Kegg Pathway: Cell cycle - yeast

J Cell Sci. 2009 Dec 1; 122(Pt 23): 4375-4382
Keating P, Rachidi N, Tanaka TU, Stark MJ

The conserved Aurora B protein kinase (Ipl1 in Saccharomyces cerevisiae) is essential for ensuring that sister kinetochores become attached to microtubules from opposite spindle poles (bi-orientation) before anaphase onset. When sister chromatids become attached to microtubules from a single pole, Aurora B/Ipl1 facilitates turnover of kinetochore-microtubule attachments. This process requires phosphorylation by Aurora B/Ipl1 of kinetochore components such as Dam1 in yeast. Once bi-orientation is established and tension is applied on kinetochores, Aurora B/Ipl1 must stop promoting this turnover, otherwise correct attachment would never be stabilised. How this is achieved remains elusive: it might be due to dephosphorylation of Aurora B/Ipl1 substrates at kinetochores, or might take place independently, for example because of conformational changes in kinetochores. Here, we show that Ipl1-dependent phosphorylation at crucial sites on Dam1 is maximal during S phase and minimal during metaphase, matching the Cell cycle window when chromosome bi-orientation occurs. Intriguingly, when we reduced tension at kinetochores through failure to establish sister chromatid cohesion, Dam1 phosphorylation persisted in metaphase-arrested Cells. We propose that Aurora B/Ipl1-facilitated bi-orientation is stabilised in response to tension at kinetochores by dephosphorylation of Dam1, resulting in termination of kinetochore-microtubule attachment turnover.

Drug Des Devel Ther. 2009; 2: 255-264
Sawa M, Masai H

Identification of novel molecular targets is critical in development of new and efficient cancer therapies. Kinases are one of the most common drug targets with a potential for cancer therapy. Cell cycle progression is regulated by a number of kinases, some of which are being developed to treat cancer. Cdc7 is a serine-threonine kinase originally discovered in budding yeast, which has been shown to be necessary to initiate the S phase. Inhibition of Cdc7 in cancer Cells retards the progression of the S phase, accumulates DNA damage, and induces p53-independent Cell death, but the same treatment in normal Cells does not significantly affect of less than viability. Low-molecular-weight compounds that inhibit Cdc7 kinase with an IC(50) 10 nM have been identified, and shown to be effective in the inhibition of tumor growth in animal models. Thus Cdc7 kinase can be recognized as a novel molecular target for cancer therapy.

Mol Syst Biol. 2009; 5: 328
Drapkin BJ, Lu Y, Procko AL, Timney BL, Cross FR

Cyclin-dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast Cell cycle. However, the ratchet model requires appropriate cyclin dose-response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose-dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing Cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2-Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions.

Mol Cell Biol. 2009 Nov 16;
Ge J, Rudnick DA, He J, Crimmins DL, Ladenson JH, Bessler M, Mason PJ

Dyskerin is a component of small nucleolar ribonucleoprotein complexes and acts as a pseudouridine synthase to modify newly synthesized ribosomal, spliceosomal and possibly other RNAs. It is encoded by the DKC1 gene, the gene mutated in X-linked dyskeratosis congenita, and is also part of the telomerase complex. The yeast ortholog, Cbf5, is an essential protein but in mammals the effect of dyskerin ablation at the Cellular level is not known. Here we show that mouse hepatocytes can survive after induction of a Dkc1 deletion. In the absence of dyskerin, ribosomal RNA processing is inhibited with the accumulation of large precursors, and fibrillarin does not accumulate in nucleoli. A low rate of apoptosis is induced in the hepatocytes, which show an induction of the p53 dependent Cell cycle checkpoint pathway. Signs of liver damage are observed including an increase in serum alanine aminotransferase activity and a disordered structure at the histological and macroscopic levels. In response to carbon tetrachloride administration, when wild type hepatocytes mount a rapid proliferative response, those without dyskerin do not divide. We conclude that hepatocytes can survive without dyskerin but that the role of dyskerin in RNA modification is essential for Cellular proliferation.

