Removal of 3′-phosphoglycolate from DNA strand-break damage in an oligonucleotide substrate by recombinant human apurinic/apyrimidinic endonuclease 1

A recombinant human AP endonuclease, HAP1, was constructed and characterized with respect to its ability to recognize and act upon a model double-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3′-phosphoglycolate and 5′-phosphate end-group chemistries. This oligodeoxy-ribonucleotide substrate exactly duplicates the chemistry and configuration of a major DNA lesion produced by ionizing radiation. HAP1 was found to recognize the strand break, and catalyze the release of the 3′-phosphoglycolate as free phosphoglycolic acid. The enzyme had a V_max of 0.1 fmole/min/pg of HAP1 protein, and a K_m of 0.05 μM for the 3′-phosphoglycolate strand break lesion. The mechanism of catalysis was hydrolysis of the phosphate ester bond between the 3′-phosphoglycolate moiety and the 3′-carbon of the adjacent dGMP moiety within the oligonucleotide. The resulting DNA contained a 3′-hydroxyl which supported nucleotide incorporation by E.coli DNA polymerase I large fragment. AP endonucleolytic activity of HAP1 was examined using an analogous double-stranded 39-mer oligodeoxyribonucleotide substrate, in which the strand break site was replaced by an apyrimidinic site. The V_max and K_m for the AP endonuclease reaction were 68 fmole/min/pg of HAP1 protein and 0.23 μM, respectively.