Bailly V, Verly W G
Department of Biochemistry, Faculty of Sciences, University of Liège, Belgium.
Nucleic Acids Res. 1988 Oct 25;16(20):9489-96. doi: 10.1093/nar/16.20.9489.
Addition of thiol compounds containing an anionic group to the 3'-terminal unsaturated sugar of the 5' fragment obtained from an oligonucleotide containing an AP site cleaved by beta-elimination, can be followed by gel electrophoresis. The technique enables to distinguish between two mechanisms of cleavage of the C3'-O-P bond 3' to an AP site: hydrolysis or beta-elimination. Addition of thiols to the double-bond of the 3'-terminal sugar resulting from beta-elimination prevents a subsequent delta-elimination. The interpretation of the action of enzymes that start by nicking 3' to AP sites must take into account the presence or absence of thiols in the reaction medium. In living cells, thiols might influence the pathways followed by the repair processes of AP site-containing DNA.
将含有阴离子基团的硫醇化合物添加到通过β-消除切割含有AP位点的寡核苷酸获得的5'片段的3'-末端不饱和糖上，随后可进行凝胶电泳。该技术能够区分AP位点3'端C3'-O-P键的两种切割机制：水解或β-消除。向β-消除产生的3'-末端糖的双键添加硫醇可防止随后的δ-消除。对从AP位点3'端切口开始作用的酶的作用进行解释时，必须考虑反应介质中硫醇的存在与否。在活细胞中，硫醇可能会影响含AP位点的DNA修复过程所遵循的途径。
