Shirley McCready

Alternative repair pathways for UV‐induced DNA damage

Ultraviolet light (UV) is thought to have had a major impact on the early evolution of life. UV is absorbed by nucleic acids and produces several types of DNA damage, which interfere with DNA replication and transcription. This damage can result in mutagenesis and cell killing. Several mechanisms for repairing UV‐induced DNA damage have been identified. Besides the widely distributed nucleotide excision repair, two alternative repair mechanisms for specific lesions in UV‐damaged DNA are known, involving photolyases and DNA glycosylases. Recently, a novel endonuclease for UV‐induced DNA damage was identified that initiates an excision repair pathway completely different from previously established repair mechanisms. The finding of this “alternative excision repair” suggests the presence of a new category of DNA repair, initiated by single‐strand breaks in DNA. Homologues of the UVDE enzyme have been found in eukaryotic microorganisms, as well as in bacteria, indicating that the enzyme originated early in evolution, and suggesting the existence of multirepair systems for UV‐induced DNA damage during early evolution. BioEssays 20:291‐297, 1998.

The plastid DNA of the malaria parasite Plasmodium falciparum is replicated by two mechanisms.

In common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two‐dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D‐loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.

The genetic control of spontaneous and UV-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe

Abstract An artificially created non-tandem heteroallelic duplication was constructed to assay mitotic intrachromosomal recombination in Schizosaccharomyces pombe. Two classes of recombinants could be distinguished: deletion-types, in which one copy of the duplicated sequence and the intervening sequence were lost, and conversion-types which retained the duplication. For spontaneous recombination, compared to wild-type cells, a rad22 mutant (corresponding to a Saccharomyces cerevisiae rad52 mutant) had wild-type levels of deletion-types, but was hypo-recombinant for conversion-types; rad16 (S. cerevisiae rad1), rad22 rad16 (S. cerevisiae rad52 rad1) and swi10 (S. cerevisiae rad10) mutants were hyper-recombinant for both types; rad22 swi10 (S. cerevisiae rad52 rad10) mutants were hypo-recombinant for both types; rhp51 (S. cerevisiae rad51) and rhp54 (S. cerevisiae rad54) mutants were hyper-recombinant for deletion-types, but almost completely lacked conversion-types. For wild-type cells, UV-irradiation induced both types of recombinant, but mainly conversion-types. All of the mutants lacked UV-induced recombination.

Repair of UV damage in the fission yeast Schizosaccharomyces pombe

This review is concerned with repair and tolerance of UV damage in the fission yeast, Schizosaccharomyces pombe and with the differences between Sch. pombe and budding yeast, Saccharomyces cerevisiae in their response to UV irradiation. Sch. pombe is not as sensitive to ultra-violet radiation as Sac. cerevisiae nor are any of its mutants as sensitive as the most sensitive Sac. cerevisiae mutants. This can be explained in part by the fact that Sch. pombe, unlike budding yeast or mammalian cells, has an extra pathway (UVER) for excision of UV photoproducts in addition to nucleotide excision repair (NER). However, even in mutants lacking this additional pathway, there are significant differences between the two yeasts. Sch. pombe mutants that lack the alternative pathway are still more UV-resistant than wild-type Sac. cerevisiae; recombination mutants are significantly UV sensitive (unlike their Sac. cerevisiae equivalents); mutants lacking the second pathway are sensitized to UV by caffeine; and checkpoint mutants are relatively more sensitive than the budding yeast equivalents. In addition, Sch. pombe has no photolyase. Thus, the response to UV in the two yeasts has a number of significant differences, which are not accounted for entirely by the existence of two alternative excision repair pathways. The long G2 in Sch. pombe, its well-developed recombination pathways and efficient cell cycle checkpoints are all significant components in survival of UV damage.

Repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in the fission yeast Schizosaccharomyces pombe

We have measured repair of both of the major lesions induced by ultraviolet irradiation (cyclobutane pyrimidine dimers and 6‐4 photoproducts) in wild‐type Schizosaccharomyces pombe and in selected rad mutants, including mutants with deletions in genes from the main phenotypic groups. We find that rad13Δ, rad15 and rad16Δ, which are the S. pombe homologues of the excision‐defective Saccharomyces cerevisiae rad2, rad3 and rad1, respectively, repair lesions somewhat more slowly than the wild type, but still have considerable repair capacity. rad2Δ, also a presumed excision‐defective mutant, behaves similarly. radS and rad9δ, which belong to different phenotypic groups, repair lesions at the same rate as wild‐type cells. These findings provide new evidence that S. pombe has a second repair system for removing ultraviolet damage, which is absent in S. cerevisiae. Surprisingly, this second mechanism repairs lesions very efficiently; its possible nature is discussed.

