Kinetic analysis of the endonuclease activity of phage lambda terminase: assembly of a catalytically competent nicking complex is rate-limiting.
The terminase enzyme from bacteriophage lambda is responsible for excision of a single genome from a concatameric DNA precursor and its insertion into an empty viral procapsid. The enzyme possesses a site-specific endonuclease activity which is responsible for excision of the viral genome and the formation of the 12 base-pair single-stranded "sticky" ends of mature lambda DNA. We have previously reported a kinetic analysis of the endonuclease activity of lambda terminase which showed an enzyme concentration-dependent change in the kinetic time course of the reaction [Tomka, M. A., & Catalano, C. E. (1993b) J. Biol. Chem. 268, 3056-3065]. We presented a model which suggested that the rate-limiting step in the nuclease reaction was the assembly of a catalytically competent prenicking complex. Here, we provide additional evidence for a slow assembly step in the nuclease reaction and demonstrate that the observed rate is affected by protein concentration, but not by the length of the DNA substrate. Consistent with our model, preincubation of terminase with DNA also yields an observable fast phase of the reaction, but only when large (> or = 3 kb) DNA substrates are used. Finally, we present data which demonstrate that phage lambda terminase can efficiently utilize DNA from the closely related phage phi21 as an endonuclease substrate and that the enzyme binds efficiently to the cosB region of both phage genomes. The implications of these results with respect to the assembly of a catalytically competent nucleoprotein complex required to initiate genome packaging are discussed.
Woods
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Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
Terpening
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Catalano
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D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D034982:Integration Host Factors; D007700:Kinetics; D017105:Pseudomonas Phages / Q000201:enzymology / Q000235:genetics; D013728:Terminator Regions, Genetic
D001426:Bacterial Proteins; D034982:Integration Host Factors; C440503:integration host factor, Pseudomonas; D004706:Endodeoxyribonucleases; C030894:terminase