Normal Replication of DNA after Repair Replication in Bacteria

Abstract

AN early step in the dark repair of ultra-violet damaged DNA in bacteria is the excision of the damaged single strand regions^1,2. It has been postulated that a later step involves the replacement of the excised segments with undamaged deoxyribonucleotides, utilizing the complementary base pairing with the intact single strand^1,2. Direct physical evidence for this repolymerization step has been obtained in our laboratory employing the procedure of DNA density labelling with 5-bromouracil and density distribution analysis in caesium chloride density gradients³ to distinguish semiconservative replication from the non-conservative mode of repair replication⁴. This non-conservative replication has been observed in bacteria after treatment with ultra-violet⁴, nitrogen mustard⁵, nitrosoguanidine (unpublished work of E. Cerda-Olmedo and P. C. Hanawalt), or thymine starvation⁶. The belief that this is the postulated repair synthesis step is strengthened by the results of a number of control experiments as follows. Non-conservative DNA replication is not observed in bacteria in which ultra-violet induced pyrimidine dimers have been repaired in situ by photo-reactivating conditions before density labelling⁴. It is not observed after ultra-violet irradiation of a strain, E. coli B_S–1, sensitive to ultra-violet light which is unable to perform the excision step in the repair sequence⁷. Non-conservative replication has also been demonstrated by the use of deuterium oxide and nitrogen-15 as density labels to prevent possible artefacts caused by the pathogenicity of 5-bromouracil⁸. We report here an additional control experiment which demonstrates that the DNA molecules damaged by ultra-violet irradiation which have undergone non-conservative replication are then capable of normal semiconservative replication.