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 oligodeoxyribonucleotide 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 mu M 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 mu M respectively.