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

Nucleic Acids Res

Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dow Street, Dundee DD1 5EH, UK.

Published: June 2023

AI Article Synopsis

  • ANKLE1 is a nuclease that resolves unresolved DNA junctions, preventing chromosomal linkages that hinder cell division.
  • The enzyme has been expressed in bacteria, showing activity with DNA Y-junctions and cleaving them unilaterally, with key active residues identified through an AlphaFold model.
  • The enzymatic activity is influenced by pH, divalent cations, and temperature, with a proposed catalytic mechanism involving general acid-base catalysis by tyrosine and histidine in conjunction with a metal ion.

Article 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 when 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10287932PMC
http://dx.doi.org/10.1093/nar/gkad416DOI Listing

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