Mammalian meiotic telomeres: composition and ultrastructure in telomerase-deficient mice

During early meiotic prophase chromosome ends become attached to the nuclear envelope, a process that is essential for faithful homologue pairing and segregation. The factors involved in this attachment are largely unknown. Here we investigated the possible involvement of telomere chromatin by using late generation (G5 and G6) Terc-/- mice. These mice lack telomerase activity and show progressive telomere shortening with increasing mouse generations. We show here that in meiotic chromosome ends of late generation Terc-/- mice telomeric TTAGGG repeats and the TRF1 telomere-binding protein are significantly reduced or below detection level. In spite of this, electron microscopy showed no apparent structural differences at the attachment sites of meiotic chromosomes to the nuclear envelope between wild-type and G6 Terc-/- meiocytes. These results suggest, as already shown in yeast, that most telomere chromatin is dispensable for proper attachment of mammalian meiotic chromosome ends to the nuclear envelope.     

An SMC-domain protein in fission yeast links telomeres to the meiotic centrosome

Abnormal centrosomal structures similar to those occurring in human cancers are induced in fission yeast by overexpression of the pericentrin homolog Pcp1p. Analysis of abnormal Pcp1p-containing structures with quantitative mass spectrometry and isotope-coded affinity tags identified a coiled-coil, structural maintenance of chromosomes (SMC) domain protein. This protein, termed Ccq1p (coiled-coil protein quantitatively enriched), localizes with Taz1p to telomeres in normal vegetative cells. Fluorescence resonance energy transfer (FRET) measurements indicate that Ccq1p also interacts with centrosomal Pcp1p in mating pheromone-stimulated cells containing centrosomally clustered telomeres. We provide evidence that the Ccq1p-Pcp1p interaction, while essential for meiosis, is deleterious when forced to occur during vegetative growth. Cells lacking one ccq1 allele exhibit a loss-of-function phenotype including abnormally long cell length, chromosome segregation failure, telomeric shortening, and defective telomeric clustering during meiotic prophase. Our data indicate a mechanism underlying meiotic chromosomal bouquet formation and suggest a recruitment model for supernumerary centrosome toxicity.     

Mammalian telomeres and telomerase

New features of mammalian telomeres and telomerase have been identified. Telomeres form t-loops, which engage the 3' single-stranded DNA overhang in an interaction with double-stranded telomeric repeats. Mammalian telomerases contain an RNA H/ACA motif and associated protein(s) shared with H/ACA family of small nucleolar ribonucleoproteins. Essential roles for telomerase in the sustained viability of cultured tumor cells and in the normal proliferative capacity of human somatic cells have been demonstrated.     

The nuclear pores of early meiotic prophase nuclei of plants

Summary Pollen mother cells at early meiotic prophase fromFritillaria lanceolata, F. mutica, Tulbaghia violacea, the lily “Formobel”,Triticum aegilopoides, T. dicoccoides, T. aestivum and synaptic and asynaptic forms ofT. durum were studied in thin sections with the electron microscope (a) in relation to distribution of nuclear pores (b) in respect of fine structure of the pore complex in those of the first four. The pores were distributed in random clusters during leptotene to pachytene in all plants, except in the two forms ofT. durum where there were either no pores or so few that they were not detectable. Probably correlated with this, the two membranes of the nuclear envelope were often widely separated and frequently sacculated. No pores were seen at leptotene in the part of the envelope to which, in theFritillarias and lily, the nucleolus was adpressed at this time. Evidence supporting a recent model which proposes that annuli are composed of three rings of eight granular subunits was obtained. These subunits as well as a dense central element, observed in most pores, were composed of filaments about 3 nm in diameter and evidently protein in character. There was evidence of a continuity between filaments in the central element and those in the rings of subunits which encircle the pore aperture at both the nuclear and cytoplasmic sides of the pore. In profiles of pores knobbed filaments were sometimes seen extending laterally from the pore wall into the perinuclear space at two sides. Questions concerning the role of the annulus are discussed. The author wish to thank Mr. R. F. Scott for construction to the model.     

