Folded chromosomes in non-cycling yeast cells

Folded chromosomes from stationary phase or ammonia-starved yeast (Saccharomyces cerevisiae) cells can be isolated as compact structures, distinct and separable by sedimentation from the folded chromosomes of pre-replicative (G₁) and post-replicative (G₂) nuclei. Such cells are in a dormant or non-cycling (G₀) stage. The folded genome from such cells is referred to as theg ₀ form and has a sedimentation velocity of about 1700S. Sedimentation analysis of mixed G₀ and G₁ and G₂ lysates indicates that theg ₀ structure is not an artifactual breakdown product of theg ₁ org ₂ structures. A comparison of the proteins fromg ₀ versusg ₁ andg ₂ structures by gel electrophoresis has revealed differences in about 10–11 non-histone and perhaps 2 histone proteins. Entry into the G₀ stage, and emergence into G₁ after G₀ arrest, are accompanied by an ordered transition fromg ₂ tog ₁ tog ₀, and fromg ₀ tog ₁ tog ₂ forms, respectively. Hence, entry into G₀ and re-emergence from G₀ can be considered as differentiative processes, not normally part of the cell cycle, and accompanied by specific changes in the tertiary organization of the genome.     

Folded chromosomes of Saccharomyces cerevisiae

Folded chromosome phenotypes have been examined and compared in four cell-division-cycle (cdc) mutants during transitions between cycling and non-cycling states. The two start mutants, cdc 28 and cdc 25, can undergo G₀ arrest at the restrictive temperature. Arrest at start, defined by the cdc 28 and cdc 25 block points, is distinguishable from G₀ arrest. Arrest at the cdc 28 and cdc 25 block points can also be distinguished from each other: folded chromosomes appear to be destabilized at the cdc 25 block, but are stable at the cdc 28 arrest point. On the other hand, folded chromosomes from cdc 28 in sporulation medium at the restrictive temperature appear unstable, while chromosomes from cdc 25 are stable. The G₁ arrest mutants, cdc 4 and cdc 7, can undergo G₀ arrest at the restrictive temperature. In sporulation medium no meiotic replication form is detected at the restrictive temperature, although incorporation of labeled precursors into nuclear DNA does take place. A schematic model incorporating these various findings is presented.     

Low levels of DNA polymerase alpha induce mitotic and meiotic instability in the ribosomal DNA gene cluster of Saccharomyces cerevisiae

The ribosomal DNA (rDNA) genes of Saccharomyces cerevisiae are located in a tandem array of about 150 repeats. Using a diploid with markers flanking and within the rDNA array, we showed that low levels of DNA polymerase alpha elevate recombination between both homologues and sister chromatids, about five-fold in mitotic cells and 30-fold in meiotic cells. This stimulation is independent of Fob1p, a protein required for the programmed replication fork block (RFB) in the rDNA. We observed that the fob1 mutation alone significantly increased meiotic, but not mitotic, rDNA recombination, suggesting a meiosis-specific role for this protein. We found that meiotic cells with low polymerase alpha had decreased Sir2p binding and increased Spo11p-catalyzed double-strand DNA breaks in the rDNA. Furthermore, meiotic crossover interference in the rDNA is absent. These results suggest that the hyper-Rec phenotypes resulting from low levels of DNA polymerase alpha in mitosis and meiosis reflect two fundamentally different mechanisms: the increased mitotic recombination is likely due to increased double-strand DNA breaks (DSBs) resulting from Fob1p-independent stalled replication forks, whereas the hyper-Rec meiotic phenotype results from increased levels of Spo11-catalyzed DSBs in the rDNA.     

Metaphase and meiotic chromosomes,synaptonemal complexes (SC) of the lizard <Emphasis Type="Italic">Zootoca vivipara</Emphasis>

Somatic mitotic and meiotic chromosomes at the pachytene and at the metaphase I of the males of the viviparous lizard, Zootoca vivipara (Lichtenstein, 1823), from northwestern Russia, belonging to the Russian form of Z. v. vivipara, are examined. The spreading of synaptonemal complexes (SC) of their chromosomes are obtained and analyzed for the first time. Eighteen SC are observed, including SC of the Z₁Z₁ (pairs 5 or 6) and the Z₂Z₂ (pair 13) sex chromosomes. Characteristics of SC are compared with the number and the shape of bivalents and with those of the karyotype structure. In the studied Russian form of Z. v. vivipara, the length ratios of bivalents correlate with that of mitotic chromosomes (2n = 36); however, some specificity in the morphology of SC of the Z₁Z₁ sex chromosomes is reported in this article.     

