Histone H2A-S122 is required for nuclear and mitochondrial genome stability

Organization and maintenance of the mitochondrial and nuclear genomes are vastly different, yet I have shown that a single serine in the H2A C-terminal tail (H2A-S122) is critical for stability of both genomes in the budding yeast, Saccharomyces cerevisiae. Phosphorylation of H2A-S 122 has previously been implicated in the spindle assembly checkpoint (SAC), however I show that by mutating the serine to an alanine (H2A-S122A), the resulting aneuploidy occurs at a much higher rate than is observed by deleting its immediate downstream kinase BUB1. Furthermore, the H2A-S122A mutant displays an increased susceptibility to DNA damaging agents that is not observed in bubΔ1 deletion cells. Our studies also implicate H2A-S122 as critical to the maintenance of the mitochondrial genome, as upon introduction of the H2A-S122A mutation, cells rapidly lose their mitochondrial genomes

Integrating multiple types of data to predict novel cell cycle-related genes

<p>Abstract</p> <p>Background</p> <p>Cellular functions depend on genetic, physical and other types of interactions. As such, derived interaction networks can be utilized to discover novel genes involved in specific biological processes. Epistatic Miniarray Profile, or E-MAP, which is an experimental platform that measures genetic interactions on a genome-wide scale, has successfully recovered known pathways and revealed novel protein complexes in <it>Saccharomyces cerevisiae</it> (budding yeast).</p> <p>Results</p> <p>By combining E-MAP data with co-expression data, we first predicted a potential cell cycle related gene set. Using Gene Ontology (GO) function annotation as a benchmark, we demonstrated that the prediction by combining microarray and E-MAP data is generally >50% more accurate in identifying co-functional gene pairs than the prediction using either data source alone. We also used transcription factor (TF)–DNA binding data (Chip-chip) and protein phosphorylation data to construct a local cell cycle regulation network based on potential cell cycle related gene set we predicted. Finally, based on the E-MAP screening with 48 cell cycle genes crossing 1536 library strains, we predicted four unknown genes (<it>YPL158C</it>, <it>YPR174C</it>, <it>YJR054W</it>, and <it>YPR045C</it>) as potential cell cycle genes, and analyzed them in detail.</p> <p>Conclusion</p> <p>By integrating E-MAP and DNA microarray data, potential cell cycle-related genes were detected in budding yeast. This integrative method significantly improves the reliability of identifying co-functional gene pairs. In addition, the reconstructed network sheds light on both the function of known and predicted genes in the cell cycle process. Finally, our strategy can be applied to other biological processes and species, given the availability of relevant data.</p

What prevents DNA replication between meiosis I and -II in yeast?

During meiosis, a single round of DNA replication is followed by two consecutive rounds of chromosome segregation. While the suppression of DNA replication between meiosis I and –II is one of the defining features of meiosis, its mechanism has remained unclear. The control of DNA replication has been studied extensively in proliferating cells in which DNA replication during S phase strictly alternates with chromosome segregation at mitosis. The mechanism ensuring that each sequence is replicated only once per cell cycle is based on the dual function of Cdk1: low Cdk1 activity after mitosis allows the establishment of prereplicative complexes at replication origins (origin licensing). Activation of Cdk1 at the onset of S phase then initiates DNA replication (origin firing) by converting the pre-replicative complex to the post-replicative complex. Since high Cdk1 activity inhibits the reformation of pre-replicative complexes, the next round of DNA replication cannot occur until after Cdk1 has been inactivated during mitosis when replicated chromosomes segregate. However, applying this concept to meiosis would trigger an additional round of DNA replication because Cdk1 activity drops and then re-appears between meiosis I and –II. Two ideas have been proposed to solve this problem: in Xenopus eggs, Cdk1 activity is reduced rather than completely destroyed between meiosis I and –II, while in yeast, a Cdk1-related kinase, called Ime2, was thought to prevent origin relicensing at anaphase I. We have tested these ideas by artificially inactivating and then reactivating Cdk1 and Ime2 at anaphase I. Remarkably, DNA replication was not induced even when both kinases were simultaneously inhibited and re-activated at anaphase I. Thus, additional mechanisms must prevent DNA replication between meiosis I and –II

