Abstract
Bacteriophage lambda is assembled from preformed viral capsids (proheads), tails, and genomes that are excised from a concatemeric DNA precursor. The enzyme responsible for insertion of the genome into the precapsid is known as terminase. This enzyme possesses site-specific endonuclease, ATPase, and DNA strand separation ("helicase") catalytic activities, which work in concert to excise and package a single viral genome during phage assembly. We have previously characterized the endonuclease [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065] and ATPase [Tomka, M. A., & Catalano, C. E. (1993) Biochemistry 32, 11992-11997] catalytic activities of lambda terminase and present here similar studies on the strand separation activity of the enzyme. Strand separation requires terminase, divalent metal, and adenosine nucleotides with a hydrolyzable beta,gamma-phosphate bond. Two apparent binding sites for ATP-mediated strand separation were identified, one of which appears to be distinct from the high- and low-affinity sites previously observed for ATP hydrolysis [Hwang, Y., Catalano, C. E., & Feiss, M. (1995) Biochemistry 35, 2796-2803]. Salt stimulates the reaction at low concentrations but is strongly inhibitory at elevated concentrations, presumably due to impaired DNA binding. The above results are identical with either a complex DNA mixture (a nicked, annealed DNA duplex in the presence of excess nonspecific DNA) or a purified DNA substrate; however, a kinetic analysis of the reaction revealed that the observed rate was approximately 5-fold greater with the purified DNA substrate. Moreover, while Escherichia coli integration host factor (IHF) stimulates terminase-mediated strand separation with both substrates, the observed stimulation is more pronounced with the complex DNA mixture (10-fold rate increase) than the purified DNA substrate (5-fold rate increase). Our data are consistent with a model where IHF binding to the terminase assembly site forms a binary protein.DNA complex readily distinguishable from bulk DNA. The implications of these results to the process of DNA packaging in bacteriophage lambda are discussed.
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