ABSTRACT
Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatids/physiology , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/physiology , DNA Replication , Proliferating Cell Nuclear Antigen/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Acetyltransferases/genetics , Acetyltransferases/metabolism , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proliferating Cell Nuclear Antigen/genetics , Replication Protein C/genetics , Replication Protein C/metabolism , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , CohesinsABSTRACT
The pairing of homologous chromosomes represents a critical step of meiosis in nearly all sexually reproducing species. In many organisms, pairing involves chromosomes that remain apparently intact. The mechanistic nature of homology recognition at the basis of such pairing is unknown. Using "meiotic silencing by unpaired DNA" (MSUD) as a model process, we demonstrate the existence of a cardinally different approach to DNA homology recognition in meiosis. The main advantage of MSUD over other experimental systems lies in its ability to identify any relatively short DNA fragment lacking a homologous allelic partner. Here, we show that MSUD does not rely on the canonical mechanism of meiotic recombination, yet it is promoted by REC8, a conserved component of the meiotic cohesion complex. We also show that certain patterns of interspersed homology are recognized as pairable during MSUD. Such patterns need to be colinear and must contain short tracts of sequence identity spaced apart at 21 or 22 base pairs. By using these periodicity values as a guiding parameter in all-atom molecular modeling, we discover that homologous DNA molecules can pair by forming quadruplex-based contacts with an interval of 2.5 helical turns. This process requires right-handed plectonemic coiling and additional conformational changes in the intervening double-helical segments. Our results 1) reconcile genetic and biophysical evidence for the existence of direct homologous double-stranded DNA (dsDNA)-dsDNA pairing, 2) identify a role for this process in initiating RNA interference, and 3) suggest that chromosomes can be cross-matched by a precise mechanism that operates on intact dsDNA molecules.
Subject(s)
Chromosomes, Fungal/physiology , DNA, Fungal/genetics , Gene Expression Regulation, Fungal/physiology , Meiosis/physiology , Neurospora crassa/physiology , Recombination, Genetic/physiology , Chromosomes, Fungal/genetics , Meiosis/genetics , Recombination, Genetic/geneticsABSTRACT
Pairing of homologous chromosomes is crucial for ensuring accurate segregation of chromosomes during meiosis. Molecular mechanisms of homologous chromosome pairing in meiosis have been extensively studied in the fission yeast Schizosaccharomyces pombe. In this organism, meiosis-specific noncoding RNA transcribed from specific genes accumulates at the respective gene loci, and chromosome-associated RNA-protein complexes mediate meiotic pairing of homologous loci through phase separation. Pairing of homologous chromosomes also occurs in somatic diploid cells in certain situations. For example, somatic pairing of homologous chromosomes occurs during the early embryogenesis in diptera, and relies on the transcription-associated chromatin architecture. Earlier models also suggest that transcription factories along the chromosome mediate pairing of homologous chromosomes in plants. These studies suggest that RNA bodies formed on chromosomes mediate the pairing of homologous chromosomes. This review summarizes lessons from S. pombe to provide general insights into mechanisms of homologous chromosome pairing mediated by phase separation of chromosome-associated RNA-protein complexes.
