Biomedical subjects

T Enoch

Publications and source records attributed to T Enoch.

At least 19 recordsLinked to original sources

PubMed1999

Mouse Hus1, a homolog of the Schizosaccharomyces pombe hus1+ cell cycle checkpoint gene.

Cell cycle checkpoints are regulatory mechanisms that arrest the cell cycle or initiate programmed cell death when critical events such as DNA replication fail to be completed or when DNA or spindle damage occurs. In fission yeast, cell cycle checkpoint responses to DNA replication blocks and DNA damage require the hus1+ gene. Mammalian homologs of hus1+ were recently identified, and here we report a detailed analysis of mouse Hus1. An approximately 4.2-kb full-length cDNA encoding the 32-kDa mouse Hus1 protein was isolated. The genomic structure and exon-intron boundary sequences of the gene were determined, and mouse Hus1 was found to consist of nine exons. Mouse Hus1 was mapped to the proximal end of chromosome 11 and is therefore a candidate gene for the mouse mutation germ cell deficient, which maps to the same genomic region. Finally, mouse Hus1 was found to be expressed in a variety of adult tissues and at several stages of embryonic development.

Amino Acid Sequence↗

PubMed1999

Requirement of sequences outside the conserved kinase domain of fission yeast Rad3p for checkpoint control.

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein-protein interactions.

Androstadienes↗

PubMed1999

Liz1p, a novel fission yeast membrane protein, is required for normal cell division when ribonucleotide reductase is inhibited.

Ribonucleotide reductase activity is required for generating deoxyribonucleotides for DNA replication. Schizosaccharomyces pombe cells lacking ribonucleotide reductase activity arrest during S phase of the cell cycle. In a screen for hydroxyurea-sensitive mutants in S. pombe, we have identified a gene, liz1(+), which when mutated reveals an additional, previously undescribed role for ribonucleotide reductase activity during mitosis. Inactivation of ribonucleotide reductase, by either hydroxyurea or a cdc22-M45 mutation, causes liz1(-) cells in G2 to undergo an aberrant mitosis, resulting in chromosome missegregation and late mitotic arrest. liz1(+) encodes a 514-amino acid protein with strong similarity to a family of transmembrane transporters, and localizes to the plasma membrane of the cell. These results reveal an unexpected G2/M function of ribonucleotide reductase and establish that defects in a transmembrane protein can affect cell cycle progression.

Carrier Proteins↗

PubMed1998

Use of gap repair in fission yeast to obtain novel alleles of specific genes.

We have adapted a method for making libraries of mutations in any specific gene for use in the fission yeast Schizosaccharomyces pombe . This elegant and simple method consists of PCR amplification of the gene of interest, followed by co-transformation of fission yeast with the PCR fragment and a linearized plasmid vector prepared such that the ends of the vector share DNA sequence with the ends of the PCR fragment. Homologous recombination between the vector and the PCR fragment occurs at a high frequency and results in a collection of yeast transformants, most harboring a mutated allele of the original gene within the vector of choice. This library can then be screened or selected for phenotypes of interest.

Alleles↗

PubMed1998

Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1.

Checkpoints maintain the order and fidelity of events of the cell cycle by blocking mitosis in response to unreplicated or damaged DNA. In most species this is accomplished by preventing activation of the cell-division kinase Cdc2, which regulates entry into mitosis. The Chk1 kinase, an effector of the DNA-damage checkpoint, phosphorylates Cdc25, an activator of Cdc2. Phosphorylation of Cdc25 promotes its binding to 14-3-3 proteins, preventing it from activating Cdc2. Here we propose that a similar pathway is required for mitotic arrest in the presence of unreplicated DNA (that is, in the replication checkpoint) in fission yeast. We show by mutagenesis that Chk1 functions redundantly with the kinase Cds1 at the replication checkpoint and that both kinases phosphorylate Cdc25 on the same sites, which include serine residues at positions 99, 192 and 359. Mutation of these residues reduces binding of 14-3-3 proteins to Cdc25 in vitro and disrupts the replication checkpoint in vivo. We conclude that both Cds1 and Chk1 regulate the binding of Cdc25 to 14-3-3 proteins as part of the checkpoint response to unreplicated DNA.

14-3-3 Proteins↗

PubMed1998

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage.

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.

Amino Acid Sequence↗

PubMed1998

The cytoplasmic zinc finger protein ZPR1 accumulates in the nucleolus of proliferating cells.

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells.

Amino Acid Sequence↗

PubMed1998

Sum1, a highly conserved WD-repeat protein, suppresses S-M checkpoint mutants and inhibits the osmotic stress cell cycle response in fission yeast.

The S-M checkpoint ensures that entry into mitosis is dependent on completion of DNA replication. In the fission yeast Schizosaccharomyces pombe, the SM checkpoint mutant cdc2-3w is thought to be defective in receiving the checkpoint signal. To isolate genes that function in the checkpoint pathway, we screened an S. pombe cDNA library for genes that, when overexpressed, could suppress the checkpoint defect of cdc2-3w. Using this approach, we have identified a novel gene, sum1+ (suppressor of uncontrolled mitosis). sum1+ encodes a highly conserved WD-transducin repeat protein with striking sequence similarity to the human transforming growth factor (TGF)-beta-receptor interacting protein TRIP-1 and to the translation initiation factor 3 subunit eIF3-p39, encoded by the TIF34 gene in Saccharomyces cerevisiae. S. pombe sum1+ is an essential gene, required for normal cell growth and division. In addition to restoring checkpoint control, overexpression of sum1+ inhibits the normal cell cycle response to osmotic stress. Furthermore, we demonstrate that inactivation of the stress-activated MAP kinase pathway, required for cell cycle stress response, restores the S-M checkpoint in cdc2-3w cells. These results suggest that Suml interacts with the stress-activated MAP kinase pathway and raise the possibility that environmental conditions may influence the checkpoint response in fission yeast.

