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.

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.

Cellular radiosensitivity of patients with papillary thyroid cancer.

Cultured skin fibroblasts from patients with papillary (differentiated) thyroid carcinomas were compared to those from healthy subjects, ataxia telangiectasia (AT) homozygotes and AT heterozygotes for colony-forming ability after low dose-rate irradiation, and post-irradiation DNA synthesis as indicated by uptake of [3H]-thymidine. The cells from the cancer patients exhibited enhanced radiosensitivity (intermediate between normal and AT) and less than normal level of radioinduced inhibition of DNA synthesis.

Exposure of organic extracts of air particulates to sunlight leads to metabolic activation independence for mutagenicity.

Air particulates were collected on Whatman, GFA glass fibre filters using a RADECO constant-flow air sampler from a car-parking basement and an open roadside adjacent to the basement. While the basement was not exposed to sunlight, the roadside from where air samples were collected was exposed to regular daylight in the month of July (peak summer month). The filters were soaked and sonicated in acetone to dislodge the particulates and then a residue was obtained after evaporation of acetone. The residues were either held in dark or exposed to natural sunlight or germicidal UV light before being tested for mutagenicity using the Salmonella tester strain TA98 with and without metabolic activation (S9 mix). The results showed that the addition of S9 mix resulted in only a slight increase in the frequency of histidine revertants/plate in the case of daylight-exposed roadside air samples. On the other hand, a considerable increase in mutagenicity was observed in the case of the basement air samples, particularly at higher concentrations of the organic extracts when S9 mix was added. However, a pre-exposure of the organic extract of air from the basement to sunlight abrogated the need for S9 mix for showing mutagenic activity. A pre-exposure of the same extracts to germicidal UV light failed to produce a similar effect. These results suggested that long wavelengths of natural sunlight could be responsible for the conversion of certain promutagens in air particulates into direct-acting mutagens. The environmental impact of solar radiation as a modifier of air particulate mutagens in high-sun countries like Saudi Arabia needs to be carefully considered for assessment of air pollution-related health risks.

DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe.

We have tested mutants corresponding to 20 DNA repair genes of the fission yeast Schizosaccharomyces pombe for their ability to arrest in G2 after DNA damage. Of the mutants tested, four are profoundly defective in this damage dependent G2 arrest. In addition, these four mutants are highly sensitive to a transient inhibition of DNA synthesis by hydroxyurea. This suggests that the pathway responsible for the recognition of DNA damage and the subsequent mitotic arrest, shares many functions with the mechanism that controls the dependency of mitosis on the completion of S phase. The phenotype of these checkpoint rad mutants in wee mutant backgrounds indicate that the G2 arrest response is mediated either through, or in parallel with, the activity of the cdc2 gene product.

rqh1+, a fission yeast gene related to the Bloom's and Werner's syndrome genes, is required for reversible S phase arrest.

In eukaryotic cells, S phase can be reversibly arrested by drugs that inhibit DNA synthesis or DNA damage. Here we show that recovery from such treatments is under genetic control and is defective in fission yeast rqh1 mutants. rqh1+, previously known as hus2+, encodes a putative DNA helicase related to the Escherichia coli RecQ helicase, with particular homology to the gene products of the human BLM and WRN genes and the Saccharomyces cerevisiae SGS1 gene. BLM and WRN are mutated in patients with Bloom's syndrome and Werner's syndrome respectively. Both syndromes are associated with genomic instability and cancer susceptibility. We show that, like BLM and SGS1, rqh1+ is required to prevent recombination and that in fission yeast suppression of inappropriate recombination is essential for reversible S phase arrest.

Molecular analysis of hus1+, a fission yeast gene required for S-M and DNA damage checkpoints.