Subject(s)
Chromosome Pairing , Chromosomes, Fungal/physiology , Chromosomes/physiology , Schizosaccharomyces/genetics , Animals , Chromosomes/genetics , Chromosomes, Fungal/genetics , DNA/chemistry , DNA/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Meiosis , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , RNA-Binding Proteins/metabolism , Schizosaccharomyces/physiology , Schizosaccharomyces pombe Proteins/metabolism , Sequence Homology, Nucleic AcidABSTRACT
The subtelomere, a telomere-adjacent chromosomal domain, contains species-specific homologous DNA sequences, in addition to various genes. However, the functions of subtelomeres, particularly subtelomeric homologous (SH) sequences, remain elusive. Here, we report the first comprehensive analyses of the cellular functions of SH sequences in the fission yeast, Schizosaccharomyces pombe. Complete removal of SH sequences from the genome revealed that they are dispensable for mitosis, meiosis and telomere length control. However, when telomeres are lost, SH sequences prevent deleterious inter-chromosomal end fusion by facilitating intra-chromosomal circularization. Surprisingly, SH-deleted cells sometimes survive telomere loss through inter-chromosomal end fusions via homologous loci such as LTRs, accompanied by centromere inactivation of either chromosome. Moreover, SH sequences function as a buffer region against the spreading of subtelomeric heterochromatin into the neighboring gene-rich regions. Furthermore, we found a nucleosome-free region at the subtelomeric border, which may be a second barrier that blocks heterochromatin spreading into the subtelomere-adjacent euchromatin. Thus, our results demonstrate multiple defense functions of subtelomeres in chromosome homeostasis and gene expression.
Subject(s)
Chromosomes, Fungal/physiology , Gene Expression , Homeostasis/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Telomere/physiology , Centromere/metabolism , Chromosomal Instability/genetics , Gene Expression Regulation, Fungal , Heterochromatin/metabolism , Organisms, Genetically Modified , Sequence Deletion , Telomere-Binding Proteins/metabolismABSTRACT
Eukaryotic chromosomes undergo movements that are involved in the regulation of functional processes such as DNA repair. To better understand the origin of these movements, we used fluorescence microscopy, image analysis and chromosome conformation capture to quantify the actin contribution to chromosome movements and interactions in budding yeast. We show that both the cytoskeletal and nuclear actin drive local chromosome movements, independently of Csm4, a putative LINC protein. Inhibition of actin polymerization reduces subtelomere dynamics, resulting in more confined territories and enrichment in subtelomeric contacts. Artificial tethering of actin to nuclear pores increased both nuclear pore complex (NPC) and subtelomere motion. Chromosome loci that were positioned away from telomeres exhibited reduced motion in the presence of an actin polymerization inhibitor but were unaffected by the lack of Csm4. We further show that actin was required for locus mobility that was induced by targeting the chromatin-remodeling protein Ino80. Correlated with this, DNA repair by homologous recombination was less efficient. Overall, interphase chromosome dynamics are modulated by the additive effects of cytoskeletal actin through forces mediated by the nuclear envelope and nuclear actin, probably through the function of actin in chromatin-remodeling complexes.
Subject(s)
Actins/physiology , Chromosomes, Fungal/physiology , Saccharomyces cerevisiae/genetics , Chromatin/genetics , Chromatin/metabolism , Membrane Proteins/physiology , Nuclear Pore/metabolism , Protein Multimerization , Protein Transport , Recombinational DNA Repair , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/physiology , Telomere/genetics , Telomere/metabolismABSTRACT
The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini-chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non-essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.
Subject(s)
Cell Division/physiology , Cell Wall/physiology , Gene Expression Regulation, Fungal/physiology , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/cytology , Schizosaccharomyces/physiology , Chromosomes, Fungal/physiology , Gene Deletion , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere-specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule-depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty-three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS-I, -II, and -III (TBZ/benomyl-sensitive regions I-III). Elevation of mono-polar attachment induced by prior nocodazole treatment was observed in H2A-I112A (TBS-I), H2A-E57A (TBS-II), and H4-L97A (TBS-III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi-orientation, occurred in H2A-I112A and H2A-E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4-L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS-I and -II, are necessary for Sgo1-mediated chromosome bi-orientation and that TBS-III is required for Htz1 function.