PLoS Genet. 2009 Nov; 5(11): e1000727
Artiles K, Anastasia S, McCusker D, Kellogg DR

The key molecular event that marks entry into the Cell cycle is transcription of G1 cyclins, which bind and activate cyclin-dependent kinases. In yeast Cells, initiation of G1 cyclin transcription is linked to achievement of a critical Cell size, which contributes to Cell-size homeostasis. The critical Cell size is modulated by nutrients, such that Cells growing in poor nutrients are smaller than Cells growing in rich nutrients. Nutrient modulation of Cell size does not work through known critical regulators of G1 cyclin transcription and is therefore thought to work through a distinct pathway. Here, we report that Rts1, a highly conserved regulatory subunit of protein phosphatase 2A (PP2A), is required for normal control of G1 cyclin transcription. Loss of Rts1 caused delayed initiation of bud growth and delayed and reduced accumulation of G1 cyclins. Expression of the G1 cyclin CLN2 from an inducible promoter rescued the delayed bud growth in rts1Delta Cells, indicating that Rts1 acts at the level of transcription. Moreover, loss of Rts1 caused altered regulation of Swi6, a key component of the SBF transcription factor that controls G1 cyclin transcription. Epistasis analysis revealed that Rts1 does not work solely through several known critical upstream regulators of G1 cyclin transcription. Cells lacking Rts1 failed to undergo nutrient modulation of Cell size. Together, these observations demonstrate that Rts1 is a key player in pathways that link nutrient availability, Cell size, and G1 cyclin transcription. Since Rts1 is highly conserved, it may function in similar pathways in vertebrates.

Proc Natl Acad Sci U S A. 2009 Nov 12;
Evrin C, Clarke P, Zech J, Lurz R, Sun J, Uhle S, Li H, Stillman B, Speck C

During pre-replication complex (pre-RC) formation, origin recognition complex (ORC), Cdc6, and Cdt1 cooperatively load the 6-subunit mini chromosome maintenance (MCM2-7) complex onto DNA. Loading of MCM2-7 is a prerequisite for DNA licensing that restricts DNA replication to once per Cell cycle. During S phase MCM2-7 functions as part of the replicative helicase but within the pre-RC MCM2-7 is inactive. The organization of replicative DNA helicases before and after loading onto DNA has been studied in bacteria and viruses but not eukaryotes and is of major importance for understanding the MCM2-7 loading mechanism and replisome assembly. Lack of an efficient reconstituted pre-RC system has hindered the detailed mechanistic and structural analysis of MCM2-7 loading for a long time. We have reconstituted Saccharomyces cerevisiae pre-RC formation with purified proteins and showed efficient loading of MCM2-7 onto origin DNA in vitro. MCM2-7 loading was found to be dependent on the presence of all pre-RC proteins, origin DNA, and ATP hydrolysis. The quaternary structure of MCM2-7 changes during pre-RC formation: MCM2-7 before loading is a single hexamer in solution but is transformed into a double-hexamer during pre-RC formation. Using electron microscopy (EM), we observed that loaded MCM2-7 encircles DNA. The loaded MCM2-7 complex can slide on DNA, and sliding is not directional. Our results provide key insights into mechanisms of pre-RC formation and have important implications for understanding the role of the MCM2-7 in establishment of bidirectional replication forks.

DNA Repair (Amst). 2009 Nov 10;
Heidenreich E, Eisler H, Lengheimer T, Dorninger P, Steinboeck F

Growing attention is paid to the concept that mutations arising in stationary, non-proliferating Cell populations considerably contribute to evolution, aging, and pathogenesis. If such mutations are beneficial to the affected Cell, in the sense of allowing a restart of proliferation, they are called adaptive mutations. In order to identify Cellular processes responsible for adaptive mutagenesis in eukaryotes, we study frameshift mutations occurring during auxotrophy-caused Cell cycle arrest in the model organism Saccharomyces cerevisiae. Previous work has shown that an exposure of Cells to UV irradiation during prolonged Cell cycle arrest resulted in an increased incidence of mutations. In the present work, we determined the influence of defects in the nucleotide excision repair (NER) pathway on the incidence of UV-induced adaptive mutations in stationary Cells. The mutation frequency was decreased in Rad16-deficient Cells and further decreased in Rad16/Rad26 double-deficient Cells. A knockout of the RAD14 gene, the ortholog of the human XPA gene, even resulted in a nearly complete abolishment of UV-induced mutagenesis in Cell cycle-arrested Cells. Thus, the NER pathway, responsible for a normally accurate repair of UV-induced DNA damage, paradoxically is required for the generation and/or fixation of UV-induced frameshift mutations specifically in non-replicating Cells.