Qualitative and quantitative Cdk control of the budding yeast cell cycle

Timely and ordered progression through the cell cycle is crucial for error-free proliferation of cells. In eukaryotes, cell division cycle is controlled by the master regulator cyclin-cyclin dependent kinase (Cdk) complexes. However, it is still unclear how cyclin-Cdk complexes ensure the order in the cell cycle so that DNA replication in S phase always precedes chromosome segregation in mitosis. Two models have been put forward to explain cell cycle ordering by cyclin-Cdk complexes. The qualitative model suggests that distinct substrate specificities of the different cyclins at successive cell cycle stages order substrate phosphorylation. In contrast, the quantitative model for Cdk control of the cell cycle suggests that the overall gradual increase in Cdk activity from G1 to mitosis orders cell cycle progression. In line with the quantitative model, a single cyclin-Cdk complex is sufficient to order the fission yeast cell cycle. However, the relative contributions of qualitative and quantitative Cdk control in other organisms is incompletely understood. In this project, I investigate cyclin specificity and redundancy in the budding yeast S. cerevisiae, which encodes three G1 (Cln1-3), two S (Clb5 and Clb6) and four G2/M (Clb1-4) cyclins that are orthologous to those found in many metazoans, including humans. With an aim to identify the minimal set of cyclins required to drive the ordered cell cycle progression in budding yeast, I have removed seven of the nine cyclins, establishing a strain harbouring one G1 cyclin, Cln2, and a mitotic cyclin, Clb2, that is expressed from an S phase cyclin CLB5 promoter in addition to its own promoter. In this strain, expressing a third copy of Clb2 under control of CLN2 promoter is sufficient to order DNA replication and chromosome segregation in the absence of Cln2. However, these cells cannot polarise or form buds. My findings indicate that the budding yeast G1 cyclin Cln2 has evolved to carry unique crucial functions to couple cell cycle progression to morphogenetic events.Open Acces

What Prevents DNA Replication between Meiosis I and II in Yeast?

During meiosis, a single round of DNA replication is followed by two consecutive rounds of chromosome segregation. While the suppression of DNA replication between meiosis I and –II is one of the defining features of meiosis, its mechanism has remained unclear. The control of DNA replication has been studied extensively in proliferating cells in which DNA replication during S phase strictly alternates with chromosome segregation at mitosis. The mechanism ensuring that each sequence is replicated only once per cell cycle is based on the dual function of Cdk1: low Cdk1 activity after mitosis allows the establishment of prereplicative complexes at replication origins (origin licensing). Activation of Cdk1 at the onset of S phase then initiates DNA replication (origin firing) by converting the pre-replicative complex to the post-replicative complex. Since high Cdk1 activity inhibits the reformation of pre-replicative complexes, the next round of DNA replication cannot occur until after Cdk1 has been inactivated during mitosis when replicated chromosomes segregate. However, applying this concept to meiosis would trigger an additional round of DNA replication because Cdk1 activity drops and then re-appears between meiosis I and –II. Two ideas have been proposed to solve this problem: in Xenopus eggs, Cdk1 activity is reduced rather than completely destroyed between meiosis I and –II, while in yeast, a Cdk1-related kinase, called Ime2, was thought to prevent origin relicensing at anaphase I. We have tested these ideas by artificially inactivating and then reactivating Cdk1 and Ime2 at anaphase I. Remarkably, DNA replication was not induced even when both kinases were simultaneously inhibited and re-activated at anaphase I. Thus, additional mechanisms must prevent DNA replication between meiosis I and –II

Sic1 plays a role in timing and oscillatory behaviour of B-type cyclins

Budding yeast cell cycle oscillates between states of low and high cyclin-dependent kinase activity, driven by association of Cdk1 with B-type (Clb) cyclins. Various Cdk1-Clb complexes are activated and inactivated in a fixed, temporally regulated sequence, inducing the behaviour known as "waves of cyclins". The transition from low to high Clb activity is triggered by degradation of Sic1, the inhibitor of Cdk1-Clb complexes, at the entry to S phase. The G(1) phase is characterized by low Clb activity and high Sic1 levels. High Clb activity and Sic1 proteolysis are found from the beginning of the S phase until the end of mitosis. The mechanism regulating the appearance on schedule of Cdk1-Clb complexes is currently unknown. Here, we analyse oscillations of Clbs, focusing on the role of their inhibitor Sic1. We compare mathematical networks differing in interactions that Sic1 may establish with Cdk1-Clb complexes. Our analysis suggests that the wave-like cyclins pattern derives from the binding of Sic1 to all Clb pairs rather than from Clb degradation. These predictions are experimentally validated, showing that Sic1 indeed interacts and coexists in time with Clbs. Intriguingly, a sic1Delta strain looses cell cycle-regulated periodicity of Clbs, which is observed in the wild type, whether a SIC1-0P strain delays the formation of Clb waves. Our results highlight an additional role for Sic1 in regulating Cdk1-Clb complexes, coordinating their appearance

