The use of thioglycolate to distinguish between 3' AP (apurinic/apyrimidinic) endonucleases and AP lyases

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Abstract
Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or g-elimination (so that the nicking enzyme should be called AP lyase). For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the presence of thioglycolate and the reaction products were analyzed on DEAE-Sephadex : deoxyribose-5-phosphate (indicating a 3' cleavage by hydrolysis) and the thioglycolate: unsaturated sugar-5-phosphate adduct (indicating a cleavage by β-elimination) are well separated allowing to eventually easily discard the hypothesis of a hydrolytic process and the appellation of 3' AP endonuclease. We have shown that addition of thioglycolate to the unsaturated sugar resulting from nicking the C(3')-O-P bond 3' to AP sites by 3-elimination is an irreversible reaction. We have also shown that the thioglycolate must be present from the beginning of the reaction with the nicking catalyst to prevent the primary 5' product of the β-elimination reaction from undergoing other modifications that complicate the interpretation of the results.