The structure of hus1+, a Schizosaccharomyces pombe gene required for S-M and DNA damage checkpoints, has been determined. Expression of hus1+ requires splicing of five exons, including a microexon that is only 13 nucleotides long. hus1+ is predicted to encode a 33 kDa protein with no similarity to sequences in any database, including the entire S. cerevisiae genome. Yeast strains disrupted for the hus1+ gene are viable but checkpoint-defective. Polyclonal antibodies were raised against bacterially expressed Hus1 protein, and used to study Hus1 regulation. Hus1 protein levels are not affected by S-phase arrest, and are not altered by mutations in other checkpoint genes, suggesting that Hus1 is not regulated at the transcriptional or translational levels.

Variation in herpes simplex virus type 1 glycoproteins produced in kidney cells from different species.

Viral glycoproteins from herpes simplex virus, type 1 (HSV-1) infected NBL-1, Vero, and BHK-21 cells were labelled with 14C-glucosamine and studied by SDS-PAGE and Con-A chromatography. SDS-PAGE analysis demonstrated differences in the number and molecular weight of glycoproteins from these cells. Con-A chromatography resulted in similar binding of glycoproteins from NBL-1 and Vero cells of 10.5 and 18.6%, respectively, whereas BHK-21 cells showed binding of 65%. These studies indicate that HSV-1 glycoprotein oligosaccharide processing is variable in kidney cells of different species.

The Schizosaccharomyces pombe hus5 gene encodes a ubiquitin conjugating enzyme required for normal mitosis.

Normal eukaryotic cells do not enter mitosis unless DNA is fully replicated and repaired. Controls called 'checkpoints', mediate cell cycle arrest in response to unreplicated or damaged DNA. Two independent Schizosaccharomyces pombe mutant screens, both of which aimed to isolate new elements involved in checkpoint controls, have identified alleles of the hus5+ gene that are abnormally sensitive to both inhibitors of DNA synthesis and to ionizing radiation. We have cloned and sequenced the hus5+ gene. It is a novel member of the E2 family of ubiquitin conjugating enzymes (UBCs). To understand the role of hus5+ in cell cycle control we have characterized the phenotypes of the hus5 mutants and the hus5 gene disruption. We find that, whilst the mutants are sensitive to inhibitors of DNA synthesis and to irradiation, this is not due to an inability to undergo mitotic arrest. Thus, the hus5+ gene product is not directly involved in checkpoint control. However, in common with a large class of previously characterized checkpoint genes, it is required for efficient recovery from DNA damage or S-phase arrest and manifests a rapid death phenotype in combination with a temperature sensitive S phase and late S/G2 phase cdc mutants. In addition, hus5 deletion mutants are severely impaired in growth and exhibit high levels of abortive mitoses, suggesting a role for hus5+ in chromosome segregation. We conclude that this novel UBC enzyme plays multiple roles and is virtually essential for cell proliferation.

p53 is dispensable for UV-induced cell cycle arrest at late G(1) in mammalian cells.

Genotoxic agents, including gamma-rays and UV light, induce transient arrest at different phases of the cell cycle. These arrests are required for efficient repair of DNA lesions, and employ several factors, including the product of the tumor suppressor gene p53 that plays a central role in the cellular response to DNA damage. p53 protein has a major function in the gamma-ray-induced cell cycle delay in G(1) phase. However, it remains uncertain as to whether p53 is also involved in the UV-mediated G(1) delay. This report provides evidence that p53 is not involved in UV-induced cellular growth arrest in late G(1) phase. This has been demonstrated in HeLa cells synchronized at the G(1)/S border by aphidicolin, followed by UV exposure. Interestingly, the length of this p53-independent G(1) arrest has been shown to be UV dose-dependent. Similar results were also obtained with other p53-deficient cell lines, including human promyelocytic leukemia HL-60 and mouse p53 knock-out cells. As expected, all of these cell lines were defective in gamma-ray-induced cell growth arrest at late G(1). Moreover, it is shown that in addition to cell cycle arrest, HL-60 cells undergo apoptosis in G(1) phase in response to UV light but not to gamma-rays. Together, these findings indicate that p53- compromised cells have a differential response following exposure to ionizing radiation or UV light.