In vivo nicking and rejoining of nuclear DNA in ultraviolet-irradiated radiation-resistant and sensitive strains of Dictyostelium discoideum

Abstract

Some aspects of DNA repair in several radiation-resistant and radiation-sensitive strains of Dictyostelium discoideum were investigated by using alkaline sucrose gradients to analyze for the production and resealing of single-strand breaks following irradiation with 254 nm UV. All radiation-resistant strains and all mutants assayed that are sensitive to both UV and 60Co gamma rays produced single-strand breaks in their nuclear DNA after a UV fluence of 15 M/m2. Mutants at the radC locus which are sensitive to UV but as resistant as their parental strains to 60Co gamma rays produced many fewer single-strand breaks in their DNA after irradiation with UV. Thus, the radC mutations alter a repair pathway specific for UV-induced DNA damage and presumably affect the activity of a UV-damage-specific endonuclease involved in excision repair. All radiation-resistant strains and all of our mutants sensitive to gamma rays rejoined much of their DNA during a three-hour post-UV-irradiation incubation, suggesting that these strains have at least a partially intact excision repair system.

Excision of pyrimidine dimers from nuclear deoxyribonucleic acid in ultraviolet-irradiated Dictyostelium discoideum.

“…This rate of dimer excision is reasonably compatible with the kinetics of ESS removal reported here, given the lower fluence used in this work. HPS104 (radC44) does not show the initially rapid removal of ESS from its nuclear DNA, in agreement with previous studies showing that few single-strand breaks appeared in the nuclear DNA of this strain immediately after UV irradiation with 15 J/m2 (26). Measurement of strand breakage and rejoining on alkaline sucrose gradients, however, does not identify the DNA lesion undergoing repair, nor does it exclude nonspecific incision events.…”

“…The ESS analysis thus confirms the suggestion that mutations belonging to complementation group C are deficient in an activity, presumably an endonuclease, responsible for incising pyrimidine dimers in UV-irradiated nuclear DNA. The kinetics of ESS removal in HPS104 also demonstrate that this strain retains a significant capacity to excise dimers from its nuclear DNA, a result which was not anticipated from the previous study (26).…”

“…DISCUSSION DNA repair after UV irradiation in NC4 (radt) is characterized by a rapid loss of ESS from nuclear DNA immediately after irradiation in buffer at 4°C, followed by a slower removal of the remaining ESS during incubation at 230C under grovs ch conditions. This rapid loss of ESS at low temperature is consistent with the immediate appearance of single-strand breaks after UV irradiation under similar conditions previously observed in this organism (26). The loss of thymine-containing dimers from trichloroacetic acid-insoluble DNA in NC4 also occurs rapidly; roughly one-third of the dimers induced by 100 J/m2 (265 nm) are removed within 30 min after irradiation (12).…”

“…The radB locus operates in the same repair pathway as radA since radA-radB double-mutant haploids are no more sensitive to UV than is radB, the more sensitive single mutant (27). Mutants in all complementation groups except radC make and rejoin singlestrand breaks in their DNA after UV irradiation; radC mutants are apparently deficient in an endonuclease activity specific for UV-irradiated DNA (26). Using the pyrimidine dimer-specifichydrochloride was a gift from Lederle Laboratories (Pearl River, N.Y.).…”

“…Strains NC4 (wild type) and HPS104 (radC44 axeAl axeBI tsgAl) were routinely grown on standard agar plates in association with E. coli B/r as previously described (5). 3H labeling of D. discoideum DNA was carried out essentially as described by Welker and Deering (26). 14C labeling of D. discoideum DNA was carried out in an analogous manner by growing slime mold cells on E. coli 15T whose DNA had been specifically labeled during prior growth on C-rninimal medium supplemented with 2.0 Ag of [2-14C]thymine per ml at a final specific activity of 0.06 Ci/mmol (Amersham Corp., Arlington Heights, Ill.).…”