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 heteromultimer of gpNu1 and gpA subunits. In an earlier investigation, a lethal mutation changing gpA residue 497 from lysine to aspartic acid (K497D) was found to cause a mild change in the high-affinity ATPase that resides in gpA and a severe defect in the endonuclease activity of terminase. The K497D terminase efficiently sponsored packaging of mature lambda DNA into proheads. In the present work, K497D terminase was found to have a severe defect in the cohesive end separation, or helicase, activity. Plaque-forming pseudorevertants of lambda A K497D were found to carry mutations in A that suppressed the lethality of the A K497D mutation. The two suppressor mutations identified, 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 the endonuclease and helicase defects caused by the K497D change. Moreover, the 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 endonuclease activity of the K497D E515G enzyme was stimulated by ATP to an extent similar to the ATP stimulation of the endonuclease activity of the wild type enzyme.
Hwang
Y
Y
Department of Microbiology, College of Medicine, Iowa City, Iowa, 52242, USA.
Hang
J Q
JQ
Neagle
J
J
Duffy
C
C
Feiss
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D010582:Bacteriophage lambda / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000821:virology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D021122:Protein Subunits; D011994:Recombinant Proteins; D004706:Endodeoxyribonucleases; C030894:terminase; D004265:DNA Helicases