Nucleic Acids Res. 2009 Nov 11;
Bauerschmidt C, Arrichiello C, Burdak-Rothkamm S, Woodcock M, Hill MA, Stevens DL, Rothkamm K

The cohesin protein complex holds sister chromatids together after synthesis until mitosis. It also contributes to post-replicative DNA repair in yeast and higher eukaryotes and accumulates at sites of laser-induced damage in human Cells. Our goal was to determine whether the cohesin subunits SMC1 and Rad21 contribute to DNA double-strand break repair in X-irradiated human Cells in the G2 phase of the Cell cycle. RNA interference-mediated depletion of SMC1 sensitized HeLa Cells to X-rays. Repair of radiation-induced DNA double-strand breaks, measured by gammaH2AX/53BP1 foci analysis, was slower in SMC1- or Rad21-depleted Cells than in controls in G2 but not in G1. Inhibition of the DNA damage kinase DNA-PK, but not ATM, further inhibited foci loss in cohesin-depleted Cells in G2. SMC1 depletion had no effect on DNA single-strand break repair in either G1 or late S/G2. Rad21 and SMC1 were recruited to sites of X-ray-induced DNA damage in G2-phase Cells, but not in G1, and only when DNA damage was concentrated in subnuclear stripes, generated by partially shielded ultrasoft X-rays. Our results suggest that the cohesin complex contributes to Cell survival by promoting the repair of radiation-induced DNA double-strand breaks in G2-phase Cells in an ATM-dependent pathway.

Sci Signal. 2009; 2(96): pe74
Shiozaki K

For decades, the fission yeast Schizosaccharomyces pombe has been used as an exCellent model with which to explore how Cellular growth is coordinated with the division cycle, a yet-unanswered question in biology. New studies in this organism show that TOR (target of rapamycin) kinase and stress-responsive MAPK (mitogen-activated protein kinase) form a signaling pathway that readjusts the timing of mitotic onset in response to poor nutrient conditions. Nutritional environment appears to be translated into graded activity of the protein kinases that influence the activation of Cdc2, a cyclin-dependent kinase driving Cell-cycle progression.

Proc Natl Acad Sci U S A. 2009 Nov 10;
Zipor G, Haim-Vilmovsky L, Gelin-Licht R, Gadir N, Brocard C, Gerst JE

Targeted mRNA trafficking and local translation may play a significant role in controlling protein localization. Here we examined for the first time the localization of all ( approximately 50) mRNAs encoding peroxisomal proteins (mPPs) involved in peroxisome biogenesis and function. By using the bacteriophage MS2-CP RNA-binding protein (RBP) fused to multiple copies of GFP, we demonstrated that >40 endogenously expressed mPPs tagged with the MS2 aptamer form fluorescent RNA granules in vivo. The use of different RFP-tagged organellar markers revealed 3 basic patterns of mPP granule localization: to peroxisomes, to the endoplasmic reticulum (ER), and nonperoxisomal. Twelve mPPs (i.e., PEX1, PEX5, PEX8, PEX11-15, DCI1, NPY1, PCS60, and POX1) had a high percentage (52%-80%) of mRNA colocalization with peroxisomes. Thirteen mPPs (i.e., AAT2, PEX6, MDH3, PEX28, etc.) showed a low percentage (30%-42%) of colocalization, and 1 mPP (PEX3) preferentially localized to the ER. The mPPs of the nonperoxisomal pattern (i.e., GPD1, PCD1, PEX7) showed <<30% colocalization. mPP association with the peroxisome or ER was verified using Cell fractionation and RT-PCR analysis. A model mPP, PEX14 mRNA, was found to be in close association with peroxisomes throughout the Cell cycle, with its localization depending in part on the 3'-UTR, initiation of translation, and the Puf5 RBP. The different patterns of mPP localization observed suggest that multiple mechanisms involved in mRNA localization and translation may play roles in the importation of protein into peroxisomes.

Biochem Cell Biol. 2009 Oct; 87(5): 747-58
Moser BA, Nakamura TM

Telomeres, the natural ends of linear chromosomes, must be protected and completely replicated to guarantee genomic stability in eukaryotic Cells. However, the protected state of telomeres is not compatible with recruitment of telomerase, an enzyme responsible for extending telomeric G-rich repeats during S-phase; thus, telomeres must undergo switches from a protected state to an accessible state during the Cell cycle. In this minireview, we will summarize recent advances in our understanding of proteins involved in the protection and replication of telomeres, and the way these factors are dynamically recruited to telomeres during the Cell cycle. We will focus mainly on recent results from fission yeast Schizosaccharomyces pombe, and compare them with results from budding yeast Saccharomyces cerevisiae and mammalian Cell studies. In addition, a model for the way in which fission yeast Cells replicate telomeres will be presented.

