Removal by human apurinic/apyrimidinic endonuclease 1 (Ape 1) and Escherichia coli exonuclease III of 3′-phosphoglycolates from DNA treated with neocarzinostatin, calicheamicin, and γ-radiation

Abstract

DNA strand breaks with terminal 3′-phosphoglycolate groups are produced by agents that can abstract the hydrogen atom from the 4′-carbon of DNA deoxyribose groups. Included among these agents are γ-radiation (via the OH radical) and enediyne compounds, such as neocarzinostatin and calicheamicin. However, while the majority of radiation-induced phosphoglycolates are found at single-strand breaks, most of the phosphoglycolates generated by these two enediynes are found at bistranded lesions, including double-strand breaks. Using a 32P-post-labelling assay, we have compared the enzyme-catalyzed removal of phosphoglycolates induced by each of these agents. Both human apurinic/apyrimidinic endonuclease 1 (Ape 1) and its Escherichia coli homolog exonuclease III rapidly removed over 80% of phosphoglycolates from γ-irradiated DNA, although there appeared to be a small resistant subpopulation. The neocarzinostatin-induced phosphoglycolates were removed more slowly, though not to completion, while the calicheamicin-induced phosphoglycolates were extremely refractory to both enzymes. These data suggest that unless other enzymes are capable of acting upon the phosphoglycolate termini at enediyne-induced double-strand breaks, such termini will be resistant to end rejoining repair pathways.