INTERACTION OF TERMINASE, THE DNA PACKAGING ENZYME OF PHAGE-LAMBDA, WITH ITS COS DNA SUBSTRATE

Terminase, the DNA packaging enzyme of phage lambda is an ATP-stimulat ed, site-specific endonuclease comprising the products of lambda genes Nu1 and A. The interaction of terminase with its specific DNA substra te cos was studied by footprinting. cos (the DNA segment R4-cosN-R3R2R 1), situated at the chromosomal junctions in a concatemer, consists of a nicking domain (cosN) where terminase nicks DNA to regenerate the 1 2-base cohesive ends of the mature lambda chromosome and a binding dom ain (cosB) that includes four 16-base-pair repeat sequences, R1, R2 an d R3 to the right of cosN and R4 (now called cosQ and not, in strict d efinition, part of cosB) to the left of cosN. We show that terminase m olecules bind asymmetrically to the two ends of the chromosome. Bindin g to the right of cosN is stimulated by ATP, whereas binding to the le ft of cosN is strictly dependent upon ATP. When cosN is deleted and AT P is withheld, terminase molecules bind exclusively to the R3, R2 and R1 sites via their gpNu1 subunits. An invariant R-site GG doublet is p rotected from methylation in both R3 and R2, showing the location of m ajor-groove close contacts upon binding. Terminase's interactions with DNAs that include all of cos are more extensive and are influenced by ATP; not only are the R sites protected, but so is the DNA between th em, as well as cosN, the cosN-R3 region, R4 and sequences to the left of R4. The pattern suggests an highly organized protein-DNA continuum involving several terminase molecules and several hundred base-pairs o f DNA, suitably named the termisome. Evidence is given that this assem bly is dependent on the interaction of ATP with the gpA subunit of ter minase. (C) 1995 Academic Press Limited