Subject(s)
Chromosomes, Fungal/physiology , Histones/physiology , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/cytology , Amino Acid Sequence , Benomyl/pharmacology , Centromere/metabolism , Centromere/ultrastructure , Chromosomal Instability , Chromosome Segregation , Drug Resistance, Fungal/genetics , Histones/genetics , Microtubules/drug effects , Models, Molecular , Molecular Sequence Data , Nocodazole/pharmacology , Nuclear Proteins/physiology , Nucleosomes/drug effects , Nucleosomes/ultrastructure , Point Mutation , Protein Conformation , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Spindle Apparatus/metabolism , Spindle Apparatus/ultrastructure , Thiabendazole/pharmacology , Tubulin Modulators/pharmacologyABSTRACT
In response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called γH2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of γH2A followed by next-generation sequencing (ChIP-seq). γH2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal γH2A localization. In contrast, γH2A is not required for H3K9 methylation or DNA methylation. Normal γH2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. γH2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.
Subject(s)
Chromosomes, Fungal/physiology , Fungal Proteins/metabolism , Heterochromatin/physiology , Histones/metabolism , Neurospora crassa/metabolism , DNA, Fungal/metabolism , Methylation , Neurospora crassa/genetics , Protein Processing, Post-TranslationalABSTRACT
In meiosis, pairing and recombination of homologous chromosomes are crucial for the correct segregation of chromosomes, and substantial movements of chromosomes are required to achieve homolog pairing. During this process, it is known that telomeres cluster to form a bouquet arrangement of chromosomes. The fission yeast Schizosaccharomyces pombe provides a striking example of bouquet formation, after which the entire nucleus oscillates between the cell poles (these oscillations are generally called horsetail nuclear movements) while the telomeres remain clustered to the spindle pole body (SPB; a centrosome-equivalent structure in fungi) at the leading edge of the moving nucleus. S. pombe mutants defective in telomere clustering frequently form aberrant spindles, such as monopolar or nonpolar spindles, leading to missegregation of the chromosomes at the subsequent meiotic divisions. Here we demonstrate that such defects in meiotic spindle formation caused by loss of meiotic telomere clustering are rescued when nuclear movement is prevented. On the other hand, stopping nuclear movement does not rescue defects in telomere clustering, nor chromosome missgregation even in cells that have formed a bipolar spindle. These results suggest that movement of the SPB without attachment of telomeres leads to the formation of aberrant spindles, but that recovering bipolar spindles is not sufficient for rescue of chromosome missegregation in mutants lacking telomere clustering.
Subject(s)
Chromosomes, Fungal/physiology , Meiosis , Schizosaccharomyces/cytology , Spindle Pole Bodies/metabolism , Cell Nucleus/physiology , Chromosome Segregation , Microscopy, Fluorescence , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Telomere/metabolism , Time-Lapse ImagingABSTRACT
Numerous studies of nucleosome positioning have shown that nucleosomes almost invariably adopt one of several alternative overlapping positions on a short DNA fragment in vitro. We define such a set of overlapping positions as a "position cluster", and the 5S RNA gene positioning sequence is presented as an example. The notable exception is the synthetic 601-sequence, which can position a nucleosome perfectly in vitro, though not in vivo. Many years ago, we demonstrated that nucleosome position clusters are present on the CUP1 and HIS3 genes in native yeast chromatin. Recently, using genome-wide paired-end sequencing of nucleosomes, we have shown that position clusters are the general rule in yeast chromatin, not the exception. We argue that, within a cell population, one of several alternative nucleosomal arrays is formed on each gene. We show how position clusters and alternative arrays can give rise to typical nucleosome occupancy profiles, and that position clusters are disrupted by transcriptional activation. The centromeric nucleosome is a rare example of perfect positioning in vivo. It is, however, a special case, since it contains the centromeric histone H3 variant instead of normal H3. Perfect positioning might be due to centromeric sequence-specific DNA binding proteins. Finally, we point out that the existence of position clusters implies that the putative nucleosome code is degenerate. We suggest that degeneracy might be a crucial point in the debate concerning the code. This article is part of a Special Issue entitled: Chromatin in time and space.
