Control of the yeast cell cycle with a photocleavable alpha-factor analogue.
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Cell Cycle↗
Biomedical subjects
Stephen J Kron
Publications and source records attributed to Stephen J Kron.
CDK Pho85 targets CDK inhibitor Sic1 to relieve yeast G1 checkpoint arrest after DNA damage.
In budding yeast, DNA damage in G1 activates a Rad9-dependent checkpoint that targets the cyclin-dependent kinase (CDK) Cdc28 to delay G1 exit. After a transient arrest, cells may enter S phase before completing DNA repair. We used genetic analysis to identify the stress-responsive CDK Pho85, the cyclin Pho80 and the targeted transcription factors Pho4 and Swi5 as determinants of G1 checkpoint adaptation. Consistent with opposing roles for the Cdc28 inhibitor Sic1 in blocking G1 exit and Pho85 in targeting Sic1 for proteolysis, mutation of Sic1 curtails G1 checkpoint delay, whereas Pho85 inhibition after DNA damage promotes Sic1 stability. G1 checkpoint delay in mutants lacking both Sic1 and Pho4 is independent of Pho85 activity. These data establish a G1 checkpoint adaptation pathway where Pho85 mediates Pho4 downregulation and Sic1 degradation to release Cdc28 activity and promote onset of S phase.
CDC28 Protein Kinase, S cerevisiae↗
Yeast G1 DNA damage checkpoint regulation by H2A phosphorylation is independent of chromatin remodeling.
Recent studies of yeast G1 DNA damage response have identified characteristic changes in chromatin adjacent to double-strand breaks (DSBs). Histone H2A (yeast H2AX) is rapidly phosphorylated on S129 by the kinase Tel1 (ATM) over a domain extending kilobases from the DSB. The adaptor protein Rad9 (53BP1) is recruited to this chromatin domain through binding of its tudor domains to histone H3 diMe-K79. Multisite phosphorylation of Rad9 by Mec1 (ATR) then activates the signaling kinase Rad53 (CHK2) to induce a delay in G1. Here, we report a previously undescribed role for Tel1 in G1 checkpoint response and show that H2A is the likely phosphorylation target, in a much as S129 mutation to Ala confers defects in G1 checkpoint arrest, Rad9 phosphorylation, and Rad53 activation. Importantly, Rad9 fails to bind chromatin adjacent to DSBs in H2A-S129A mutants. Previous work showed that H2A phosphorylation allows binding of NuA4, SWR, and INO80 chromatin remodeling complexes, perhaps exposing H3 diMe-K79. Yet, mutants lacking SWR or INO80 remain checkpoint competent, whereas loss of NuA4-dependent histone acetylation leads to G1 checkpoint persistence, suggesting that H2A phosphorylation promotes two independent events, rapid Rad9 recruitment to DSBs and subsequent remodeling by NuA4, SWR, and INO80.
Acetyltransferases↗
Photocleavable peptide hydrogel arrays for MALDI-TOF analysis of kinase activity.
We have developed an acrylamide copolymerization strategy to immobilize acrylamide labeled peptides and proteins into a hydrogel surface and detect their modifications using MALDI-TOF mass spectrometry. Copolymerization into hydrogels is robust, compatible with "off-the-shelf" chemistry, and yields materials and surfaces that are stable to aqueous or organic solvents, drying, high or low temperature, high or low pH, oxidizing agents, sonication, mechanical contact, etc. The use of acrylamide hydrogels allows immobilization of substrates in a hydrated environment that can be used both as a biological reaction matrix and as a MALDI target. In our strategy, a substrate peptide was designed in a modular fashion to include both modification site and affinity domains. It was labeled with an acrylamide functionality using a generalized chemistry and covalently attached to the surface with a photocleavable linker, allowing for aggressive washing to remove any fouling, followed by selective release for MALDI-TOF analysis. Using this system we were able to analyze and compare v-Abl (truncated) and c-Abl (full-length) kinase activity on a peptide substrate with an affinity domain specific for the full-length kinase, observing excellent overall reproducibility in the extent of phosphorylation detected. This work serves as proof of principle for modular substrate design strategies for mass spectrometry-readable biosensors.