Amino Acid Sequence↗

PubMed1998

Suppressors of cdc25p overexpression identify two pathways that influence the G2/M checkpoint in fission yeast.

Checkpoints maintain the order of cell-cycle events. At G2/M, a checkpoint blocks mitosis in response to damaged or unreplicated DNA. There are significant differences in the checkpoint responses to damaged DNA and unreplicated DNA, although many of the same genes are involved in both responses. To identify new genes that function specifically in the DNA replication checkpoint pathway, we searched for high-copy suppressors of overproducer of Cdc25p (OPcdc25(+)), which lacks a DNA replication checkpoint. Two classes of suppressors were isolated. One class includes a new gene encoding a putative DEAD box helicase, suppressor of uncontrolled mitosis (sum3(+)). This gene negatively regulates the cell-cycle response to stress when overexpressed and restores the checkpoint response by a mechanism that is independent of Cdc2p tyrosine phosphorylation. The second class includes chk1(+) and the two Schizosaccharomyces pombe 14-3-3 genes, rad24(+) and rad25(+), which appear to suppress the checkpoint defect by inhibiting Cdc25p. We show that rad24Delta mutants are defective in the checkpoint response to the DNA replication inhibitor hydroxyurea at 37 degrees and that cds1Delta rad24Delta mutants, like cds1Delta chk1Delta mutants, are entirely checkpoint deficient at 29 degrees. These results suggest that chk1(+) and rad24(+) may function redundantly with cds1(+) in the checkpoint response to unreplicated DNA.

Amino Acid Sequence↗

PubMed1997

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.

Adenosine Triphosphatases↗

PubMed1997

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.

Amino Acid Sequence↗

PubMed1996

S-phase and DNA-damage checkpoints: a tale of two yeasts.

Many genes required for the S-phase and DNA-damage checkpoints have been identified in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. This year many checkpoint genes have been sequenced, providing new information about the mechanism of checkpoint control. Several of these genes are conserved between the two yeasts but others are species-specific.

Cell Division↗

PubMed1996

Identification of residues in fission yeast and human p34cdc2 required for S-M checkpoint control.

In fission yeast, regulation of p34cdc2 plays an important role in the checkpoint coupling mitosis to completion of DNA replication. The cdc2 mutations cdc2-3w (C67Y) and cdc2-4w (C67F) abolish checkpoint control without seriously affecting normal cell proliferation. However the molecular basis of this phenotype is not known. To better understand the role of p34cdc2 in checkpoint control, we have screened for more mutations in Schizosaccharomyces pombe cdc2 with this phenotype. We have isolated cdc2-3w and cdc2-4w, as well as three new cdc2 alleles: cdc2-6w (N66I), cdc2-7w (E8V) and cdc2-8w (K9E). The altered residues map to two different regions on opposite faces of the protein, suggesting that the interaction between p34cdc2 and components of the checkpoint pathway may be complex. In contrast to cdc2-3w and cdc2-4w, the new mutations alter residues that are conserved between the fission yeast cdc2+ and other cdks, including the human CDC2 protein. Expression of the equivalent human CDC2 mutants in fission yeast abolishes checkpoint control, suggesting that these residues could be involved in checkpoint-dependent regulation of other eukaryotic cdks.

Amino Acid Sequence↗

PubMed1995

Cell-cycle checkpoints. Keeping mitosis in check.

Mutations in an essential yeast gene, encoding DNA polymerase epsilon, abolish the dependence of mitosis on the completion of DNA replication, suggesting that the replication complex provides the checkpoint signal.

Animals↗

PubMed1995

Cellular responses to DNA damage: cell-cycle checkpoints, apoptosis and the roles of p53 and ATM.

'Checkpoint' controls arrest the cell cycle after DNA damage, allowing repair to take place before mutations can be perpetuated. In multicellular organisms, DNA damage can also induce apoptotic cell death, protecting the organism at the expense of the individual cell. How does a cell 'choose' between cycle arrest and death? Analysis of two human tumour suppressor proteins, p53 and the ATM (ataxia-telangiectasia mutated) gene product, may provide some answers.

Apoptosis↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1994

mik1+ encodes a tyrosine kinase that phosphorylates p34cdc2 on tyrosine 15.

mik1+ and wee1+ function to regulate the tyrosine phosphorylation of p34cdc2 in Schizosaccharomyces pombe (Lundgren, K., Walworth, N., Booher, R., Dembski, M., Kirschner, M., and Beach, D. (1991) Cell 64, 1111-1122). wee1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15, resulting in the inactivation of the cyclin B/p34cdc2 complex. We have overproduced the mik1+ gene product in insect cells and in S. pombe in order to characterize it biochemically. Immunoprecipitates of Mik1 from both sources catalyzed the phosphorylation of p34cdc2 on tyrosine 15 whereas immunoprecipitates of a kinase-deficient mutant of Mik1 were negative in this assay. Mik1 overproduced in insect cells was partially purified by column chromatography, and column fractions were assayed for their ability to phosphorylate p34cdc2 on tyrosine 15. Two major peaks of Mik1 protein were detected by gel filtration chromatography. One peak eluted in the void volume, and a second peak eluted with an apparent molecular mass expected for monomeric Mik1 (approximately 68 kDa). The tyrosine 15 kinase activity co-eluted with the 68 kDa form of Mik1. These results indicate that mik1+ encodes a tyrosine kinase that directly phosphorylates p34cdc2 on tyrosine 15.

Amino Acid Sequence↗

PubMed1993

Checkpoint check.

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Genes, Fungal↗