Kinetochore Structural Plasticity and its Regulation in <i>Saccharomyces cerevisiae</i>

The kinetochore is a multi-protein complex that connects the chromosome with microtubule and pulls the chromosome during chromosome segregation. In yeast, a single kinetochore assembles on a Cse4 nucleosome and attaches to a single microtubule, making it an ideal system for studying the architecture of kinetochore-microtubule interaction, function, and regulation. The inner kinetochore assembles on the centromere and anchors the outer kinetochore that attaches to the dynamic microtubule. The interplay between the kinetochore structure and microtubule dynamics in a living cell has not been studied. I have used a combination of in-house developed quantitative microscopy, genetic techniques and mathematical simulation in the budding yeast, S.cerevisiae, to elucidate a novel structural transition of the kinetochore. During anaphase, the microtubule undergoes rapid depolymerization. Kinetochore tracking of microtubule during this phase is critical for chromosome segregation, but how this tracking is achieved is not well understood. A combination of FRAP and photoconversion on kinetochore proteins have revealed that the kinetochore consists of highly dynamic sub-modules and stable sub-modules. I show that microtubule dynamics significantly affect the dynamic kinetochore sub-modules but not the stable ones. Mathematical modeling of microtubule dynamics and kinetochore attachment has revealed the importance of kinetochore structural transition for microtubule attachment. In addition, I have discovered that kinetochore structural transitions are regulated by known metaphase-anaphase specific pathways. Scm3 is the chaperone responsible for assembling the centromeric nucleosome that contains a specific histone variant Cse4. I helped to identify and characterize a new chaperone for Cse4. I demonstrated that recombinant yCAF-1 can promote the assembly of Cse4 nucleosomes in vitro and at non-centromeric positions, causing problems for chromatin-based processes. In summary, my thesis work has significantly contributed to our understanding of centromere and kinetochore function in budding yeast. I speculate that many of the findings will inform our perspectives on centromere and kinetochore function in higher eukaryotes

Structural plasticity of the living kinetochore

The kinetochore is a large, evolutionarily conserved protein structure that connects chromosomes with microtubules. During chromosome segregation, outer kinetochore components track depolymerizing ends of microtubules to facilitate the separation of chromosomes into two cells. In budding yeast, each chromosome has a point centromere upon which a single kinetochore is built, which attaches to a single microtubule. This defined architecture facilitates quantitative examination of kinetochores during the cell cycle. Using three independent measures-calibrated imaging, FRAP, and photoconversion-we find that the Dam1 submodule is unchanged during anaphase, whereas MIND and Ndc80 submodules add copies to form an "anaphase configuration" kinetochore. Microtubule depolymerization and kinesin-related motors contribute to copy addition. Mathematical simulations indicate that the addition of microtubule attachments could facilitate tracking during rapid microtubule depolymerization. We speculate that the minimal kinetochore configuration, which exists from G1 through metaphase, allows for correction of misattachments. Our study provides insight into dynamics and plasticity of the kinetochore structure during chromosome segregation in living cells

What prevents DNA replication between meiosis I and -II in yeast?

During meiosis, a single round of DNA replication is followed by two consecutive rounds of chromosome segregation. While the suppression of DNA replication between meiosis I and –II is one of the defining features of meiosis, its mechanism has remained unclear. The control of DNA replication has been studied extensively in proliferating cells in which DNA replication during S phase strictly alternates with chromosome segregation at mitosis. The mechanism ensuring that each sequence is replicated only once per cell cycle is based on the dual function of Cdk1: low Cdk1 activity after mitosis allows the establishment of prereplicative complexes at replication origins (origin licensing). Activation of Cdk1 at the onset of S phase then initiates DNA replication (origin firing) by converting the pre-replicative complex to the post-replicative complex. Since high Cdk1 activity inhibits the reformation of pre-replicative complexes, the next round of DNA replication cannot occur until after Cdk1 has been inactivated during mitosis when replicated chromosomes segregate. However, applying this concept to meiosis would trigger an additional round of DNA replication because Cdk1 activity drops and then re-appears between meiosis I and –II. Two ideas have been proposed to solve this problem: in Xenopus eggs, Cdk1 activity is reduced rather than completely destroyed between meiosis I and –II, while in yeast, a Cdk1-related kinase, called Ime2, was thought to prevent origin relicensing at anaphase I. We have tested these ideas by artificially inactivating and then reactivating Cdk1 and Ime2 at anaphase I. Remarkably, DNA replication was not induced even when both kinases were simultaneously inhibited and re-activated at anaphase I. Thus, additional mechanisms must prevent DNA replication between meiosis I and –II