Acrylamides↗
Assaying Bcr-Abl kinase activity and inhibition in whole cell extracts by phosphorylation of substrates immobilized on agarose beads.
There is a current and increasing demand for simple, robust, nonradioactive assays of protein tyrosine kinase activity with applications for clinical diagnosis and high-throughput screening of potential molecularly targeted therapeutic agents. One significant challenge is to detect and measure the activity of specific kinases with key roles in cell signaling as an approach to distinguish normal cells from cancer cells and as a means of evaluating targeted drug efficacy and resistance in cancer cells. Here, we describe a method in which kinase substrates fused to glutathione-S-transferase and immobilized on glutathione agarose beads are phosphorylated, eluted, and then assayed to detect kinase activity. The activity of recombinant, purified c-Abl kinase or Bcr-Abl kinase in whole cell extracts can be detected with equivalent specificity, sensitivity, and reproducibility. Similarly, inhibition of recombinant c-Abl or Bcr-Abl in cells or cell extracts by imatinib mesylate and other Bcr-Abl targeted kinase inhibitors is readily assayed. This simple kinase assay is sufficiently straightforward and robust for use in clinical laboratories and is potentially adaptable to high-throughput assay formats.
Amino Acid Sequence↗
Role of Dot1-dependent histone H3 methylation in G1 and S phase DNA damage checkpoint functions of Rad9.
We screened radiation-sensitive yeast mutants for DNA damage checkpoint defects and identified Dot1, the conserved histone H3 Lys 79 methyltransferase. DOT1 deletion mutants (dot1Delta) are G1 and intra-S phase checkpoint defective after ionizing radiation but remain competent for G2/M arrest. Mutations that affect Dot1 function such as Rad6-Bre1/Paf1 pathway gene deletions or mutation of H2B Lys 123 or H3 Lys 79 share dot1Delta checkpoint defects. Whereas dot1Delta alone confers minimal DNA damage sensitivity, combining dot1Delta with histone methyltransferase mutations set1Delta and set2Delta markedly enhances lethality. Interestingly, set1Delta and set2Delta mutants remain G1 checkpoint competent, but set1Delta displays a mild S phase checkpoint defect. In human cells, H3 Lys 79 methylation by hDOT1L likely mediates recruitment of the signaling protein 53BP1 via its paired tudor domains to double-strand breaks (DSBs). Consistent with this paradigm, loss of Dot1 prevents activation of the yeast 53BP1 ortholog Rad9 or Chk2 homolog Rad53 and decreases binding of Rad9 to DSBs after DNA damage. Mutation of Rad9 to alter tudor domain binding to methylated Lys 79 phenocopies the dot1Delta checkpoint defect and blocks Rad53 phosphorylation. These results indicate a key role for chromatin and methylation of histone H3 Lys 79 in yeast DNA damage signaling.
Blotting, Western↗
Cellular response to DNA damage.
Eukaryotic cells, from yeast to man, possess evolutionarily conserved mechanisms to accurately and efficiently repair the overwhelming majority of DNA damage, thereby ensuring genomic integrity. Important repair pathways include base excision repair, nucleotide excision repair, mismatch repair, non-homologous end-joining, and homologous recombination. Defects in DNA repair processes generally result in susceptibility to cancer and, often, abnormalities in multiple organ systems. While signal transduction pathways have been intensely studied, epigenetic changes occurring in response to DNA damage are rapidly increasing in importance. Effective radiation and chemotherapy sensitization could result from selective inhibition of DNA repair in tumor cells. DNA damage repair is a dynamic field of research where the fruits of basic research often have important clinical implications.
