An intron is required for dihydrofolate reductase protein stability

We compared the expression of dihydrofolate reductase minigenes with and without an intron. The levels of protein were significantly higher in the presence of dihydrofolate reductase intron 1. However, mRNA levels in both constructs were comparable. In addition, the RNA transcribed from either construct was correctly polyadenylated and exported to the cytoplasm. The intron-mediated increase in dihydrofolate reductase protein levels was position-independent and was also observed when dihydrofolate reductase intron 1 was replaced by heterologous introns. The translational rate of dihydrofolate reductase protein was increased in transfectants from the intron-containing minigene. In addition, the protein encoded by the intronless construct was unstable and subject to lysosomal degradation, thus showing a shorter half-life than the protein encoded by the intron-containing minigene. We conclude that an intron is required for the translation and stability of dihydrofolate reductase protein.     

The Saccharomyces cerevisiae centromere protein Slk19p is required for two successive divisions during meiosis

Meiotic cell division includes two separate and distinct types of chromosome segregation. In the first segregational event the sister chromatids remain attached at the centromere; in the second the chromatids are separated. The factors that control the order of chromosome segregation during meiosis have not yet been identified but are thought to be confined to the centromere region. We showed that the centromere protein Slk19p is required for the proper execution of meiosis in Saccharomyces cerevisiae. In its absence diploid cells skip meiosis I and execute meiosis II division. Inhibiting recombination does not correct this phenotype. Surprisingly, the initiation of recombination is apparently required for meiosis II division. Thus Slk19p appears to be part of the mechanism by which the centromere controls the order of meiotic divisions.     

Solution structure of the p53 regulatory domain of the p19Arf tumor suppressor protein

Arf is a tumor suppressor that regulates p53 function and is a frequent target for loss in human cancers. Through two novel mechanisms, Arf inhibits the oncoprotein Hdm2, a negative regulator of p53. (1) Arf inhibits the E3 ubiquitin ligase activity of Hdm2 that leads to p53 degradation, and (2) Arf sequesters Hdm2 within nucleoli. These activities of Arf promote p53-mediated cell cycle arrest and apoptosis. Fundamental to these processes are interactions between Arf and Hdm2. Here we show that a peptide containing the 37 N-terminal amino acids of mouse Arf (mArfN37) localizes to nucleoli, sequesters Hdm2 within nucleoli, and causes cell cycle arrest. Circular dichroism and NMR spectroscopy show that mArfN37 is largely unstructured under aqueous conditions; however, the peptide adopts two alpha-helices (helix 1, residues 4-14; and helix 2, residues 20-29) in 2,2,2-trifluoroethanol (TFE). Each helix contains an amino acid motif that is repeated twice in mArfN37, once in each helix. The two helices, however, do not interact but are connected by an apparently flexible linker. The repeated motif contains Arg residues spaced by a hydrophobic segment that may be involved in Hdm2 recognition and binding. The RRPR nucleolar localization signal, contained within residues 31-34, appears to be disordered under all conditions. The identification of two Arf structural modules suggests that short peptides containing the repeated motif may function as Arf mimics and may allow the design of small molecule Arf mimics in the future.     

Schizosaccharomyces pombe Hsk1p is a potential cds1p target required for genome integrity

The fission yeast Hsk1p kinase is an essential activator of DNA replication. Here we report the isolation and characterization of a novel mutant allele of the gene. Consistent with its role in the initiation of DNA synthesis, hsk1(ts) genetically interacts with several S-phase mutants. At the restrictive temperature, hsk1(ts) cells suffer abnormal S phase and loss of nuclear integrity and are sensitive to both DNA-damaging agents and replication arrest. Interestingly, hsk1(ts) mutants released to the restrictive temperature after early S-phase arrest in hydroxyurea (HU) are able to complete bulk DNA synthesis but they nevertheless undergo an abnormal mitosis. These findings indicate a second role for hsk1 subsequent to HU arrest. Consistent with a later S-phase role, hsk1(ts) is synthetically lethal with Deltarqh1 (RecQ helicase) or rad21ts (cohesin) mutants and suppressed by Deltacds1 (RAD53 kinase) mutants. We demonstrate that Hsk1p undergoes Cds1p-dependent phosphorylation in response to HU and that it is a direct substrate of purified Cds1p kinase in vitro. These results indicate that the Hsk1p kinase is a potential target of Cds1p regulation and that its activity is required after replication initiation for normal mitosis.     

