Vectors for the expression of tagged proteins in Schizosaccharomyces pombe.

A series of vectors is described which enables the episomal expression of proteins fused to different tag sequences in Schizosaccharomyces pombe. Proteins can be expressed with their amino termini fused to GFP/EGFP, three copies of the HA or Pk epitopes or a combined tag which contains two copies of the myc epitope and six histidine residues (MH). Fusion of the carboxyl terminus of a protein to a tag is possible with GFP/EGFP or Pk. Expression of the fusion proteins is controlled by the medium strength mutant version of the regulatable nmt1 promoter.

14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast.

During the cell cycle, DNA is replicated and segregated equally into two daughter cells. The DNA damage checkpoint ensures that DNA damage is repaired before mitosis is attempted. Genetic studies of the fission yeast Schizosaccharomyces pombe have identified two genes, rad24 and rad25, that are required for this checkpoint. These genes encode 14-3-3 protein homologs that together provide a function that is essential for cell proliferation. In addition, S. pombe rad24 null mutants, and to a lesser extent rad25 null mutants, enter mitosis prematurely, which indicates that 14-3-3 proteins have a role in determining the timing of mitosis.

Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage.

The rad2 mutant of Schizosaccharomyces pombe is sensitive to UV irradiation and deficient in the repair of UV damage. In addition, it has a very high degree of chromosome loss and/or nondisjunction. We have cloned the rad2 gene and have shown it to be a member of the Saccharomyces cerevisiae RAD2/S. pombe rad13/human XPG family. Using degenerate PCR, we have cloned the human homolog of the rad2 gene. Human cDNA has 55% amino acid sequence identity to the rad2 gene and is able to complement the UV sensitivity of the rad2 null mutant. We have thus isolated a novel human gene which is likely to be involved both in controlling the fidelity of chromosome segregation and in the repair of UV-induced DNA damage. Its involvement in two fundamental processes for maintaining chromosomal integrity suggests that it is likely to be an important component of cancer avoidance mechanisms.

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

The rad10, rad16, rad20, 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 rad16, 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 RAD1 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 rad16 and swi10 genes of S. pombe, which mirrors that reported for RAD1 and RAD10 in S. cerevisiae. We have identified the mutations in the four alleles of rad16. They mapped to the N-terminal (rad10), central (rad20), and C-terminal (rad16 and swi9) regions. The rad10 and rad20 mutations are in the splice donor sequences of introns 2 and 4, respectively. The plasmid correcting the UV sensitivity of the rad20 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.

Identification and characterization of new elements involved in checkpoint and feedback controls in fission yeast.

To investigate the mechanisms that ensure the dependency relationships between cell cycle events and to investigate the checkpoints that prevent progression through the cell cycle after DNA damage, we have isolated mutants defective in the checkpoint and feedback control pathways. We report the isolation and characterization of 11 new loci that define distinct classes of mutants defective in one or more of the checkpoint and feedback control pathways. Two mutants, rad26.T12 and rad27.T15, were selected for molecular analysis. The null allele of the rad26 gene (rad26.d) shares the phenotype reported for the "checkpoint rad" mutants rad1, rad3, rad9, rad17, and hus1, which are defective in the radiation checkpoint and in the feedback controls that ensure the order of cell cycle events. The null allele of the rad27 gene (rad27.d) defines a new class of Schizosaccharomyces pombe mutant. The rad27 complementing gene codes for a putative protein kinase that is required for cell cycle arrest after DNA damage but not for the feedback control that links mitosis to the completion of prior DNA synthesis (the same gene has recently been described by Walworth et al. (1993) as chk1). These properties are similar to those of the rad9 gene of Saccharomyces cerevisiae. A comparative analysis of the radiation responses in rad26.d, rad26.T12, and rad27.d cells has revealed the existence of two separable responses to DNA damage controlled by the "checkpoint rad" genes. The first, G2 arrest, is defective in rad27.d and rad26.d but is unaffected in rad26.T12 cells. The second response is not associated with G2 arrest after DNA damage and is defective in rad26.d and rad26.T12 but not rad27.d cells. A study of the radiation sensitivity of these mutants through the cell cycle suggests that this second response is associated with S phase and that the checkpoint rad mutants, in addition to an inability to arrest mitosis after radiation, are defective in an S phase radiation checkpoint.

Feedback controls and G2 checkpoints: fission yeast as a model system.

Dependency relationships within the cell cycle allow cells to arrest the cycle reversibly in response to agents or conditions that interfere with specific aspects of its normal progression. In addition, overlapping pathways exist which also arrest the cell cycle in response to DNA damage. Collectively, these control mechanisms have become known as checkpoints. Analysis of checkpoints is facilitated by the fact that dependency relationships within the cell cycle, such as the dependency of mitosis on the completion of DNA synthesis, and the DNA damage checkpoint can be separated genetically. In fission yeast, Schizosaccharomyces pombe, the dependency of mitosis on prior completion of DNA synthesis is mediated through tyrosine-15 phosphorylation of the ubiquitous mitotic regulator p34cdc2. In contrast, the arrest of mitosis caused by DNA damage acts through a separate mechanism that appears to be independent of tyrosine-15 phosphorylation. Despite these distinct interactions with the mitotic machinery, the majority of fission yeast mutants that are deficient in mitotic arrest after DNA damage are also unable to respond to inhibition of DNA synthesis. In this essay we survey the current knowledge concerning feedback controls and checkpoints within fission yeast and relate this to information derived from other systems.

Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene.

Cells mutated at the rad13 locus in the fission yeast, Schizosaccharomyces pombe are deficient in excision-repair of UV damage. We have cloned the S.pombe rad13 gene by its ability to complement the UV sensitivity of a rad13 mutant. The gene is not essential for cell proliferation. Sequence analysis of the cloned gene revealed an open reading-frame of 1113 amino acids with structural homology to the RAD2 gene of the distantly related Saccharomyces cerevisiae. The sequence similarity is confined to three domains, two close to the N-terminus of the encoded protein, the third being close to the C-terminus. The central region of about 500 amino acids shows little similarity between the two organisms. The first and third domains are also found in a related yet distinct pair of homologous S.pombe/S.cerevisiae DNA repair genes (rad2/YKL510), which have only a very short region between these two conserved domains. Using the polymerase chain reaction with degenerate primers, we have isolated fragments from a gene homologous to rad13/RAD2 from Aspergillus nidulans. These findings define new functional domains involved in excision-repair, as well as identifying a conserved family of genes related to RAD2.