DNA↗
Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites.
Yeast histone H2A is phosphorylated on Ser129 upon DNA damage, an event required for efficient repair. We show that phosphorylation occurs rapidly over a large region around DNA double-strand breaks (DSBs). Histone H4 acetylation is also important for DSB repair, and we found that the NuA4 HAT complex associates specifically with phospho-H2A peptides. A single NuA4 subunit, Arp4, is responsible for the interaction. The NuA4 complex is recruited to a DSB concomitantly with the appearance of H2A P-Ser129 and Arp4 is important for this binding. Arp4 is also a subunit of the Ino80 and Swr1 chromatin remodeling complexes, which also interact with H2A P-Ser129 and are recruited to DSBs. This association again requires Arp4 but also prior NuA4 recruitment and action. Thus, phosphorylation of H2A at DNA damage sites creates a mark recognized by different chromatin modifiers. This interaction leads to stepwise chromatin reconfiguration, allowing efficient DNA repair.
Acetyltransferases↗
Yeast cell death during DNA damage arrest is independent of caspase or reactive oxygen species.
CDC13 encodes a telomere-binding protein that prevents degradation of telomeres. cdc13-1 yeast grown at the nonpermissive temperature undergo G2/M arrest, progressive chromosome instability, and subsequent cell death. Recently, it has been suggested that cell death in the cdc13-1 mutant is an active process characterized by phenotypic hallmarks of apoptosis and caspase activation. In this work, we show that cell death triggered by cdc13-1 is independent of the yeast metacaspase Yca1p and reactive oxygen species but related to cell cycle arrest per se. Inactivating YCA1 or depleting reactive oxygen species does not increase viability of cdc13-1 cells. In turn, caspase activation does not precede cell death in the cdc13-1 mutant. Yca1p activity assayed by cell binding of mammalian caspase inhibitors is confounded by artifactual labeling of dead yeast cells, which nonspecifically bind fluorochromes. We speculate that during a prolonged cell cycle arrest, cdc13-1 cells reach a critical size and die by cell lysis.
Apoptosis↗
Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity.
We report the development and characterization of a polyacrylamide-based protein immobilization strategy for surface-bound protein assays, including concentration detection, binding affinity, and enzyme kinetics. Glutathione S-transferase (GST) fusion proteins have been labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through copolymerization with acrylic monomer. The specific attachment of GST-green fluorescent protein (GFP) fusion protein was more than sevenfold greater than the nonspecific attachment of nonacrylic-labeled GST-GFP; 0.32 ng/mm(2) of surface-attached GST-GFP was detectable by direct measurement of GFP fluorescence and this lower detection limit was reduced to 0.080 ng/mm(2) using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src. Michaelis-Menten kinetic constants for the reaction occurring in solution were K(m) = 2.7 +/- 1.0 microM and V(max) = 8.1 +/- 3.1 (arbitrary units). Kinetic values for the reaction utilizing surface-immobilized substrate were K(m) = 0.36 +/- 0.033 microM and V(max) = 9.7 +/- 0.63 and were found to be independent of the acrylamide concentration within the copolymer. Such a surface attachment strategy should be applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.
Acrylic Resins↗
Monitoring changes in the subcellular location of proteins in S. cerevisiae.
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Biochemistry↗
Histone H2AX phosphorylation as a predictor of radiosensitivity and target for radiotherapy.
Based on the role of phosphorylation of the histone H2A variant H2AX in recruitment of DNA repair and checkpoint proteins to the sites of DNA damage, we have investigated gammaH2AX as a reporter of tumor radiosensitivity and a potential target to enhance the effectiveness of radiation therapy. Clinically relevant ionizing radiation (IR) doses induced similar patterns of gammaH2AX focus formation or immunoreactivity in radiosensitive and radioresistant human tumor cell lines and xenografted tumors. However, radiosensitive tumor cells and xenografts retained gammaH2AX for a greater duration than radioresistant cells and tumors. These results suggest that persistence of gammaH2AX after IR may predict tumor response to radiotherapy. We synthesized peptide mimics of the H2AX carboxyl-terminal tail to test whether antagonizing H2AX function affects tumor cell survival following IR. The peptides did not alter the viability of unirradiated tumor cells, but both blocked induction of gammaH2AX foci by IR and enhanced cell death in irradiated radioresistant tumor cells. These results suggest that H2AX is a potential molecular target to enhance the effects of radiotherapy.
