The rad16 gene of Schizosaccharomyces pombe: a homolog of the RAD1 gene of Saccharomyces cerevisiae.

Abstract

The radiO, radl6, rad2O, and swi9 mutants of the fission yeast Schizosaccharomyces pombe, isolated by their radiation sensitivity or abnormal mating-type switching, have been shown previously to be allelic. We have cloned DNA correcting the UV sensitivity or mating-type switching phenotype of these mutants and shown that the correcting DNA is encompassed in a single open reading frame. The gene, which we will refer to as radi6, is approximately 3 kb in length, contains seven introns, and encodes a protein of 892 amino acids. It is not essential for viability of S. pombe. The predicted protein is the homolog of the Saccharomyces cerevisiae RADI protein, which is involved in an early step in excision-repair of UV damage from DNA. The approximately 30% sequence identity between the predicted proteins from the two yeasts is distributed throughout the protein. Two-hybrid experiments indicate a strong protein-protein interaction between the products of the radl6 and swilO genes of S. pombe, which mirrors that reported for RADJ and RADIO in S. cerevisiae. We have identified the mutations in the four alleles of radl6. They mapped to the N-terminal (radiO), central (rad2O), and C-terminal (radi6 and swi9) regions. The radiO and rad2O mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad2O mutation was missing the sequence corresponding to the 335 N-terminal amino acids of the predicted protein. Neither smaller nor larger truncations were, however, able to correct its UV sensitivity.DNA repair is a process of fundamental importance in maintaining the integrity of the genome. In humans, deficiencies in DNA repair result in severe multisystem genetic disorders such as xeroderma pigmentosum and ataxia telangiectasia. In order to counteract the deleterious effects of DNA damage, cells have evolved a series of complex pathways whereby damage of different types can be reversed, removed, or tolerated. Many genes involved in these repair pathways have been cloned from humans and from both budding and fission yeasts. Genes involved in nucleotide excision repair show a striking level of cross-species sequence conservation (18,19). Thus, the cloned human ERCCJ, ERCC2 (XPD), ERCC3 (XPB), ERCC5 (XPG), XPA and XPC genes all share homologies with yeast excision repair genes (3,19,(21)(22)(23)37).Although DNA repair processes have been most extensively studied in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe is also proving to be an excellent model for higher eukaryotes. Early work on radiation-sensitive mutants of S. pombe suggested the existence of at least two phenotypic groups (29). Mutants in the first group, thought to be involved in excision-repair, are characterized by sensitivity to UV but not y-radiation, caffeine sensitization to UV damage, and pronounced UV-hypermutability. Mutants in the second group are sensitive to both UV and y-radiation and have lost the caffeine sensitization to UV damage. Their mutabil...

The rad18 Gene of Schizosaccharomyces pombe Defines a New Subgroup of the SMC Superfamily Involved in DNA Repair

“…The rad18 gene was cloned by complementation of the UV sensitivity of the rad18 mutant using a S. pombe genomic library constructed in the S. pombe shuttle vector pUR19 (5) as described in our earlier work (9,10,12). DNA was extracted from a complementing clone and used to transform DH5␣FЈ by using standard procedures.…”

“…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).…”

“…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.…”

Induction of a G 1 -S checkpoint in fission yeast

“…2). In analogy to NER in human and budding yeast cells the XPA/Rad14 homolog Rhp14 has a role in damage recognition, whereafter the endonucleases Rad16 and Rad13 make cuts on either side of the lesion (31)(32)(33). Then the oligonucleotide containing the lesion is removed by a helicase, followed by resynthesis and ligation.…”

DNA repair, DNA replication and human disorders: A personal journey

“…We had the good fortune to be able to hire Tony Carr in the CMU in 1988 and within about 6 months Tony together with Jo Murray and Felicity Watts cloned about ten S pombe DNA repair genes and together, we spent the next few years characterising them and assigning them to different pathways. These included genes involved in classical NER [47,48], in a second repair pathway peculiar to S. pombe and a few other organisms [49], and in cell cycle checkpoints (e.g. [50]).…”