Keywords :
Oligonucleotides; Polynucleotides; Sulfhydryl Compounds; Thioglycolates; Apurinic Acid; Spermine; Deoxyribose; Mercaptoethanol; Deoxyribonucleases, Type III Site-Specific; Apurinic Acid/metabolism; DNA Damage; Micrococcus/enzymology; Polynucleotides/metabolism; T-Phages/enzymology; DNA Repair; Genetics
Abstract :
[en] 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.
Funding text :
Acknowledgments We thank Dr. M. Liuzzi from Edmonton (Canada) and Dr. 3. Laval from Paris for the gift of M.luteus UV endonuclease and E.coli endonuclease III, respectively. The T4 UV endonuclease overproducer was received from Dr. A.A. Van Zeeland from Leiden (The Netherlands). This work was supported by grants from the Fonds Cancerologique de la Caisse Generale d'Epargne et de Retraite and by the Fonds de la Recherche Scientifique Medicale.
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