binding site that is responsible for terminase holoenzyme formation.

Kinetic and mutational dissection of the two ATPase activities of terminase, the DNA packaging enzyme of bacteriophage Chi.

The results indicate the presence of a low-affinity, DNA-stimulated ATPase center in gpNul, and a high-Affinity site in gpA, which is not restored by increased DNA.

Kinetic characterization of the ATPase activity of the DNA packaging enzyme from bacteriophage lambda.

Terminases are enzymes common to all of the complex double-stranded DNA viruses and are required for viral assembly. These enzymes function to excise a single viral genome from a concatemeric DNA