In vitro repair of radiation-induced strand breaks in DNA

Abstract

DNA, γ-irradiated in vitro or in isolated thymocytes was treated with several enzymes to achieve repair of the radiation-induced single strand braks. Whereas an incubation with polynucleotide ligase can join only 25% of the single strand breaks, a combined treatment with exonuclease III (EC 3.1.4.1), DNA polymerase I (EC 2.7.7.7), and polynucleotide ligase leads to repair of 80% of the breaks. For this in vitro repair the exonuclease III has to remove several, probably damaged, nucleotides from the 3′-terminal producing a single-stranded gap, which will be filled in by DNA polymerase I and joined by ligase. Tests for successful rejoining of the strand breaks were performed by showing the loss of primer 3′-OH sites for DNA polymerase I, by the resistance of incorporated nucleotides in the gap to removal by a second exonuclease III treatment, and by strand break determination in the analytical ultracentrifuge. 20% of the radiation-induced strand breaks will not be repaired by this combined treatment possibly due to an incomplete binding of the ligase on the 5′-terminals and/or an incomplete removal of the damaged 3′-terminals by exonuclease III.