Nuclear and mitochondrial DNA of the primate malarial parasite Plasmodium knowlesi

Restriction analyses and DNA/DNA hybridisation of parasite DNA isolated from monkeys infected with the malarial parasite Plasmodium knowlesi has permitted unambiguous identification of the nuclear DNA of this species. Its (G+C) content, as determined by estimations of buoyant density as well as by direct analysis, is about 38%, essentially indistinguishable from that of its primate laboratory host, and grossly different from that of the major human malaria parasite, P. falciparum, which has a (G+C) content of approx. 19%. In addition, gradient fractionation of total P. knowlesi DNA revealed a minor DNA component (approx. 1% of the total) with a (G+C) content of about 19%. This DNA comprises covalently closed circular molecules which have a contour length about 11.6 microns, carry a small cruciform structure, and are thought to originate in the parasites mitochondria.

Antisuppressors in yeast

Summary1. Twenty-three mutations, mapping at eight different loci, were shown to prevent detectable suppression of ade2-1 and can1-100 by the ochre suppressor, ocSUPQ2. 2. Some reduce the efficiency of suppression of his5-2 and lys1-1 also. 3. Mutants from each of the eight loci prevented the lethal phenotype of ocSUPQ2 in [psi+] strains. They are, therefore, antisuppressor mutations. 4. One of the loci, asu3, is centromere-linked; two of the loci, asu2 and asu6, are linked to each other.

The repair of ultraviolet light-induced DNA damage in the halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii

Extremely halophilic archaebacteria have been reported to have no capacity for dark repair (excision repair) of ultraviolet damage and to rely on very efficient photoreactivation for recovery after UVC irradiation. Post-UV incubation in the light restores 100% survival in these organisms. This has been taken to indicate that cyclobutane dimers are the only significant UV-induced lesions and that they are completely repaired by photoreactivation. However, in all organisms studied to date, pyrimidine (6-4) pyrimidone photoproducts are a significant cytotoxic and mutagenic lesion and constitute 10-30% of UV photoproducts. The question arises, therefore--are 6-4 photoproducts induced in the halophilic archaebacteria and, if they are, how are they repaired? This paper shows that both cyclobutane dimers and 6-4 photoproducts are induced in the extremely halophilic archaebacteria, Halobacterium cutirubrum, Halobacterium halobium and Haloferax volcanii, at similar levels as in other organisms. Furthermore, contrary to previous reports, there is dark repair of both lesions. As in other organisms, 6-4 photoproducts are removed more efficiently than cyclobutane dimers in the dark. In the light, cyclobutane dimers are repaired very rapidly and there is also photoenhanced repair of 6-4 photoproducts. This work confirms that organisms such as Halobacterium and Haloferax which live in conditions of high exposure to sunlight have very efficient rates of repair of UV lesions in the light.

Repair of 6-4 photoproducts in Sacchromyces cerevisiae

We have developed a dot blot immunoassay for UV photoproducts which distinguishes 6-4 photoproducts from cyclobutane dimers. The assay uses a polyclonal antiserum that is specific for UV-irradiated DNA. Cyclobutane dimers are measured in DNA samples which have been treated with hot alkali to destroy 6-4 photoproducts. 6-4 Photoproducts are measured using blots that have been incubated in photoreactivating enzyme to eliminate cyclobutane dimers. A combination of the two treatments leaves no detectable antigenic lesions. Wild-type S. cerevisiae repairs 6-4 photoproducts, in the genome overall, more rapidly than cyclobutane dimers. The most sensitive alleles of rad1, rad2, rad3 and rad4 are completely unable to repair either kind of photoproduct. We conclude that 6-4 photoproducts are repaired by essentially the same mechanism as are cyclobutane dimers.

The Saccharomyces cerevisiae RAD2 gene complements a Schizosaccharomyces pombe repair mutation

SummaryTwo Saccharomyces cerevisiae genes necessary for excision repair of UV damage in DNA, RAD1 and RAD2, were introduced individually, on a yeast shuttle vector, into seven Schizosaccharomyces pombe mutants — rads1, 2, 5, 13, 15,16 and 17. The presence of the cloned RAD1 gene did not affect survival of any of the S. pombe mutants. The RAD2 gene increased survival of S. pombe rad13 to near the wild-type level after UV irradiation and had no effect on any of the other mutants tested. S. pombe rad13 mutants are somewhat defective in removal of pyrimidine dimers so complementation by the S. cerevisiae RAD2 gene suggests that the genes may code for equivalent proteins in the two yeasts.