In vitro repair of radiation-induced strand breaks in DNA.
DNA, gamma-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 treatemnt 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.
D000818:Animals; D004247:DNA / Q000528:radiation effects*; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000528:radiation effects; D003851:Deoxyribonucleases; D004926:Escherichia coli / Q000201:enzymology; D005092:Exonucleases / Q000378:metabolism; D007700:Kinetics; D011118:Polynucleotide Ligases / Q000378:metabolism; D011830:Radiation Effects; D051381:Rats; D013698:Templates, Genetic; D013950:Thymus Gland / Q000378:metabolism
D004277:DNA, Single-Stranded; D004247:DNA; D004254:DNA Nucleotidyltransferases; D003851:Deoxyribonucleases; D005092:Exonucleases; D011118:Polynucleotide Ligases