The Importance of the N-Terminus of T7 Endonuclease I in the Interaction with DNA Junctions

• Published in: Journal of molecular biology Vol. 425; no. 2; pp. 395 - 410
• Main Authors: Freeman, Alasdair D.J., Déclais, Anne-Cécile, Lilley, David M.J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 23.01.2013
• Subjects: amino acids; Bacteriophage T7; Bacteriophage T7 - enzymology; Binding Sites; chemistry; Deoxyribonuclease I; Deoxyribonuclease I - chemistry; Deoxyribonuclease I - genetics; Deoxyribonuclease I - metabolism; DNA; DNA Primers; DNA Primers - chemistry; DNA repair; DNA, Bacterial; DNA, Bacterial - chemistry; DNA, Bacterial - metabolism; DNA–protein interactions; enthalpy; enzymology; fluorescence; genetics; junction-resolving enzymes; metabolism; Models, Molecular; nucleases; Nucleic Acid Conformation; Peptide Fragments; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding; structure-selective recognition; BSA; TEV; structure-selective recognition; nucleases; DNA–protein interactions; EDTA; junction-resolving enzymes; 2-AP; DNA repair
• ISSN: 0022-2836; 1089-8638; 1089-8638
• DOI: 10.1016/j.jmb.2012.11.029

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. [Display omitted] ► Endonuclease I selectively binds and cleaves four-way DNA junctions. ► Its N-terminal peptides are not visible in crystal structures with or without DNA. ► The peptides facilitate a central opening of the junction required for coordinated bilateral cleavage. ► In their absence, cleavage rates are slowed 3 orders of magnitude. ► The N-termini are essential for productive resolution of DNA junctions.