Silver staining in transcriptionally active NORs of meiotic and mitotic cells in Acheta domesticus (L.) (Orthoptera)

The behaviour of the NOR material in mitotic and meiotic cells of Acheta domesticus was studied by silver staining. — In mitotic chromosomes black silver staining is observed in the centromeric region of 2 pairs of acrocentric chromosomes. Additionally a polymorphic silver positive region is found at the telomere of a large submetacentric chromosome. — The Ag-pattern of the amplified rDNA material in various stages of oogenesis was followed. During pachytene the extra DNA body shows dark brownish staining and only a few black spots. One distinct black precipitate, however, is found in association with meiotic chromosomes. In early diplotene the central core of the extra DNA body is heavily stained with silver. The outer shell shows only brown staining. In the following stages of diplotene the compact structure of the outer shell is loosened and small brown extra nucleoli are found in the remaining nucleus. These nucleoli show black Ag-precipitates in their centres. During the desintegration of the extra DNA body the nucleus becomes filled with small extra nucleoli. The black stained central core is reduced in size and finally disappears.     

Timing of DNA replication during the meiotic process in monokaryotic basidiocarps of Coprinus macrorhizus

By applying the method of fluorescent microscopy to propidium iodide stained cells, change in the relative amount of DNA in a basidium was examined during the meiotic process in Coprinus macrorhizus. In the monokaryotic basidiocarp of the mutant strain Fis^c, mitotic DNA replication was first induced soon after a 3h-illumination period on the 10th day of culture, and subsequently meiotic DNA replication occurred after karyogamy.     

Condensin restructures chromosomes in preparation for meiotic divisions

The production of haploid gametes from diploid germ cells requires two rounds of meiotic chromosome segregation after one round of replication. Accurate meiotic chromosome segregation involves the remodeling of each pair of homologous chromosomes around the site of crossover into a highly condensed and ordered structure. We showed that condensin, the protein complex needed for mitotic chromosome compaction, restructures chromosomes during meiosis in Caenorhabditis elegans. In particular, condensin promotes both meiotic chromosome condensation after crossover recombination and the remodeling of sister chromatids. Condensin helps resolve cohesin-independent linkages between sister chromatids and alleviates recombination-independent linkages between homologues. The safeguarding of chromosome resolution by condensin permits chromosome segregation and is crucial for the formation of discrete, individualized bivalent chromosomes.     

Atm-dependent interactions of a mammalian Chk1 homolog with meiotic chromosomes

Background: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest.Results: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1^Hu/Mo has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22–23, a region marked by frequent deletions and loss of heterozygosity in human tumors.Conclusions: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22–23 indicates that the CHK1 gene is a candidate tumor suppressor gene.     

Properties of a membrane-attached form of the folded chromosome of Escherichia coli

Two types of DNA-containing particles are released from lysozyme-produced Escherichia coli spheroplasts after gentle lysis with non-ionic detergents in 1.-0 m-NaCl. Lysis at 25 °C releases the folded chromosomes (1300 S to 2200 S particles). Lysis at 10 °C results in faster sedimenting structures (3000 S to 4000 S). Both types of particles coexist in extracts of cells lysed at intermediate temperatures, i.e. 15 °C.The 3000 S to 4000 S particles are folded chromosomes attached to membrane fragments; they contain membrane proteins and phospholipids in addition to the folded DNA and nascent RNA chains. Incubation of the membrane-attached chromosomes with 1% Sarkosyl releases the folded chromosomes; this Sarkosyl treatment removes the membrane proteins and phospholipids, and halves the sedimentation velocity of the particles, but has no effect on the folded DNA and nascent RNA chains.Membrane-attached chromosomes cannot be isolated from amino acid-starved cells which have completed their rounds of DNA replication; all of the DNA then appears as released folded chromosomes. After resumption of protein synthesis, chromosome attachment to the membrane precedes the initiation of DNA replication. Controls strongly suggest that the changes observed, i.e. the attachment and release from the membrane of the folded chromosome, are related to the act of DNA replication itself.     

