In Vitro Excision-Repair of Ultraviolet-Irradiated Transforming DNA from Bacillus subtilis

Abstract

The biological activity of UV-inactivated Bacillus subtilis DNA is partly restored after incubation with a UV-specific endonuclease from Micrococcus luteus in conjunction with DNA polymerase and DNA ligase, both isolated from Escherichia coli. The restored activity is not further increased by photoreactivation. Pyrimidine dimers are specifically liberated when irradiated DNA is exposed to the three enzymes. None of these effects is observed when pancreatic DNase is used instead of UV-specific endonuclease.Since the demonstration by RMrsch et al. (1) and by Elder and Beers (2) that dark-repair of UV-irradiated DNA is enzymatic in nature, much effort has been given to elucidate its mechanism. Using UV-irradiated OX 174 replicative form type I (RFI) DNA and extracts from Micrococcus luteus, Rorsch et al. (3) showed that simultaneously with an increase in biological activity (assayed as the ability to form virus particles in spheroplasts of Escherichia coli), single-strand breaks are introduced in RFI-DNA, suggesting that an endonuclease is involved in the reactivation process.Setlow and Carrier (4) -and Boyce and Howard-Flanders (5) observed that thymine dimers are excised from UV-irradiated DNA in the in vivo dark-repair process. They proposed a repair mechanism in which dimers are removed by the combined action of an endonuclease, which recognizes dimers, and an exonuclease, which excises the UV-damaged region. After repair-replication (6), the physical continuity of the DNA strand is restored by DNA ligase (7).Results of in vitro experiments support this model: Carrier and Setlow (8) were able to demonstrate excision of pyrimidine dimers from UV-irradiated DNA in extracts from M. luteus. Both a UV-specific endonuclease and an exonuclease have been purified from this organism (9, 10). The endonuclease specifically recognizes dimers (11,12). Recently, Kelly et al. (13) demonstrated that DNA polymerase from E. coli is able to excise pyrimidine dimers and to insert new nucleotides into the DNA chain in the 5' to 3' direction, thus 'translating' single-strand nicks.These results prompted us to investigate whether UV-irradiated DNA can be repaired in vitro with UV-specific endonuclease, DNA polymerase, and DNA ligase. To detect repair, we measured the recovery of biological activity of UVinactivated Bacillus subtilis transforming DNA.The B. subtilis transformation system is particularly well suited for this purpose. First, mutants of B. subtilis are known in which in vivo dark-repair of UV-inactivated transforming DNA is blocked (14) §, so that in vitro repair of UV-damage is not obscured by repair occurring in vivo. Second, in UVsensitive mutants of B. subtilis, biological activity of UVinactivated transforming DNA is further decreased after it is nicked with UV-specific endonuclease (14), implying that complete dark-repair would have to occur in vitro before an increase in biological activity could be observed. This is in contrast with other biological test systems, such as Haemophilus influenzae transforma...

Ultraviolet-Sensitive Mutator mutU4 of Escherichia coli Inviable with polA

“…Kelly et al (16) found that the enzyme could excise thymine dimers from UV-irradiated DNA in vitro, suggesting that it could function in both excision and the subsequent repair replication. DNA polymerase I has been successfully used for repair replication in the in vitro repair of UV-irradiated transforming DNA (9). In toluene-treated cells, Moses and Richardson (22) were able to distinguish replicative from repair synthesis.…”

Enzymatic Repair of DNA

“…Heijneker et al (170) partially restored the transforming activity of UV-irradiated B. subtilis DNA by incising the DNA with the M. luteus correndonuclease II and then incubating the incised DNA with a mixture of E. coli polymerase 1 and polynucleotide ligase. Roughly 20 nucleotides per pyrimidine dimer were excised at 34°C.…”

Physico-chemical and biological study of excision-repair of UV - irradiated øX174 RF DNA in vitro

“…Excision-repair has been carried out in vitro by measuring the restoration of the biological activity of B.subtilis transforming DNA (Heyneker et al, 1971), (Hamilton et al,1974). In this system the biological activity is restored only when all the steps of the excision-repair process are carried out in vitro (Strauss, 1966).…”