title: The in vitro endonuclease activity of gene product A, the large subunit of the bacteriophage lambda terminase, and its relationship to the endonuclease activity of the holoenzyme.
abstract: The reaction requirements and kinetic properties of the in vitro endonuclease activity of the bacteriophage lambda terminase and its large subunit, gene product (gp) A, have been analyzed. Optimal cleavage reaction activity for both proteins requires Mg2+, a pH between 8.5 and 9.0, and is enhanced by ATP or ATP analogs. Under these conditions both terminase and gpA generate aberrant nicks in and around cosN. Optimal nicking specificity of terminase is observed under conditions of 50-100 mM salt, 5 mM spermidine, 1.5 mM ATP, and a pH between 7.0 and 7.5. Specific activity of terminase is greatly reduced under these conditions, and gpA is completely inactive at all protein concentrations tested. Under optimal reaction conditions, gpA endonuclease activity differs from that of the holoenzyme in that it can only be detected at high concentrations, is strongly protein concentration-dependent, and can not be stimulated by the Escherichia coli protein integration host factor. Addition of purified gpNu1 partially, but not completely, minimized these differences, suggesting that the role of gpNu1 in the holoenzyme is to modulate the basal endonuclease of gpA.
authors.affiliation: Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Rubinchik
authors.forename: W
authors.initials: W
authors.lastname: Parris
authors.forename: M
authors.initials: M
authors.lastname: Gold
mesh_terms: D010582:Bacteriophage lambda / Q000201:enzymology*; D001483:Base Sequence; D004279:DNA, Viral; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004279:DNA, Viral; D010446:Peptide Fragments; D004706:Endodeoxyribonucleases; C030894:terminase