Thioglycollate reacts with the 5' product of AP lyase activity on apurinic/apyrimidinic (AP) sites in DNA. The 3'-terminal thioglycollate-unsaturated sugar 5-phosphate adduct can be released by the use of Escherichia coli endonuclease IV or endonuclease VI, and identified by DEAE-Sephadex chromatography. In contrast, the mammalian AP endonuclease is unable to excise a 3'-terminal thiol-unsaturated sugar adduct; this lesion, which must sometimes occur in vivo, might be irreparable and have pathological consequences.
View Article and Find Full Text PDFAddition 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 beta-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 beta-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 beta-elimination is an irreversible reaction.
View Article and Find Full Text PDFIn vitro, in the presence of Mg++, the 3'-phosphoglycolatase activity of endonuclease IV is about 4-times smaller than that of exonuclease III for the same AP endonuclease activity. It thus seems that endonuclease IV has only a minor role in the repair of strand breaks limited by 3'-phosphoglycolate ends in Escherichia coli even after the amount of enzyme has been increased by induction with O2 -generating agents.
View Article and Find Full Text PDFUsing [32P]DNA alkylated with [3H]methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks. The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of [3H]methyl groups and by reaction with [14C]methoxyamine. The 32P label of DNA was used to measure the acid-solubility.
View Article and Find Full Text PDFThe interstrand crosslinks that appear in stored depurinated DNA interfere with the counting of apurinic sites and strand breaks by sucrose gradient analysis. They could not be cleaved at acid or alkaline pH, or by treatment with methoxyamine.
View Article and Find Full Text PDFBiochim Biophys Acta
January 1971
Arch Int Physiol Biochim
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