The DNA Maturation Domain of gpA, the DNA Packaging Motor Protein of Bacteriophage Lambda, Contains an ATPase Site Associated with Endonuclease Activity

Abstract

SummaryTerminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N-terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in E. coli. Biochemical characterization of gpA-ΔN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N-terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A "P-loop" sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme -DNA maturation and DNA packaging -are discussed.

Packaging of a Unit-Length Viral Genome: The Role of Nucleotides and the gpD Decoration Protein in Stable Nucleocapsid Assembly in Bacteriophage λ

“…28 Furthermore, the C 1/2 obtained in this experiment is essentially identical with the K m for the packaging ATPase catalytic site in gpA (∼5 μM). 13,39,40 The data are well described by a cooperative binding (Hill) model, and nonlinear least squares analysis affords a Hill coefficient of 3.3 ± 0.5. This suggests that the functional packaging motor is composed, minimally, of four ATPase catalytic sites that act in a cooperative manner to package DNA.…”

“…1b). 13 This affords complex I, a packaging intermediate in which the left "sticky" end of the viral genome (D L ) is tightly bound and protected by terminase (Fig. 1a).…”

Energy-Independent Helicase Activity of a Viral Genome Packaging Motor

“…The nicked, annealed duplex is G-C rich and strand separation requires the so-called “helicase” activity of TerL (24, 25). Both reactions, which in combination represent DNA maturation (Figure 1), require a dedicated maturation ATPase site in a C-terminal domain in gpA (26-28). DNA maturation affords complex I, a stable intermediate composed of terminase tightly and specifically bound to the mature left end of the λ genome (D L ).…”

Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

“…Terminase possesses three discrete ATPase catalytic activities that are involved putatively in (i) assembly of the motor at cos (gpNu1 ATPase), (ii) modulation of the endonuclease/strand-separation activities of the enzyme, and (iii) fueling motor translocation (packaging ATPase) (6,31). The packaging ATPase activity can be monitored selectively by using low concentrations of ATP (50 M) in the reaction mixture because the K m for the packaging ATPase is significantly lower than the other two sites (5 M versus 500 M) (29,32).…”