Postreplication repair and PCNA modification in Schizosaccharomyces pombe

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays a crucial role in regulating replication past DNA damage in eukaryotes, but the detailed mechanisms appear to vary in different organisms. We have examined the modification of PCNA in Schizosaccharomyces pombe. We find that, in response to UV irradiation, PCNA is mono- and poly-ubiquitinated in a manner similar to that in Saccharomyces cerevisiae. However in undamaged Schizosaccharomyces pombe cells, PCNA is ubiquitinated in S phase, whereas in S. cerevisiae it is sumoylated. Furthermore we find that, unlike in S. cerevisiae, mutants defective in ubiquitination of PCNA are also sensitive to ionizing radiation, and PCNA is ubiquitinated after exposure of cells to ionizing radiation, in a manner similar to the response to UV-irradiation. We show that PCNA modification and cell cycle checkpoints represent two independent signals in response to DNA damage. Finally, we unexpectedly find that PCNA is ubiquitinated in response to DNA damage when cells are arrested in G2.     

Candida albicans exoglucanase as a reporter gene in Schizosaccharomyces pombe

The Candida albicans XOG1 gene, previously shown to be a good reporter gene in Saccharomyces cerevisiae and C. albicans, was tested in Schizosaccharomyces pombe. Unlike the budding yeast, S. pombe does not produce exoglucanase activity and hence this system would be applicable to any given strain of this organism. The XOG1 gene was located under the control of the nmt1 promoter and its functionality could be demonstrated even at high temperatures (37 degrees C). The exoglucanase activity can be measured both in vivo and in vitro by either a simple biochemical reaction (on cells or media) or by flow cytometry, because the cells remain viable after the assay.     

The Schizosaccharomyces pombe fusion gene hal3 encodes three distinct activities

Saccharomyces cerevisiae Hal3 and Vhs3 are moonlighting proteins, forming an atypical heterotrimeric decarboxylase (PPCDC) required for CoA biosynthesis, and regulating cation homeostasis by inhibition of the Ppz1 phosphatase. The Schizosaccharomyces pombe ORF SPAC15E1.04 (renamed as Sp hal3) encodes a protein whose amino‐terminal half is similar to Sc Hal3 whereas its carboxyl‐terminal half is related to thymidylate synthase (TS). We show that Sp Hal3 and/or its N‐terminal domain retain the ability to bind to and modestly inhibit in vitro S. cerevisiae Ppz1 as well as its S. pombe homolog Pzh1, and also exhibit PPCDC activity in vitro and provide PPCDC function in vivo, indicating that Sp Hal3 is a monogenic PPCDC in fission yeast. Whereas the Sp Hal3 N‐terminal domain partially mimics Sc Hal3 functions, the entire protein and its carboxyl‐terminal domain rescue the S. cerevisiae cdc21 mutant, thus proving TS function. Additionally, we show that the 70 kDa Sp Hal3 protein is not proteolytically processed under diverse forms of stress and that, as predicted, Sp hal3 is an essential gene. Therefore, Sp hal3 represents a fusion event that joined three different functional activities in the same gene. The possible advantage derived from this surprising combination of essential proteins is discussed.     

Efflux system for pyridoxine in Schizosaccharomyces pombe

Pyridoxine-charged Schizosaccharomyces pombe released pyridoxine rapidly at 30 degrees C: very low amounts of three other B6 vitamers were also released. The rate of efflux was temperature-dependent. The initial rate of efflux was dependent on the concentration of pyridoxine in the cells: the rate was almost zero at lower than 0.02 mM and became saturated at higher than 0.2 mM. Na+, sodium azide, and dinitrophenol increased the rate in both the presence and absence of D-glucose. Mg++, thiamine, and menadione inhibited the efflux. The intracellular concentration of ATP did not significantly affect the efflux rate. The system may be dependent on a membrane potential of the yeast cells. It was found that the fission yeast cells have a gate or carrier system for efflux of pyridoxine, which was distinct from that in Saccharomyces cerevisiae.     

Radiation resistance in Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe serves as an excellent alternative and complementary model system for the analysis of genes and gene products involved in DNA repair. This brief review outlines the advantages of S. pombe and describes the radiation-sensitive mutants available for the analysis of DNA repair and recombination mechanisms in this organism. The progress in the cloning and characterization of representative genes is also described.     

Single point mutations located outside the inter-monomer domains abolish trimerization of Schizosaccharomyces pombe PCNA.

We have generated proliferating cell nuclear antigen (PCNA) mutants by low fidelity PCR and screened for lethal mutations by testing for lack of complementation of a Schizosaccharomyces pombe strain disrupted for the pcn1 + gene. We thus identified eight lethal mutants out of the 50 cDNAs tested. Six were truncated in their C-terminal region due to the introduction of a stop codon within their coding sequences. Two were full-length with a single point mutation at amino acid 68 or 69. The two latter mutants were overexpressed in insect cells via a recombinant baculovirus and were purified. They were unable to stimulate DNA polymerase delta DNA replication activity on a poly(dA).oligo(dT) template. Cross-linking experiments showed that this was due to their inability to form trimers. Since these two mutations are adjacent and not located in a domain of the protein putatively involved in inter-monomer interactions, our results show that the beta-sheet betaF1 to which they belong must play an essential role in maintaining the 3-dimensional structure of S.pombe PCNA.     

