Analysis of Conformational Changes in the DNA Junction-Resolving Enzyme T7 Endonuclease I on Binding a Four-Way Junction Using EPR

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    The four-way (Holiday) DNA junction is the central intermediate in homologous recombination. It is ultimately resolved into two nicked-duplex species by the action of a junction-resolving enzyme. These enzymes are highly selective for the structure of branched DNA, yet as a class these proteins impose significant distortion on their target junctions. Bacteriophage T7 endonuclease I selectively binds and cleaves DNA four-way junctions. The protein is an extremely stable dimer, comprising two globular domains joined by a beta-strand bridge with each active site including amino acids from both polypeptides. The crystal structure of endonuclease I has been solved both as free protein and in complex with a DNA junction, showing that the protein, as well as the junction, becomes distorted on binding. We have therefore used site-specific spin-labeling in conjunction with EPR distance measurements to analyze induced fit in the binding of endonuclease I to a DNA four-way junction. The results support the change in protein structure as it binds to the junction. In addition, we have examined the structure of wild type and catalytically inactive mutants alone and in complex with DNA. We demonstrate the presence of hitherto undefined metastable conformational states within endonuclease I, showing how these states can be influenced by DNA-junction binding or mutations within the active sites. In addition, we demonstrate a previously unobserved instability in the N-terminal alpha 1-helix upon active site mutation. These studies reveal that structural changes in both DNA and protein occur in the action of this junction-resolving enzyme.

    Original languageEnglish
    Pages (from-to)9963-9972
    Number of pages10
    JournalBiochemistry
    Volume50
    Issue number46
    DOIs
    Publication statusPublished - 22 Nov 2011

    Keywords

    • ACTIVE-SITE
    • SPIN-LABELS
    • RECOMBINATION
    • SPECTROSCOPY
    • RNA
    • BACTERIOPHAGE-T7
    • RECOGNITION
    • CONVERSION
    • MECHANISM
    • PACKAGE

    Cite this