Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase.
Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated 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 K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D 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.
Hwang
Y
Y
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Feiss
M
M
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000595:Amino Acid Sequence; D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D004279:DNA, Viral / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D019065:Virus Assembly / Q000235:genetics*
D004279:DNA, Viral; D000255:Adenosine Triphosphate; D004706:Endodeoxyribonucleases; C030894:terminase; D000251:Adenosine Triphosphatases