Mutations Affecting the High Affinity ATPase Center of gpA, the Large Subunit of Bacteriophage λ Terminase, Inactivate the Endonuclease Activity of Terminase

Abstract

Phage λ terminase carries out the coscleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimu lated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K 497A and K 497D were found to be lethal, and phages carrying these mutations were defective in coscleavage, in vivo. Purified K 497A and K 497D enzymes cleaved cos in vitroat rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in coscleavage are sufficient to explain the lethality of the K 497A and K 497D defects. In in vitropackaging studies using mature (cleaved) phage DNA, the K 497A enzyme was indistinguishable from the wild-type enzyme, and the K 497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K 497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 μM ATP, indicating that the K 497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.