Catalytic and binding mutants of the junction-resolving enzyme endonuclease I of bacteriophage t7: role of acidic residues.
Endonuclease I is a 149 amino acid protein of bacteriophage T7 that is a Holliday junction-resolving enzyme, i.e. a four-way junction-selective nuclease. We have performed a systematic mutagenesis study of this protein, whereby all acidic amino acids have been individually replaced by other residues, mainly alanine. Out of 21 acidic residues, five (Glu20, Glu35, Glu65, Asp55 and Asp74) are essential. Replacement of these residues by other amino acids leads to a protein that is inactive in the cleavage of DNA junctions, but which nevertheless binds selectively to DNA junctions. The remaining 16 acidic residues can be replaced without loss of activity. The five critical amino acids are located within one section of the primary sequence. It is rather likely that their function is to bind one or more metal ions that coordinate the water molecule that brings about hydrolysis of the phosphodiester bond. We have also constructed a mutant of endonuclease I that lacks nine amino acids (six of which are arginine or lysine) at the C-terminus. Unlike the acidic point mutants, the C-terminal truncation is unable to bind to DNA junctions. It is therefore likely that the basic C-terminus is an important element in binding to the DNA junction.
Parkinson
M J
MJ
CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, Dundee DD1 4HN, UK.
Pöhler
J R
JR
Lilley
D M
DM
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000201:enzymology*; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010446:Peptide Fragments; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C035571:endodeoxyribonuclease VII; D000409:Alanine