The active site of the junction-resolving enzyme T7 endonuclease I.
Endonuclease I is a junction-resolving enzyme encoded by bacteriophage T7, that selectively binds and cleaves four-way DNA junctions. We have recently solved the structure of this dimeric enzyme at atomic resolution, and identified the probable catalytic residues. The putative active site comprises the side-chains of three acidic amino acids (Glu20, Asp55 and Glu65) together with a lysine residue (Lys67), and shares strong similarities with a number of type II restriction enzymes. However, it differs from a typical restriction enzyme as the proposed catalytic residues in both active sites are contributed by both polypeptides of the dimer. Mutagenesis experiments confirm the importance of all the proposed active site residues. We have carried out in vitro complementation experiments using heterodimers formed from mutants in different active site residues, showing that Glu20 is located on a different monomer from the remaining amino acid residues comprising the active site. These experiments confirm that the helix-exchanged architecture of the enzyme creates a mixed active site in solution. Such a composite active site structure should result in unilateral cleavage by the complemented heterodimer; this has been confirmed by the use of a cruciform substrate. Based upon analogy with closely similar restriction enzyme active sites and our mutagenesis experiments, we propose a two-metal ion mechanism for the hydrolytic cleavage of DNA junctions.
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CRC Nucleic Acid Structure Research Group, Department of Biochemistry, The University of Dundee, Dundee, DD1 4HN, UK.
Hadden
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J
Phillips
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SE
Lilley
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DM
D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D004278:DNA, Superhelical / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005816:Genetic Complementation Test; D007700:Kinetics; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D003850:Deoxyribonuclease I