δ-Elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA

Abstract

[5'-32P]pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and [d(-)-1',2'-3H, 5'-32P]DNA, containing AP sites labelled with 3H in the 1' and 2' positions of the base-free deoxyribose [d(-)] and with 32P 5' to this deoxyribose, were used to investigate the yields of the β-elimination and δ-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3'-phosphatase activity of T4 polynucleotide kinase, of the 3'-phosphate resulting from the βδ-elimination. Phage-ΦX174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: β-elimination, δ-elimination, hydrolysis of 3'-phosphate, DNA polymerization and ligation. Spermine, in one experiment, and Escherichia coli formamidopyrimidine:DNA glycosylase, in another experiment, were used to catalyse the first and second steps (β-elimination and δ-elimination). These repair pathways, involving a δ-elimination step, may be operational not only in E. coli repairing its DNA containing a formamidopyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.