Endonuclease and helicase activities of bacteriophage lambda terminase: Changing nearby residue 515 restores activity to the gpA K497D mutant enzyme

Terminase, the DNA packaging enzyme of bacteriophage lambda, is a heteromul timer of gpNu1 and gpA subunits. In an earlier investigation, a lethal muta tion changing gpA residue 497 from lysine to aspartic acid (K497D) was foun d to cause a mild change in the high-affinity ATPase that resides in gpA an d a severe defect in the endonuclease activity of terminase. The K497D term inase efficiently sponsored packaging of mature lambda DNA into proheads. I n the present work, K497D terminase was found to have a severe defect in th e cohesive end separation, or helicase, activity. Plaque-forming pseudoreve rtants of lambda A K497D were found to carry mutations in A that suppressed the lethality of the A K497D mutation. The two suppressor mutations identi fied, A E515G and A E515K, affected residue 515, which is located near the putative P-loop of gpA. A codon substitution study of codon 515 showed that hydrophobic and basic residues suppress the K497D defect, but hydrophilic and acidic residues do not. The E515G change was demonstrated to reverse th e endonuclease and helicase defects caused by the K497D change. Moreover, t he gpA K497D E515G enzyme was found to have kinetic constants for the high- affinity ATPase center similar to those of the wild type enzyme, and the en donuclease activity of the K497D E515G enzyme was stimulated by ATP to an e xtent similar to the ATP stimulation of the endonuclease activity of the wi ld type enzyme. (C) 2000 Academic Press.