Conversion of Phosphoglycolate to Phosphate Termini on 3′ Overhangs of DNA Double Strand Breaks by the Human Tyrosyl-DNA Phosphodiesterase hTdp1

Mammalian cells contain potent activity for removal of 3′-phosphoglycolates from single-stranded oligomers and from 3′ overhangs of DNA double strand breaks, but no specific enzyme has been implicated in such removal. Fractionated human whole-cell extracts contained an activity, which in the presence of EDTA, catalyzed removal of glycolate from phosphoglycolate at a single-stranded 3′ terminus to leave a 3′-phosphate, reminiscent of the human tyrosyl-DNA phosphodiesterase hTdp1. Recombinant hTdp1, as well asSaccharomyces cerevisiae Tdp1, catalyzed similar removal of glycolate, although less efficiently than removal of tyrosine. Moreover, glycolate-removing activity could be immunodepleted from the fractionated extracts by antiserum to hTdp1. When a plasmid containing a double strand break with a 3′-phosphoglycolate on a 3-base 3′ overhang was incubated in human cell extracts, phosphoglycolate processing proceeded rapidly for the first few minutes but then slowed dramatically, suggesting that the single-stranded overhangs gradually became sequestered and inaccessible to hTdp1. The results suggest a role for hTdp1 in repair of free radical-mediated DNA double strand breaks bearing terminally blocked 3′ overhangs.