Biochemical and mechanistic analysis of the cleavage of branched DNA by human ANKLE1

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    Abstract

    ANKLE1 is a nuclease that provides a final opportunity to process unresolved junctions in DNA that would otherwise create chromosomal linkages blocking cell division. It is a GIY-YIG nuclease. We have expressed an active domain of human ANKLE1 containing the GIY-YIG nuclease domain in bacteria, that is monomeric in solution and bound to a DNA Y-junction, and unilaterally cleaves a cruciform junction. Using an AlphaFold model of the enzyme we identify the key active residues, and show that mutation of each leads to impairment of activity. There are two components in the catalytic mechanism. Cleavage rate is pH dependent, corresponding to a pKa of 6.9, suggesting an involvement of the conserved
    histidine in proton transfer. The reaction rate depends on the nature of the divalent cation, likely bound by glutamate and asparagine side chains, and is log-linear with the metal ion pKa. We propose that the reaction is subject to general acid-base catalysis, using a combination of tyrosine and histidine acting as general base and water directly coordinated to the metal ion as general acid. The reaction is temperature dependent; activation energy Ea = 37 kcal mol-1, suggesting that cleavage is coupled to opening of DNA in the transition state.
    Original languageEnglish
    Article numbergkad416
    Pages (from-to)5743–5754
    Number of pages12
    JournalNucleic Acids Research
    Volume51
    Issue number11
    Early online date22 May 2023
    DOIs
    Publication statusPublished - 23 Jun 2023

    Keywords

    • DNA junctions
    • GIY-YIG nucleases
    • general acid-base catalysis
    • metal ion catalysis

    ASJC Scopus subject areas

    • Genetics

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