TopBP1 and ATR colocalization at meiotic chromosomes: role of TopBP1/Cut5 in the meiotic recombination checkpoint

Mammalian TopBP1 is a BRCT domain-containing protein whose function in mitotic cells is linked to replication and DNA damage checkpoint. Here, we study its possible role during meiosis in mice. TopBP1 foci are abundant during early prophase I and localize mainly to histone gamma-H2AX-positive domains, where DNA double-strand breaks (required to initiate recombination) occur. Strikingly, TopBP1 showed a pattern almost identical to that of ATR, a PI3K-like kinase involved in mitotic DNA damage checkpoint. In the synapsis-defective Fkbp6(-/-) mouse, TopBP1 heavily stains unsynapsed regions of chromosomes. We also tested whether Schizosaccharomyces pombe Cut5 (the TopBP1 homologue) plays a role in the meiotic recombination checkpoint, like spRad3, the ATR homologue. Indeed, we found that a cut5 mutation suppresses the checkpoint-dependent meiotic delay of a meiotic recombination defective mutant, indicating a direct role of the Cut5 protein in the meiotic checkpoint. Our findings suggest that ATR and TopBP1 monitor meiotic recombination and are required for activation of the meiotic recombination checkpoint.     

A probe into nuclear events during the cell cycle of Saccharomyces cerevisiae: studies of folded chromosomes in cdc mutants which arrest in G1

The sedimentation behavior of folded chromosomes from celldivision-cycle (cdc) mutants which arrest in g ₁ was examined. At the restrictive temperature the folded genome of cdc 7, which arrests after spindle pole body (SPB) separation and spindle formation, cosediments with a standard g ₁ structure, indicating that by the cdc 7 step the g ₁ form of the folded genome has been assembled. In the mutant, cdc 4, which arrests before SPB separation but after SPB duplication, a standard g ₁ structure is not formed, cdc 4 cells, however, are able to enter G₀ at the restrictive temperature, and the corresponding g ₀ structure is stable. These results indicate that the cdc 4 gene product may be involved in the development of folded genome conformation which leads to the g ₁ structure. Since the cdc 4 gene product is required for SPB separation, the g ₁ structure may be defined by an association between chromosomes and spindle components. The folded chromosomes of the start mutants cdc 25 and cdc 28 are unstable at the restrictive temperature. In contrast to cdc 4, neither cdc 25 nor cdc 28 are able to enter the G₀ stage in a normal manner, i.e., the g ₀ structure is unstable at the restrictive temperature. The inference is that both the cdc 25 and cdc 28 gene products are required for the functional integrity of the folded genome at both a stage early in G₁ and in the pathway to G₀.     

Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.     

Properties of membrane-associated folded chromosomes of E. coli related to initiation and termination of DNA replication

Analysis of folded chromosomes prepared from amino acid-starved E. coli cells or from a dnaC initiation mutant indicates that a unique structure is associated with completion or near completion of rounds of chromosome replication in E. coli. Chromosomes remain associated with portions of the bacterial cell envelope throughout the DNA replication cycle, but become more rapidly sedimenting as replication proceeds in the absence of reinitiation. Before reinitiation of chromosome replication occurs after restoring required amino acids to amino acid-starved cells or after lowering the temperature in a thermosensitive dnaC mutant, sedimentation velocities of the membrane-associated folded chromosomes decrease substantially. The decrease in sedimentation velocity does not depend on renewed DNA synthesis, but does require the activity of at least the dnaC gene product.     

