The importance of the N-terminus of T7 endonuclease I in the interaction with DNA junctions

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    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. (C) 2012 Elsevier Ltd. All rights reserved.

    Original languageEnglish
    Pages (from-to)395-410
    Number of pages16
    JournalJournal of Molecular Biology
    Volume425
    Issue number2
    DOIs
    Publication statusPublished - 23 Jan 2013

    Keywords

    • BACTERIOPHAGE T7
    • ESCHERICHIA-COLI
    • RESOLVING ENZYME CCE1
    • STRUCTURAL RECOGNITION
    • REPLICATION FORKS
    • HOMOLOGOUS RECOMBINATION
    • GENETIC-RECOMBINATION
    • HOLLIDAY JUNCTION
    • SULFOLOBUS-SOLFATARICUS
    • SACCHAROMYCES-CEREVISIAE

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