Investigation of Schizosaccharomyces pombe as a cloning host for human telomere and alphoid DNA

The fission yeast Schizosaccharomyces pombe (Sch. pombe) has been proposed as a possible cloning host for both mammalian artificial chromosomes (MACs) and mammalian genomic libraries, due to the large size of its chromosomes and its similarity to higher eukaryotic cells. Here, it was investigated for its ability to form telomeres from human telomere sequence and to stably maintain long stretches of alphoid DNA. Using linear constructs terminating in the telomere repeat, T2AG3, human telomere DNA was shown to efficiently seed telomere formation in Sch. pombe. Much of the human telomeric sequence was removed on addition of Sch. pombe telomeric sequence, a process similar to that described in S. cerevisiae. To investigate the stability of alphoid DNA in fission yeast, bacterial artificial chromosomes (BACs) containing 130 and 173 kb of alphoid DNA were retrofitted with the Sch. pombe ars1 element and ura4+ marker using Cre-lox recombination. These alphoid BACs were found to be highly unstable in Sch. pombe deleting down to less than 40 kb, whilst control BACs of 96 and 202 kb, containing non-repetitive DNA, were unrearranged. Alphoid DNA has been shown to be sufficient for human centromere function, and this marked instability excludes Sch. pombe as a useful cloning host for mammalian artificial chromosomes. In addition, regions containing repetitive DNA from mammalian genomes may not be truly represented in libraries constructed in Sch. pombe.     

Cellular inequivalence in a culture of Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe was grown in the chemostat at D = 0.03, 0.05, 0.1, 0.15 and 0.20 h-1. The dry weight and substrate quantities, the number of cells and their morphological characteristics were determined in the steady state. The curves for the cell number and dry weight demonstrate changes in the coordination between the processes of cell growth and division at various growth rates. The cell division was shown to be asymmetric under the conditions of substrate limitation.     

Schizosaccharomyces pombe ypt5: a homologue of the rab5 endosome fusion regulator.

The ypt/rab proteins are a family of small GTP-binding proteins thought to be required for different stages of membrane traffic. From the fission yeast Schizosaccharomyces pombe we have isolated and characterized ypt5, a gene encoding a homologue of rab5, a mammalian protein apparently involved in regulating fusion of early endosomes. Recombinant ypt5 protein bound GTP. The ypt5 gene was found to be essential for viability on minimal media, but ypt5-disrupted cells grew slowly on some rich media and accumulated a population of small vesicles not observed in wild-type cells. Canine rab5 cDNA could replace the ypt5 gene in S. pombe and restore normal growth and viability. Ypt5 protein expressed in mammalian cells colocalized with the transferrin receptor to early endosomes. Thus, molecular aspects of the early endocytic pathway may be conserved between mammalian cells and S. pombe and hence may be amenable to genetic analysis.     

Rough membranes in Schizosaccharomyces pombe protoplasts

Regenerating protoplasts of Schizosaccharomyces pombe have been examined in the electron microscope after glutaraldehyde fixation. Membrane-bound ribosomes were seen not only on the nuclear envelope and endoplasmic reticulum but also on the outer membranes of mitochondria and on sub-surface cysternae. Functions which have been suggested for sub-surface cysternae in other plant and animal material are considered in relation to this yeast, and the implications for protein synthesis of four different sites for membrane-bound ribosomes are discussed.     

Malate transport in Schizosaccharomyces pombe.

The transport of malate was studied in a Schizosaccharomyces pombe wild-type strain and in mutant strains unable to utilize malic acid. Two groups of such mutants, i.e., malic enzyme-deficient and malate transport-defective mutants, were differentiated by a 14C-labeled L-malate transport assay and by starch gel electrophoresis followed by activity staining for malic enzyme (malate dehydrogenase [oxaloacetate decarboxylating] [NAD+]; 1.1.1.38) and malate dehydrogenase (1.1.1.37). Transport of malate in S. pombe was constitutive and strongly inhibited by inhibitors of oxidative phosphorylation and of the formulation of proton gradients. Transport was a saturable function of the malate concentration. The apparent Km and Vmax values for transport by the parent were 3.7 mM and 40 nmol/min per mg of protein, respectively, while those of the malic enzyme-deficient mutant were 5.7 mM and 33 nmol/min per mg of protein, respectively. Malate transport was pH and temperature dependent. The specificity of transport was studied with various substrates, including mono- and dicarboxylic acids, and the possibility of a common transport system for dicarboxylic acids is discussed.     

Protein prenylation in Schizosaccharomyces pombe.

S. pombe is shown to be a powerful system for studies concerning attachment of polyisoprenoid moieties to proteins, due to its ability to take up exogenous mevalonic acid efficiently. The fission yeast can take up about 5% of the exogenously added mevalonic acid and incorporate approximately 10% of this into protein. By contrast, the uptake obtained with the budding yeast S. cerevisiae is less than 0.5%. HPLC analysis of total S. pombe protein-bound isoprenoids revealed that approximately 55% of the counts co-migrated with the geranylgeraniol standard, while approximately 45% of the counts co-migrated with farnesol. We could not detect any effects of mevinolin or other HMG-CoA reductase inhibitors in S. pombe.