DNA Repair (Amst). 2009 Nov 6;
Kainov DE, Selth LA, Svejstrup JQ, Egly JM, Poterzsman A

TFIIH is an evolutionary conserved eukaryotic multi-protein complex composed of ten subunits. It is involved in transcription, Cell cycle regulation, RNA splicing and the nucleotide excision DNA repair pathway (NER). Depending on the process in which it is functioning, the composition of TFIIH varies and activities of its subunits are differentially regulated. Here we focused on interplay between the Ssl2, Tfb2 and Tfb5 subunits of TFIIH from Saccharomyces cerevisiae. We found that Tfb2 bridges the Ssl2 helicase and the NER-specific Tfb5 subunit. Moreover, the Tfb5-interacting domain of Tfb2 also binds nucleic acids (NA), although the addition of Tfb5 triggers dissociation of NA from Tfb2. In yeast Cells, deletion of TFB5 is more detrimental to NER than loss of the Tfb5/NA-interacting domain of Tfb2, while combining these mutations resulted in suppression of the UV sensitivity of tfb5Delta. The implications of our findings in regards to TFIIH function and group A trichothiodystrophy, an inherited disease associated with mutations in the human TFB5 gene, are discussed.

Methods Enzymol. 2009; 467: 335-56
Shmulevich I, Aitchison JD

Traditionally molecular biology research has tended to reduce biological pathways to composite units studied as isolated parts of the Cellular system. With the advent of high throughput methodologies that can capture thousands of data points, and powerful computational approaches, the reality of studying Cellular processes at a systems level is upon us. As these approaches yield massive datasets, systems level analyses have drawn upon other fields such as engineering and mathematics, adapting computational and statistical approaches to decipher relationships between molecules. Guided by high quality datasets and analyses, one can begin the process of predictive modeling. The findings from such approaches are often surprising and beyond normal intuition. We discuss four classes of dynamical systems used to model genetic regulatory networks. The discussion is divided into continuous and discrete models, as well as deterministic and stochastic model classes. For each combination of these categories, a model is presented and discussed in the context of the yeast Cell cycle, illustrating how different types of questions can be addressed by different model classes.

Cell. 2009 Nov 13; 139(4): 719-30
Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JF

The licensing of eukaryotic DNA replication origins, which ensures once-per-Cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1*Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.

PLoS Genet. 2009 Nov; 5(11): e1000710
Hickman MA, Rusche LN

Deacetylases of the Sir2 family regulate lifespan and response to stress. We have examined the evolutionary history of Sir2 and Hst1, which arose by gene duplication in budding yeast and which participate in distinct mechanisms of gene repression. In Saccharomyces cerevisiae, Sir2 interacts with the SIR complex to generate long-range silenced chromatin at the cryptic mating-type loci, HMLalpha and HMRa. Hst1 interacts with the SUM1 complex to repress sporulation genes through a promoter-specific mechanism. We examined the functions of the non-duplicated Sir2 and its partners, Sir4 and Sum1, in the yeast Kluyveromyces lactis, a species that diverged from Saccharomyces prior to the duplication of Sir2 and Hst1. KlSir2 interacts with both KlSir4 and KlSum1 and represses the same sets of target genes as ScSir2 and ScHst1, indicating that Sir2 and Hst1 subfunctionalized after duplication. However, the KlSir4-KlSir2 and KlSum1-KlSir2 complexes do not function as the analogous complexes do in S. cerevisiae. KlSir4 contributes to an extended repressive chromatin only at HMLalpha and not at HMRa. In contrast, the role of KlSum1 is broader. It employs both long-range and promoter-specific mechanisms to repress cryptic mating-type loci, Cell-type-specific genes, and sporulation genes and represents an important regulator of Cell identity and the sexual cycle. This study reveals that a single repressive complex can act through two distinct mechanisms to regulate gene expression and illustrates how mechanisms by which regulatory proteins act can change over evolutionary time.

