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-stimulated, site-specific endonuclease comprising the products of lambda genes Nu1 and A. The interaction of terminase with its specific DNA substrate cos was studied by footprinting. cos (the DNA segment R4-cosN-R3R2R1), situated at the chromosomal junctions in a concatemer, consists of a nicking domain (cosN) where terminase nicks DNA to regenerate the 12-base cohesive ends of the mature lambda chromosome and a binding domain (cosB) that includes four 16-base-pair repeat sequences, R1, R2 and R3 to the right of cosN and R4 (now called cosQ and not, in strict definition, part of cosB) to the left of cosN. We show that terminase molecules bind asymmetrically to the two ends of the chromosome. Binding to the right of cosN is stimulated by ATP, whereas binding to the left of cosN is strictly dependent upon ATP. When cosN is deleted and ATP is withheld, terminase molecules bind exclusively to the R3, R2 and R1 sites via their gpNu1 subunits. An invariant R-site GG doublet is protected from methylation in both R3 and R2, showing the location of major-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 them, 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 of DNA, suitably named the termisome. Evidence is given that this assembly is dependent on the interaction of ATP with the gpA subunit of terminase.
Higgins
R R
RR
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Becker
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels; D010582:Bacteriophage lambda / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014764:Viral Proteins / Q000378:metabolism
D000345:Affinity Labels; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C030894:terminase