Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes.

Abstract

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.

Processing of Targeted Psoralen Cross-Links in Xenopus Oocytes

“…Psoralen adducts are processed in oocytes, and, as in other cell types (14,15,41), mutations are often created at the site of the lesion. Psoralen cross-links are removed in oocytes, but at rates that are much slower than published rates for repair of UV-induced damage (16,21,37). The oocyte provides a particularly favorable setting in which to study DSB-induced recombination because of its high capacity for SSA events and the absolute dependence of homologous recombination on DSBs (1,2,35).…”

“…Our expectation that DSBs might be produced was based on the observation that cross-link-stimulated DSBs in yeast are dependent on NER activities (7,18) and the demonstration of NER at UV lesions in oocytes (16,21,37). Our results make it likely that the processing of psoralen adducts in Xenopus differs from that in yeast.…”

“…In COS cells, the target plasmid was replication competent, and pains were taken to ensure that only replicated DNAs were included in the analysis (8,40,41). Xenopus oocytes do not replicate injected DNA (9, 13), but they are capable of repair synthesis and NER (6,16,21). It is conceivable that different patterns of misincorporation would occur during replicative and repairtype synthesis.…”

“…Full-grown Xenopus oocytes have been shown to possess an active NER system with a capacity several orders of magnitude greater than that of mammalian cells (16,21,37). Therefore, it seemed reasonable to expect that psoralen crosslinks would be repaired in oocytes and that DSBs would be produced during this process.…”

Regulation of translation initiation following stress

“…It is expected that the DNA damage induced by genotoxic stress in mammalian cells may also be repaired by the error-prone DNA repair/ replication mechanisms (Hays et al, 1990;Viswanathan et al, 1999 and refs. therein) which can ®x mutations that may also alter the translation initiation codon.…”