Yeast Nap1-binding protein Nbp2p is required for mitotic growth at high temperatures and for cell wall integrity

Nbp2p is a Nap1-binding protein in Saccharomyces cerevisiae identified by its interaction with Nap1 by a two-hybrid system. NBP2 encodes a novel protein consisting of 236 amino acids with a Src homology 3 (SH3) domain. We showed that NBP2 functions to promote mitotic cell growth at high temperatures and cell wall integrity. Loss of Nbp2 results in cell death at high temperatures and in sensitivity to calcofluor white. Cell death at high temperature is thought not to be due to a weakened cell wall. Additionally, we have isolated several type-2C serine threonine protein phosphatases (PTCs) as multicopy suppressors and MAP kinase-kinase (MAPKK), related to the yeast PKC MAPK pathway, as deletion suppressors of the nbp2Delta mutant. Screening for deletion suppressors is a new genetic approach to identify and characterize additional proteins in the Nbp2-dependent pathway. Genetic analyses suggested that Ptc1, which interacts with Nbp2 by the two-hybrid system, acts downstream of Nbp2 and that cells lacking the function of Nbp2 prefer to lose Mkk1, but the PKC MAPK pathway itself is indispensable when Nbp2 is deleted at high temperature.     

Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes

The dramatic condensation of chromosomes that occurs during mitosis is widely thought to be largely controlled by a protein complex termed condensin. Here, we describe a conditional knockout of the condensin subunit ScII/SMC2 in chicken DT40 cells. In cells lacking this condensin subunit, chromosome condensation is delayed, but ultimately reaches near-normal levels. However, these chromosomes are structurally compromised. Kinetochores appear normal, but the localization of nonhistone proteins such as topoisomerase II and INCENP is aberrant. Both proteins also fail to partition into the chromosome scaffold fraction, which appears to be largely missing in the absence of condensin. Furthermore, the chromosomes lack structural integrity, as defined by an assay that tests the stability of the chromosomal higher-order structure. Thus, a major function of condensin is to promote the correct association of nonhistone proteins with mitotic chromosomes, and this is essential for establishment of a robust chromosome structure.     

Mmm1p, a mitochondrial outer membrane protein, is connected to mitochondrial DNA (mtDNA) nucleoids and required for mtDNA stability

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p-green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.     

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.     

Schizosaccharomyces pombe Pmr1p is essential for cell wall integrity and is required for polarized cell growth and cytokinesis

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.     

p53 Binding protein 53BP1 is required for DNA damage responses and tumor suppression in mice

53BP1 is a p53 binding protein of unknown function that binds to the central DNA-binding domain of p53. It relocates to the sites of DNA strand breaks in response to DNA damage and is a putative substrate of the ataxia telangiectasia-mutated (ATM) kinase. To study the biological role of 53BP1, we disrupted the 53BP1 gene in the mouse. We show that, similar to ATM(-/-) mice, 53BP1-deficient mice were growth retarded, immune deficient, radiation sensitive, and cancer prone. 53BP1(-/-) cells show a slight S-phase checkpoint defect and prolonged G(2)/M arrest after treatment with ionizing radiation. Moreover, 53BP1(-/-) cells feature a defective DNA damage response with impaired Chk2 activation. These data indicate that 53BP1 acts downstream of ATM and upstream of Chk2 in the DNA damage response pathway and is involved in tumor suppression.     

The vesicular transport protein Cgp1p/Vps54p/Tcs3p/Luv1p is required for the integrity of the actin cytoskeleton

The CGP1 gene was identified in a screen for mutations that were synthetic lethal in combination with a deletion of the gene (CPF1) for centromere and promoter factor 1. Cells deleted for CGP1 showed reduced viability, were temperature sensitive for growth and exhibited altered sensitivity to microtubule-destabilizing drugs. Furthermore, Deltacgp1 cells showed increased rates of loss of a circular minichromosome and defects in the positioning of the short mitotic spindle. Further phenotypic analysis of Deltacgp1 cells revealed that loss of Cgp1p function led to severe depolarization of the actin cytoskeleton. In addition, cells deleted for CGP1 were hypersensitive to the actin-disrupting compound Latrunculin-A, exhibited strongly reduced polarized localization of the unconventional myosin Myo2p, and showed defects in other actin-related processes, such as shmoo formation and cell wall integrity. Cgp1p was recently identified by several groups as Vps54p, which is a member of the VFT complex that is involved in vesicular protein transport at the level of the late Golgi, acting as a tethering factor. Our data show for the first time that Cgp1p/Vps54p links aspects of vesicular protein transport with the organization of the actin cytoskeleton.     

Mammalian DNA2 helicase/nuclease cleaves G‐quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G‐quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2‐deficient mice develop aneuploidy‐associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.