Animals↗
Science education. Educating future scientists.
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Biological Science Disciplines↗
Asynchronous cell cycle and asymmetric vacuolar inheritance in true hyphae of Candida albicans.
Candida albicans forms unconstricted hyphae in serum-containing medium that are divided into discrete compartments. Time-lapse photomicroscopy, flow cytometry, and a novel three-dimensional imaging system were used to demonstrate that the kinetics and cell cycle events accompanying hyphal development were correlated with dynamic changes in vacuole morphology and the pattern of vacuole inheritance. Apical cells of hyphae underwent continuous extension before and after the first cytokinesis event. However, the resulting mother cell and sub-apical compartments did not immediately reenter the cell cycle and instead underwent cell cycle arrest before reentering the cycle. Vacuole was inherited asymmetrically at cytokinesis so that the distal, arrested compartments inherited most vacuole and the growing apical cell inherited most cytoplasm. Hydroxyurea release experiments demonstrated that the arrested, vacuolated hyphal compartments were in the G(1) phase of the cycle. The period of cell cycle arrest was decreased by the provision of assimilatable forms of nitrogen, suggesting that the hyphal cell cycle is regulated by nitrogen limitation that results in sup-apical cell cycle arrest. This pattern of growth is distinct from that of the synchronous, symmetrical development of pseudohyphae of C. albicans and other yeast species. These observations suggest that the cellular vacuole space correlates with alterations in the cell cycles of different cell types and that the total organelle space may influence size-regulated functions and hence the timing of the eukaryotic cell cycle.
Animals↗
Design and implementation of algorithms for focus automation in digital imaging time-lapse microscopy.
BACKGROUND: Digital time-lapse microscopy using Nomarski-DIC requires that an autofocusing system adapt to changes in cell shape, size, and position while contending with drift, noise, and hysteresis in the microscope and imager. We have designed and implemented an autofocusing system that tracks subjects under dynamic conditions and maintains focus within a threshold of discriminability. METHODS: With the use of proven and novel algorithms for autofocusing in Nomarski, we performed "virtual" experiments on recorded image stacks to simulate drift and sudden displacements and test the search algorithm response. RESULTS: We found that combining a simple [1, -1] contrast function with an adaptive "warmer-colder" focusing algorithm yields a reasonable compromise between focusing precision and noise tolerance. This method was implemented to record growth kinetics of yeast cells in single and multiple fields of view over several hours. CONCLUSIONS: We have implemented a robust digital autofocus that maintains focus on optically complex samples imaged at high resolution. The tolerance of this system of drift and vibration suggests that it is a practical system for time-lapse imaging in many biological applications.
Algorithms↗
An essential function of yeast cyclin-dependent kinase Cdc28 maintains chromosome stability.
