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 exci sion of a single genome from a concatameric DNA precursor and its inse rtion into an empty viral procapsid. The enzyme possesses a site-speci fic endonuclease activity which is responsible for excision of the vir al genome and the formation of the 12 base-pair single-stranded ''stic ky'' 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 o f the reaction [Tomka, M. A., Br 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 catalytic ally competent prenicking complex. Here, we provide additional evidenc e for a slow assembly step in the nuclease reaction and demonstrate th at the observed rate is affected by protein concentration, but not by the length of the DNA substrate. Consistent with our model, preincubat ion of terminase with DNA also yields an observable fast phase of the reaction, but only when large (greater than or equal to 3 kb) DNA subs trates are used. Finally, we present data which demonstrate that phage lambda terminase can efficiently utilize DNA from the closely related phage phi 21 as an endonuclease substrate and that the enzyme binds e fficiently to the cosB region of both phage genomes. The implications of these results with respect to the assembly of a catalytically compe tent nucleoprotein complex required to initiate genome packaging are d iscussed.