Subject(s)
Nucleosomes/physiology , Yeasts/genetics , Animals , Base Sequence , Centromere/metabolism , Centromere/physiology , Chromatin Assembly and Disassembly , Chromosomes, Fungal/metabolism , Chromosomes, Fungal/physiology , Fungal Proteins/metabolism , Fungal Proteins/physiology , Gene Expression Regulation, Fungal , Genome, Fungal , Histones/metabolism , Histones/physiology , Humans , Nucleic Acid Conformation , Nucleosomes/metabolismABSTRACT
Sets of overlapping microtubules support the segregation of chromosomes by linking the poles of mitotic spindles. Recent work examines the effect of putting these linkages under pressure by the activation of dicentric chromosomes and sheds new light on the structural role of several well-known spindle midzone proteins.
Subject(s)
Chromosome Segregation/physiology , Spindle Apparatus/physiology , Chromosomes, Fungal/physiology , Microtubules/physiology , Saccharomyces cerevisiaeABSTRACT
During the past year important progress has been made in refining our understanding of how chromosomes become equally distributed to daughter cells during mitosis. Unlike the situation in diatoms and yeast, it now appears that spindle pole (centrosome) separation during spindle formation and anaphase B is mediated in vertebrates primarily by an astral pulling, and not a pushing, mechanism. Kinetochore motility is directionally unstable, which has important consequences for how chromosomes move to the equator of the forming spindle. Finally, the observation that sister chromatid disjunction occurs even in the presence of high levels of maturation promoting factor reveals that the series of biochemical events responsible for this phenomenon is not an obligatory part of the pathway by which the cell exits mitosis.
Subject(s)
Chromosomes, Fungal/physiology , Chromosomes/physiology , Kluyveromyces/physiology , Mitosis/physiology , Saccharomyces cerevisiae/physiology , Schizosaccharomyces/physiology , Anaphase , Base Sequence , Chromatids/physiology , Conserved Sequence , Genes, Fungal , Kluyveromyces/ultrastructure , Saccharomyces cerevisiae/ultrastructure , Schizosaccharomyces/ultrastructure , Spindle Apparatus/physiologyABSTRACT
The past year saw the molecular characterization of components of the Saccharomyces cerevisiae kinetochore and spindle pole body. In Schizosaccharomyces pombe, new cytological methods have been described for detection of centromeric DNA by light microscopy and probable kinetochores by electron microscopy.
Subject(s)
Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae/ultrastructure , Schizosaccharomyces/physiology , Schizosaccharomyces/ultrastructure , Spindle Apparatus/physiology , Chromosomes, Fungal/physiology , Chromosomes, Fungal/ultrastructure , DNA, Fungal/analysis , Genes, Fungal , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/genetics , Spindle Apparatus/ultrastructureABSTRACT
Homologue pairing mediates both recombination and segregation of chromosomes at meiosis I. The recognition of nucleic-acid-sequence homology within the somatic nucleus has an impact on DNA repair and epigenetic control of gene expression. Here we investigate interchromosomal interactions using a non-invasive technique that allows tagging and visualization of DNA sequences in vegetative and meiotic live yeast cells. In non-meiotic cells, chromosomes are ordered in the nucleus, but preferential pairing between homologues is not observed. Association of tagged chromosomal domains occurs irrespective of their genomic location, with some preference for similar chromosomal positions. Here we describe a new phenomenon that promotes associations between sequence-identical ectopic tags with a tandem-repeat structure. These associations, termed interchromosome trans-associations, may underlie epigenetic phenomena.
