The rad18 mutant of Schizosaccharomyces pombe is very sensitive to killing by both UV and ␥ radiation. We have cloned and sequenced the rad18 gene and isolated and sequenced its homolog from Saccharomyces cerevisiae, designated RHC18. The predicted Rad18 protein has all the structural properties characteristic of the SMC family of proteins, suggesting a motor function-the first implicated in DNA repair. Gene deletion shows that both rad18 and RHC18 are essential for proliferation. Genetic and biochemical analyses suggest that the product of the rad18 gene acts in a DNA repair pathway for removal of UV-induced DNA damage that is distinct from classical nucleotide excision repair. This second repair pathway involves the products of the rhp51 gene (the homolog of the RAD51 gene of S. cerevisiae) and the rad2 gene.Cells of all organisms have evolved an intricate series of DNA repair pathways to counteract the deleterious effects of all types of DNA damage. In Escherichia coli, nucleotide excision repair (NER) of UV damage requires the products of six genes. A complex of the UvrA and UvrB proteins binds to DNA and translocates to the site of the damage. The UvrC product then attaches to the complex, displacing UvrA, and the damaged DNA strand is nicked on both sides of the damaged site. The helicase activity of the UvrD product releases the oligonucleotide containing the damage, and DNA polymerase I and ligase complete the repair process (24). In eukaryotes, NER requires considerably more gene products, most of which are highly conserved (20). In Saccharomyces cerevisiae, the products of the RAD1, -2, -3, -4, -10, -14, and -25 genes are absolutely required for excision repair of UV damage, whereas there is only a partial requirement for RAD7, -16, and -23. There is evidence that the products of many of these genes form a multisubunit complex (e.g., see reference 53). In Schizosaccharomyces pombe, genes encoding highly homologous proteins have been identified (9,10,30,39), demonstrating the conservation of the classical NER pathway in this yeast.Null mutations in the S. cerevisiae NER genes RAD1, -2, -3, -10, and -14 result in a total deficiency in excision repair of UV-induced cyclobutane dimers and 6-4 photoproducts (28). Null mutants of the S. pombe homologs of RAD1 and RAD2 (rad16 and rad13, respectively), while showing many properties expected of excision repair-deficient mutants, are still able to excise UV-induced cyclobutane dimers and 6-4 photoproducts at a significant rate (7, 27). These results suggest that, in contrast to S. cerevisiae, there is a second pathway in S. pombe for removal of UV photoproducts.Most of the rad mutants of S. pombe are sensitive to both UV and ␥ irradiation. Some of these are involved in checkpoint control of the cell cycle to radiation (2, 3, 41). Others, which are particularly sensitive to ionizing radiation, are deficient in recombination repair (6,29,33,54). No mutant which is sensitive to ionizing but not to UV irradiation has yet been identified. The S. pombe rad18-X mutant ...
contributed equally to this work 4Corresponding author Following DNA damage or a block to DNA synthesis, checkpoint pathways act to arrest mitosis and prevent the attempted segregation of damaged or unreplicated DNA. The radl7 locus of Schizosaccharomyces pombe is one of seven known radiation-sensitive (rad) loci which are absolutely required to prevent mitosis following DNA damage in fission yeast. Six of these (radi, rad3, rad9, radl 7, rad26 and husi) are also required for the checkpoint which prevents mitosis from occurring before DNA replication is complete. We report here that the predicted radl7 gene product is a basic hydrophilic protein of 606 amino acids which contains five domains with sequence homology to replication factor C (RF-C)/activator 1 subunits. Western analysis and fusion with Green Fluorescent Protein indicate that the abundance and electrophoretic mobility of Radl7 is not significantly modified following a block to DNA synthesis or following DNA damage, and that Radl7 is localized in the nucleus. Radl7 function is not essential for growth, but is required for the function of the DNA structure-dependent checkpoints. Sitedirected mutagenesis has been used to demonstrate the biological significance of the RF-C/activator 1-related domains. These studies have also defined an element of the radiation sensitivity caused by loss of Radl7 function which is not associated with the radiationinduced G2 arrest defect seen in the radl7.d null mutant cells.
Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers. In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4–5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle. We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids. The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers.
The Ty element of yeast represents a class of eukaryotic transposons that show remarkable structural similarity to retroviral proviruses. Recently, these comparisons have been strengthened by a series of observations on the yeast Ty element: Ty transposes via an RNA intermediate; it contains a sequence (Fig. 1) which, when translated, is homologous to a conserved region found in all reverse transcriptases; a fusion protein encoded by Ty is produced by a frameshift event that is directly analogous to the production of Pr180gag-pol in a retrovirus such as Rous sarcoma virus. Here we identify the reverse transcriptase activity that, until now, has been presumed to mediate Ty transposition and show that it is sequestered in virus-like particles that also contain Ty RNA.
Schizosaccharomyces pombe cells deficient in nucleotide excision repair (NER) are still able to remove photoproducts from cellular DNA, showing that there is a second pathway for repair of UV damage in this organism. We have characterized this repair pathway by cloning and disruption of the genomic gene encoding UV damage endonuclease (UVDE). Although uvde gene disruptant cells are only mildly UV sensitive, a double disruptant of uvde and rad13 (a S. pombe mutant defective in NER) was synergistically more sensitive than either single disruptant and was unable to remove any photoproducts from cellular DNA. Analysis of the kinetics of photoproduct removal in different mutants showed that the UVDE-mediated pathway operates much more rapidly than NER. In contrast to a previous report, our genetic analysis showed that rad12 and uvde are not the same gene. Disruption of the rad2 gene encoding a structure- specific flap endonuclease makes cells UV sensitive, but much of this sensitivity is not observed if the uvde gene is also disrupted. Further genetic and immunochemical analyses suggest that DNA incised by UVDE is processed by two separate mechanisms, one dependent and one independent of flap endonuclease.
An artificially created non-tandem hetero-allelic 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.
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