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 charact erized with respect to its ability to recognize and act upon a model d ouble-stranded 39-mer oligodeoxyribonucleotide substrate containing a strand break site with 3'-phosphoglycolate and 5'-phosphate end-group chemistries. This oligodeoxyribonucleotide substrate exactly duplicate s the chemistry and configuration of a major DNA lesion produced by io nizing radiation. HAP1 was found to recognize the strand break, and ca talyze 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. Th e mechanism of catalysis was hydrolysis of the phosphate ester bond be tween 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 po lymerase I large fragment. AP endonucleolytic activity of HAP1 was exa mined using an analogous double-stranded 39-mer oligodeoxyribonucleoti de substrate, in which the strand break site was replaced by an apyrim idinic site. The V-max and K-m for the AP endonuclease reaction were 6 8 fmole/min/pg of HAP1 protein and 0.23 mu M respectively.