Subject(s)
Chromosomes, Fungal/physiology , DNA, Fungal/physiology , Meiosis/physiology , Saccharomyces cerevisiae/genetics , Genome, Fungal , Image Processing, Computer-Assisted/methodsABSTRACT
Accurate chromosome segregation during mitotic division of budding yeast depends on the multiprotein kinetochore complex, Dam1 (also known as DASH). Purified Dam1 heterodecamers encircle microtubules (MTs) to form rings that can function as "couplers," molecular devices that transduce energy from MT disassembly into the motion of a cargo. Here we show that MT depolymerization develops a force against a Dam1 ring that is sixfold larger than the force exerted on a coupler that binds only one side of an MT. Wild-type rings slow depolymerization fourfold, but rings that include a mutant Dam1p with truncated C terminus slow depolymerization less, consistent with the idea that this tail is part of a strong bond between rings and MTs. A molecular-mechanical model for Dam1-MT interaction predicts that binding between this flexible tail and the MT wall should cause a Dam1 ring to wobble, and Fourier analysis of moving, ring-attached beads corroborates this prediction. Comparison of the forces generated against wild-type and mutant complexes confirms the importance of tight Dam1-MT association for processive cargo movement under load.
Subject(s)
Chromosomes, Fungal/physiology , Microtubule-Associated Proteins/physiology , Microtubules/physiology , Biomechanical Phenomena , Chromosome Segregation , Kinetochores/physiology , Kinetochores/ultrastructure , Models, Biological , Saccharomycetales/metabolismABSTRACT
Many plant pathogenic fungi contain conditionally dispensable (CD) chromosomes that are associated with virulence, but not growth in vitro. Virulence-associated CD chromosomes carry genes encoding effectors and/or host-specific toxin biosynthesis enzymes that may contribute to determining host specificity. Fusarium oxysporum causes devastating diseases of more than 100 plant species. Among a large number of host-specific forms, F. oxysporum f. sp. conglutinans (Focn) can infect Brassicaceae plants including Arabidopsis (Arabidopsis thaliana) and cabbage. Here we show that Focn has multiple CD chromosomes. We identified specific CD chromosomes that are required for virulence on Arabidopsis, cabbage, or both, and describe a pair of effectors encoded on one of the CD chromosomes that is required for suppression of Arabidopsis-specific phytoalexin-based immunity. The effector pair is highly conserved in F. oxysporum isolates capable of infecting Arabidopsis, but not of other plants. This study provides insight into how host specificity of F. oxysporum may be determined by a pair of effector genes on a transmissible CD chromosome.
Subject(s)
Chromosomes, Fungal/genetics , Fusarium/genetics , Plant Diseases/microbiology , Arabidopsis/immunology , Arabidopsis/microbiology , Brassicaceae/immunology , Brassicaceae/microbiology , Chromosomes, Fungal/physiology , Fusarium/pathogenicity , Fusarium/physiology , Genome, Fungal/genetics , Host-Pathogen Interactions/immunology , Plant Diseases/immunologyABSTRACT
Long-range chromosome organization is known to influence nuclear function. Budding yeast centromeres cluster near the spindle pole body, whereas telomeres are grouped in five to eight perinuclear foci. Using live microscopy, we examine the relative positions of right and left telomeres of several yeast chromosomes. Integrated lac and tet operator arrays are visualized by their respective repressor fused to CFP and YFP in interphase yeast cells. The two ends of chromosomes 3 and 6 interact significantly but transiently, forming whole chromosome loops. For chromosomes 5 and 14, end-to-end interaction is less frequent, yet telomeres are closer to each other than to the centromere, suggesting that yeast chromosomes fold in a Rabl-like conformation. Disruption of telomere anchoring by deletions of YKU70 or SIR4 significantly compromises contact between two linked telomeres. These mutations do not, however, eliminate coordinated movement of telomere (Tel) 6R and Tel6L, which we propose stems from the territorial organization of yeast chromosomes.
