Asparagine 212 is essential for abasic site recognition by the human DNA repair endonuclease HAP1.
HAP1 is a divalent cation-dependent endonuclease from human cells with specificity for apurinic/apyrimidinic (AP) sites in DNA. Extraction of the essential metal ion from purified HAP1 stabilized its binding to an oligonucleotide containing a single AP site, permitting AP site binding studies to be undertaken using gel retardation assays. Binding of HAP1 to such an oligonucleotide was dependent upon the presence of an AP site. Previous structural and modelling studies have suggested a role for Asn212 (Asn153 in exonuclease III, the bacterial homologue of HAP1) in substrate recognition. Substitution of alanine for Asn212 abolished the AP endonuclease activity of purified recombinant HAP1 protein. More conservative substitutions of aspartate or glutamine for Asn212 still led to a reduction in specific activity of at least 300-fold. Moreover, none of the three Asn212 substitution mutants of HAP1 possessed detectable AP site binding activity in vitro. This study indicates that chelation of the active site metal ion in HAP1 stabilizes the complex of the protein with AP sites and identifies an active site asparagine residue as an important component of AP site recognition by the HAP1 protein.
Rothwell
D G
DG
Imperial Cancer Research Fund Laboratories, University of Oxford, John Radcliffe Hospital, UK.
Hickson
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ID
D001216:Asparagine / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004492:Edetic Acid / Q000378:metabolism; D006801:Humans; D008190:Lyases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D001216:Asparagine; D004247:DNA; D004492:Edetic Acid; D043223:Deoxyribonuclease IV (Phage T4-Induced); D008190:Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase