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 en donuclease (Ape) in double-strand break repair, the substrate specific ity of this enzyme was investigated using short DNA duplexes and parti al duplexes, each having a single 3'-phosphoglycolate terminus. Phosph oglycolate removal by Ape was detected as a shift in mobility of 5'-en d-labeled DNA strands on polyacrylamide sequencing gels, and was quant ified by phosphorimaging. Recombinant Ape efficiently removed phosphog lycolates from the 3'-terminus of an internal 1 base gap in a 38mer du plex, but acted more slowly on 3'-phosphoglycolates at a 19 base-reces sed 3'-terminus, at an internal nick with no missing bases, and at a d ouble-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 su ggest that both a single-base internal gap, and duplex DNA on each sid e of the gap are important binding/recognition determinants for Ape. W hile 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.