Binding of the junction-resolving enzyme bacteriophage T7 endonuclease I to DNA: separation of binding and catalysis by mutation.
Bacteriophage T7 endonuclease I is a resolving enzyme that selectively cleaves four-way DNA junctions, and related branched species. We have isolated mutants of this protein that retain full structural selectivity of binding to four-way junctions, but which are completely inactive as nucleases. This is consistent with a divisibility of structure-selective binding and catalysis. The mutations that inactivate endonuclease I as a nuclease are clustered into the second quarter of the primary sequence, a region that displays some sequence similarity with the related junction-resolving enzyme endonuclease VII from bacteriophage T4. This suggests that these residues may form the active site of these enzymes. The configuration of the helical arms of the junction bound by mutant endonuclease I has been investigated by gel electrophoretic methods. We find that the junction is bound in the presence or absence of magnesium ions, and that the global structure of the bound form is apparently identical with or without cations. The patterns of mobilities suggest that the structure of the junction becomes perturbed by the binding of the protein.
Duckett
D R
DR
Department of Biochemistry The University, Dundee, U.K.
Panis
M J
MJ
Lilley
D M
DM
D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D004252:DNA Mutational Analysis; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation / Q000502:physiology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic / Q000235:genetics; D013205:Staphylococcal Protein A / Q000235:genetics; D013329:Structure-Activity Relationship
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D006639:Histidine; D004247:DNA; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C035571:endodeoxyribonuclease VII; D008274:Magnesium