Subject(s)
Chromosomes, Fungal/physiology , Saccharomyces cerevisiae/physiology , Telomere/physiology , Cell Nucleolus/physiology , Cell Nucleus/physiology , DNA-Binding Proteins/genetics , Fluorescence Recovery After Photobleaching , G1 Phase/physiology , Gene Deletion , Genotype , Interphase/physiology , Luminescent Proteins/genetics , Microscopy, Fluorescence , Recombinant Fusion Proteins/genetics , Repressor Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , Spindle Apparatus/physiologyABSTRACT
In diploid organisms, meiosis reduces the chromosome number by half during the formation of haploid gametes. During meiotic prophase, telomeres transiently cluster at a limited sector of the nuclear envelope (bouquet stage) near the spindle pole body (SPB). Cohesin is a multisubunit complex that contributes to chromosome segregation in meiosis I and II divisions. In yeast meiosis, deficiency for Rec8 cohesin subunit induces telomere clustering to persist, whereas telomere cluster-SPB colocalization is defective. These defects are rescued by expressing the mitotic cohesin Scc1 in rec8delta meiosis, whereas bouquet-stage exit is independent of Cdc5 pololike kinase. An analysis of living Saccharomyces cerevisiae meiocytes revealed highly mobile telomeres from leptotene up to pachytene, with telomeres experiencing an actin- but not microtubule-dependent constraint of mobility during the bouquet stage. Our results suggest that cohesin is required for exit from actin polymerization-dependent telomere clustering and for linking the SPB to the telomere cluster in synaptic meiosis.
Subject(s)
Actins/physiology , Cell Cycle Proteins/physiology , Fungal Proteins/physiology , Meiosis , Nuclear Proteins/physiology , Saccharomyces cerevisiae/physiology , Telomere/physiology , Cell Cycle Proteins/metabolism , Cell Nucleus/genetics , Cell Nucleus/physiology , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Fungal/physiology , Green Fluorescent Proteins/genetics , Microtubules/physiology , Mutation , Nuclear Envelope/physiology , Phosphorylation , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Shelterin Complex , Spindle Apparatus , Telomere/genetics , Telomere-Binding Proteins/genetics , Telomere-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , CohesinsABSTRACT
We have shown previously that perturbation of origin firing in chromosome replication causes DNA lesions and triggers DNA damage checkpoint control, which ensures genomic integrity by stopping cell cycle progression until the lesions are repaired or by inducing cell death if they are not properly repaired. This was based on the observation that the temperature-sensitive phenotype of orc1-4 and orc2-1 mutants required a programmed action of the RAD9-dependent DNA damage checkpoint. Here, we report that DNA lesions in the orc mutants are induced much more quickly and frequently within the rRNA gene (rDNA) locus than at other chromosomal loci upon temperature shift. orc mutant cells with greatly reduced rDNA copy numbers regained the ability to grow at restrictive temperatures, and the checkpoint response after the temperature shift became weak in these cells. In orc2-1 cells, completion of chromosomal duplication was delayed specifically on chromosome XII, where the rDNA array is located, and the delay was partially suppressed when the rDNA copy number was reduced. These results suggest that the rDNA locus primarily signals abnormalities in the initiation program to the DNA damage checkpoint and that the rDNA copy number modulates the sensitivity of this monitoring function.
Subject(s)
Chromosomes, Fungal/physiology , DNA Replication , Genes, rRNA , Origin Recognition Complex/metabolism , Saccharomyces cerevisiae/physiology , Chromosomes, Fungal/genetics , DNA Damage , Gene Dosage , Genome, Fungal , Mutation , Origin Recognition Complex/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The dynamic reorganization of chromatin into rigid and compact mitotic chromosomes is of fundamental importance for faithful chromosome segregation. Owing to the difficulty of investigating this process under physiological conditions, the exact morphological transitions and the molecular machinery driving chromosome condensation remain poorly defined. Here, we review how imaging-based methods can be used to quantitate chromosome condensation in vivo, focusing on yeast and animal tissue culture cells as widely used model systems. We discuss approaches how to address structural dynamics of condensing chromosomes and chromosome segments, as well as to probe for mechanical properties of mitotic chromosomes. Application of such methods to systematic perturbation studies will provide a means to reveal the molecular networks underlying the regulation of mitotic chromosome condensation.