Multiple surveillance pathways maintain genomic integrity in yeast during mitosis. Although the cyclin-dependent kinase Cdc28 is a well established regulator of mitotic progression, evidence for a direct role in mitotic surveillance has been lacking. We have now implicated a conserved sequence in the Cdc28 carboxyl terminus in maintaining chromosome stability through mitosis. Six temperature-sensitive mutants were isolated via random mutagenesis of 13 carboxyl-terminal residues. These mutants identify a Cdc28 domain necessary for proper mitotic arrest in the face of kinetochore defects or microtubule inhibitors. These chromosome stability-defective cdc28(CST) mutants inappropriately continue mitosis when the mitotic spindle is disrupted at 23 degrees C, display high rates of spontaneous chromosome loss at 30 degrees C, and suffer catastrophic aneuploidy at 35 degrees C. A dosage suppression screen identified Cak1, a kinase known to phosphorylate and activate Cdc28, as a specific high copy suppressor of cdc28(CST) temperature sensitivity and chromosome instability. Suppression is independent of the kinase activity of Cak1, suggesting that Cak1 may bind to the carboxyl terminus to serve a non-catalytic role in assembly and/or stabilization of active Cdc28 complexes. Significantly, these studies implicate Cdc28 and Cak1 in an essential surveillance function required to maintain genetic stability through mitosis.
Amino Acid Sequence↗
Bcl-x(L) complements Saccharomyces cerevisiae genes that facilitate the switch from glycolytic to oxidative metabolism.
All eukaryotic organisms have mechanisms to adapt to changing metabolic conditions. The mammalian cell survival gene Bcl-x(L) enables cells to adapt to changes in cellular metabolism. To identify genes whose function can be substituted by Bcl-x(L) in a unicellular eukaryote, a genetic screen was performed using the yeast Saccharomyces cerevisiae. S. cerevisiae grows by anaerobic glycolysis when glucose is available, switching to oxidative phosphorylation when carbohydrate in the media becomes limiting (diauxic shift). Given that Bcl-x(L) appears to facilitate the switch from glycolytic to oxidative metabolism in mammalian cells, a library of yeast mutants was tested for the ability to efficiently undergo diauxic shift in the presence and absence of Bcl-x(L). Several mutants were identified that have a defect in growth when switched from a fermentable to a nonfermentable carbon source that is corrected by the expression of Bcl-x(L). These genes include the mitochondrial chaperonin TCM62, as well as previously uncharacterized genes. One of these uncharacterized genes, SVF1, promotes cell survival in mammalian cells in response to multiple apoptotic stimuli. The finding that TCM62 and the analogous human prohibitin gene also inhibit mammalian cell death following growth factor withdrawal implicates mitochondrial chaperones as regulators of apoptosis. Further characterization of the genes identified in this screen may enhance our understanding of Bcl-x(L) function in mammalian cells, and of cell survival pathways in general.
Animals↗
Robust G1 checkpoint arrest in budding yeast: dependence on DNA damage signaling and repair.
Although most eukaryotes can arrest in G1 after ionizing radiation, the existence or significance of a G1 checkpoint in S. cerevisiae has been challenged. Previous studies of G1 response to chemical mutagens, X-ray or UV irradiation indicate that the delay before replication is transient and may reflect a strong intra-S-phase checkpoint. We examined the yeast response to double-stranded breaks in G1 using gamma irradiation. G1 irradiation induces repair foci on chromosome spreads and a Rad53 band shift characteristic of activation, which suggest an active DNA damage response. Consistent with a G1 arrest, bud emergence, spindle pole duplication and DNA replication are each delayed in a dose-dependent manner. Sensitivity to mating pheromone is prolonged to over 18 hours when G1 cells are lethally gamma or UV irradiated. Strikingly, G1 delay is the predominant response to continuous gamma irradiation at a dose that confers no loss of viability but delays cell division. Like the G2/M checkpoint, G1 delay is completely dependent on both RAD9 and RAD24 epistasis groups but independent of POL(epsilon). Lethally irradiated rad9 mutants rapidly exit G1 but perform a slow S phase, whereas rad17 and rad24 mutants are completely arrest deficient. Distinct from gamma irradiation, G1 arrest after UV is RAD14 dependent, suggesting that DNA damage processing is required for checkpoint activation. Therefore, as in the yeast G2/M checkpoint response, free DNA ends and/or single-stranded DNA are necessary and sufficient to induce a bona fide G1 checkpoint arrest.
Cell Cycle Proteins↗