Mol Syst Biol. 2009; 5: 312
Omberg L, Meyerson JR, Kobayashi K, Drury LS, Diffley JF, Alter O

This report provides a global view of how gene expression is affected by DNA replication. We analyzed synchronized cultures of Saccharomyces cerevisiae under conditions that prevent DNA replication initiation without delaying Cell cycle progression. We use a higher-order singular value decomposition to integrate the global mRNA expression measured in the multiple time courses, detect and remove experimental artifacts and identify significant combinations of patterns of expression variation across the genes, time points and conditions. We find that, first, approximately 88% of the global mRNA expression is independent of DNA replication. Second, the requirement of DNA replication for efficient histone gene expression is independent of conditions that elicit DNA damage checkpoint responses. Third, origin licensing decreases the expression of genes with origins near their 3' ends, revealing that downstream origins can regulate the expression of upstream genes. This confirms previous predictions from mathematical modeling of a global causal coordination between DNA replication origin activity and mRNA expression, and shows that mathematical modeling of DNA microarray data can be used to correctly predict previously unknown biological modes of regulation.

Cell cycle. 2009 Dec 1; 8(23):
Guacci VA

J Cell Sci. 2009 Dec 1; 122(Pt 23): 4330-4340
Doyle A, Martín-García R, Coulton AT, Bagley S, Mulvihill DP

Class V myosins are dimeric actin-associated motor proteins that deliver Cellular cargoes to discrete Cellular locations. Fission yeast possess two class V myosins, Myo51 and Myo52. Although Myo52 has been shown to have roles in vacuole distribution, cytokinesis and Cell growth, Myo51 has no as yet discernible function in the vegetative life cycle. Here, we uncover distinct functions for this motor protein during mating and meiosis. Not only does Myo51 transiently localise to a foci at the site of Cell fusion upon conjugation, but overexpression of the Myo51 globular tail also leads to disruption of Cell fusion. Upon completion of meiotic prophase Myo51 localises to the outside of the spindle pole bodies (SPBs), where it remains until completion of meiosis II. Association of Myo51 with SPBs is not dependent upon actin or the septation initiation network (SIN); however, it is dependent on a stable microtubule cytoskeleton and the presence of the Cdc2-CyclinB complex. We observe a rapid and dynamic exchange of Myo51 at the SPB during meiosis I but not meiosis II. Finally, we show that Myo51 has an important role in regulating spore formation upon completion of meiosis.

Int J Bioinform Res Appl. 2009; 5(6): 625-46
Chen T, Li F

In this paper, we integrate transcriptional regulatory modelling with temporal correlation analysis between one Transcription Factor (TF) and its corresponding Cell cycle-regulated targets to investigate Cell cycle Regulators (CCRs) and combinatorial interactions among TFs across the Cell cycle in Saccharomyces cerevisiae. Our method is developed based on the rationale that if one TF or one TF combinatorial interaction takes more possibilities of sharing common Cell cycle-regulated targets with other TFs, this TF or TF combinatorial interaction is more likely to control the Cell cycle. Our results reveal abundant CCRs and TF co-operativities supported by biological experiments or other computational methods.

Curr Genomics. 2009 May; 10(3): 198-205
Xiao L, Grove A

Cells grow in response to nutrients or growth factors, whose presence is detected and communicated by elaborate signaling pathways. Protein kinases play crucial roles in processes such as Cell cycle progression and gene expression, and misregulation of such pathways has been correlated with various diseased states. Signals intended to promote Cell growth converge on ribosome biogenesis, as the ability to produce Cellular proteins is intimately tied to Cell growth. Part of the response to growth signals is therefore the coordinate expression of genes encoding ribosomal RNA (rRNA) and ribosomal proteins (RP). A key player in regulating Cell growth is the Target of Rapamycin (TOR) kinase, one of the gatekeepers that prevent Cell cycle progression from G1 to S under conditions of nutritional stress. TOR is structurally and functionally conserved in all eukaryotes. Under favorable growth conditions, TOR is active and Cells maintain a robust rate of ribosome biogenesis, translation initiation and nutrient import. Under stress conditions, TOR signaling is suppressed, leading to Cell cycle arrest, while the failure of TOR to respond appropriately to environmental or nutritional signals leads to uncontrolled Cell growth. Emerging evidence from Saccharomyces cerevisiae indicates that High Mobility Group (HMGB) proteins, non-sequence-specific chromosomal proteins, participate in mediating responses to growth signals. As HMGB proteins are distinguished by their ability to alter DNA topology, they frequently function in the assembly of higher-order nucleoprotein complexes. We review here recent evidence, which suggests that HMGB proteins may function to coordinate TOR-dependent regulation of rRNA and RP gene expression.