Keywords :
DNA, Viral; Polynucleotides; apyrimidinic acid; Apurinic Acid; Spermine; Polynucleotide 5'-Hydroxyl-Kinase; DNA Polymerase I; DNA Ligases; Bacteriophage phi X 174/genetics; DNA Ligases/physiology; DNA Polymerase I/physiology; DNA, Viral/drug effects; Polynucleotide 5'-Hydroxyl-Kinase/metabolism; Spermine/pharmacology; DNA Repair; Biochemistry; Molecular Biology; Cell Biology
Abstract :
[en] [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 beta-elimination and delta-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 beta delta-elimination. Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-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 (beta-elimination and delta-elimination). These repair pathways, involving a delta-elimination step, may be operational not only in E. coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.
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