title: The importance of the N-terminus of T7 endonuclease I in the interaction with DNA junctions.
abstract: T7 endonuclease I is a dimeric nuclease that is selective for four-way DNA junctions. Previous crystallographic studies have found that the N-terminal 16 amino acids are not visible, neither in the presence nor in the absence of DNA. We have now investigated the effect of deleting the N-terminus completely or partially. N-terminal deleted enzyme binds more tightly to DNA junctions but cleaves them more slowly. While deletion of the N-terminus does not measurably affect the global structure of the complex, the presence of the peptide is required to generate a local opening at the center of the DNA junction that is observed by 2-aminopurine fluorescence. Complete deletion of the peptide leads to a cleavage rate that is 3 orders of magnitude slower and an activation enthalpy that is 3-fold higher, suggesting that the most important interaction of the peptide is with the reaction transition state. Taken together, these data point to an important role of the N-terminus in generating a central opening of the junction that is required for the cleavage reaction to proceed properly. In the absence of this, we find that a cruciform junction is no longer subject to bilateral cleavage, but instead, just one strand is cleaved. Thus, the N-terminus is required for a productive resolution of the junction.
authors.affiliation: Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee DD1 5EH, UK.
authors.forename: Alasdair D J
authors.initials: AD
authors.lastname: Freeman
authors.forename: Anne-Cécile
authors.initials: AC
authors.lastname: Déclais
authors.forename: David M J
authors.initials: DM
authors.lastname: Lilley
mesh_terms: D017123:Bacteriophage T7 / Q000201:enzymology*; D001665:Binding Sites; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D010446:Peptide Fragments; D003850:Deoxyribonuclease I