Mammalian BLM helicase is critical for integrating multiple pathways of meiotic recombination

Bloom’s syndrome (BS) is an autosomal recessive disorder characterized by growth retardation, cancer predisposition, and sterility. BS mutated (Blm), the gene mutated in BS patients, is one of five mammalian RecQ helicases. Although BLM has been shown to promote genome stability by assisting in the repair of DNA structures that arise during homologous recombination in somatic cells, less is known about its role in meiotic recombination primarily because of the embryonic lethality associated with Blm deletion. However, the localization of BLM protein on meiotic chromosomes together with evidence from yeast and other organisms implicates a role for BLM helicase in meiotic recombination events, prompting us to explore the meiotic phenotype of mice bearing a conditional mutant allele of Blm. In this study, we show that BLM deficiency does not affect entry into prophase I but causes severe defects in meiotic progression. This is exemplified by improper pairing and synapsis of homologous chromosomes and altered processing of recombination intermediates, resulting in increased chiasmata. Our data provide the first analysis of BLM function in mammalian meiosis and strongly argue that BLM is involved in proper pairing, synapsis, and segregation of homologous chromosomes; however, it is dispensable for the accumulation of recombination intermediates.     

CRL2LRR-1 E3-Ligase Regulates Proliferation and Progression through Meiosis in the Caenorhabditis elegans Germline

The ubiquitin-proteolytic system controls the stability of proteins in space and time. In this study, using a temperature-sensitive mutant allele of the cul-2 gene, we show that CRL2^LRR-1 (CUL-2 RING E3 ubiquitin-ligase and the Leucine Rich Repeat 1 substrate recognition subunit) acts at multiple levels to control germline development. CRL2^LRR-1 promotes germ cell proliferation by counteracting the DNA replication ATL-1 checkpoint pathway. CRL2^LRR-1 also participates in the mitotic proliferation/meiotic entry decision, presumably controlling the stability of meiotic promoting factors in the mitotic zone of the germline. Finally, CRL2^LRR-1 inhibits the first steps of meiotic prophase by targeting in mitotic germ cells degradation of the HORMA domain-containing protein HTP-3, required for loading synaptonemal complex components onto meiotic chromosomes. Given its widespread evolutionary conservation, CUL-2 may similarly regulate germline development in other organisms as well.     

Separation of DNA replication from the assembly of break-competent meiotic chromosomes

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not strictly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms.     

Cyclin B-cdk activity stimulates meiotic rereplication in budding yeast

Haploidization of gametes during meiosis requires a single round of premeiotic DNA replication (meiS) followed by two successive nuclear divisions. This study demonstrates that ectopic activation of cyclin B/cyclin-dependent kinase in budding yeast recruits up to 30% of meiotic cells to execute one to three additional rounds of meiS. Rereplication occurs prior to the meiotic nuclear divisions, indicating that this process is different from the postmeiotic mitoses observed in other fungi. The cells with overreplicated DNA produced asci containing up to 20 spores that were viable and haploid and demonstrated Mendelian marker segregation. Genetic tests indicated that these cells executed the meiosis I reductional division and possessed a spindle checkpoint. Finally, interfering with normal synaptonemal complex formation or recombination increased the efficiency of rereplication. These studies indicate that the block to rereplication is very different in meiotic and mitotic cells and suggest a negative role for the recombination machinery in allowing rereplication. Moreover, the production of haploids, regardless of the genome content, suggests that the cell counts replication cycles, not chromosomes, in determining the number of nuclear divisions to execute.     

Assembly of correct kinetochore architecture in Xenopus egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying the formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one uses a direct chromatin assembly from sperm nuclei in cytostatic factor (CSF)-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step with subsequent reestablishment of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: The amounts of outer kinetochore proteins such as Bub1, BubR1, and Dynactin subunit p150^glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. On the contrary, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results indicate that the transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

On the mechanism of premeiotic DNA synthesis in the yeast Saccharomyces cerevisiae

Premeiotic DNA synthesis in synchronously sporulating cultures of the yeast, Saccharomyces cerevisiae, was analysed by sedimentation in alkaline sucrose gradients and by DNA-fibre autoradiography. The gradient profiles of cells pulse-labelled for varying times were essentially identical with those obtained with mitotic cultures, revealing a close resemblance between the meiotic and mitotic replication mechanisms. This was supported by the finding that exposure of meiotic cells to a specific concentration of hydroxyurea led to the accumulation of completed, but unjoined replicons, just as it does in mitotic cells. The results of DNA-fibre autoradiography confirmed that replicons in meiotic cells are the same size (20–180 Kb, averaging around 90 Kb) as in mitotic cells, and assuming replication is bi-directional, replication forks must move at round the same rate as in mitosis, i.e. about 0.7 μm/min.