3'-phosphodiesterase activity of human apurinic/apyrimidinic endonuclease at DNA double-strand break ends.
In order to assess the possible role of human apurinic/apyrimidinic endonuclease (Ape) in double-strand break repair, the substrate specificity of this enzyme was investigated using short DNA duplexes and partial duplexes, each having a single 3'-phosphoglycolate terminus. Phosphoglycolate removal by Ape was detected as a shift in mobility of 5'-end-labeled DNA strands on polyacrylamide sequencing gels, and was quantified by phosphorimaging. Recombinant Ape efficiently removed phosphoglycolates from the 3'-terminus of an internal 1 base gap in a 38mer duplex, but acted more slowly on 3'-phosphoglycolates at a 19 base-recessed 3'-terminus, at an internal nick with no missing bases, and at a double-strand break end with either blunt or 2 base-recessed 3'-termini. There was no detectable activity of Ape toward 3'-phosphoglycolates on 1 or 2 base protruding single-stranded 3'-overhangs. The results suggest that both a single-base internal gap, and duplex DNA on each side of the gap are important binding/recognition determinants for Ape. While Ape may play a role in repair of terminally blocked double-strand breaks, there must also be additional factors involved in removal of at least some damaged 3'-termini, particularly those on 3'-overhangs.
Suh
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Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA.
Wilson
D M
DM
Povirk
L F
LF
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D006016:Glycolates / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity
D006016:Glycolates; D009838:Oligodeoxyribonucleotides; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); D010727:Phosphoric Diester Hydrolases; D008190:Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C013193:phosphoglycolate