Mammalian meiotic recombination: a reexamination

Recombination nodules (RNs) are small electron-dense structures associated with the synaptonemal complex. Two types have been identified: early RNs present during zygonema-early pachynema, which are thought to be involved in gene conversion and synaptic initiation, and late RNs present during mid-to-late pachynema, which are thought to be involved in reciprocal recombination leading to chiasma formation. In organisms as diverse as Sodaria, Drosophila, and plants there is indeed a close correlation between the observed number of late RNs and crossovers, or their cytogenetic manifestation, chiasmata. However, as this reexamination of the human data shows, there is not a similar correlation in mammals. Instead, there is a severe deficiency in RNs in eutherian males and marsupial females near chromosome ends and other recombinational hot spots (defined genetically), or localized chiasmata (defined cytogenetically). Many of these sites of hyper-recombination correspond to sites of telomere or telomere-associated sequences. Together these observations suggest the possibility of a second, mechanistically different, recombination pathway that does not involve RNs, but may directly involve telomere or telomere-associated sequences. This pathway may be responsible for sex-specific hot-spots of recombination observed at highly localized sites throughout the genome.     

MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks.

The yeast Sir2/3/4p complex is found in abundance at telomeres, where it participates in the formation of silent heterochromatin and telomere maintenance. Here, we show that Sir3p is released from telomeres in response to DNA double-strand breaks (DSBs), binds to DSBs, and mediates their repair, independent of cell mating type. Sir3p relocalization is S phase specific and, importantly, requires the DNA damage checkpoint genes MEC1 and RAD9. MEC1 is a homolog of ATM, mutations in which cause ataxia telangiectasia (A-T), a disease characterized by various neurologic and immunologic abnormalities, a predisposition for cancer, and a cellular defect in repair of DSBs. This novel mode by which preformed DNA repair machinery is mobilized by DNA damage sensors may have implications for human diseases resulting from defective DSB repair.     

Metastable network model of protein transport through nuclear pores

To reconcile the observed selectivity and the high rate of translocation of cargo-importin complexes through nuclear pores, we propose that the core of the nuclear pore complex is blocked by a metastable network of phenylalanine and glycine nucleoporins. Although the network arrests the unfacilitated passage of objects larger than its mesh size, cargo-importin complexes act as catalysts that reduce the free energy barrier between the cross-linked and the dissociated states of the Nups, and open the network. Using Brownian dynamics simulations we calculate the distribution of passage times through the network for inert particles and cargo-importin complexes of different sizes and discuss the implications of our results for experiments on translocation of proteins through the nuclear pore complex.     

Mammalian telomeres and telomerase: why they matter for cancer and aging

Chromosome ends, or telomeres, are formed by a special chromatin structure that protects them from recombination and degradation, thus preventing end-to-end chromosome fusions and other chromosomal aberrations. The functionality of telomeres, and that of the cellular activity that synthesizes them, telomerase, has been shown to impact on both cancer and aging, as well as on the organismal sensitivity to ionizing radiation. This review focuses on the analysis of different mouse models for proteins that are important for telomere function, which have highlighted the importance of telomeres and telomerase for cancer and aging.     

Ribosome biogenesis factors bind a nuclear envelope SUN domain protein to cluster yeast telomeres

Two interacting ribosome biogenesis factors, Ebp2 and Rrs1, associate with Mps3, an essential inner nuclear membrane protein. Both are found in foci along the nuclear periphery, like Mps3, as well as in the nucleolus. Temperature-sensitive ebp2 and rrs1 mutations that compromise ribosome biogenesis displace the mutant proteins from the nuclear rim and lead to a distorted nuclear shape. Mps3 is known to contribute to the S-phase anchoring of telomeres through its interaction with the silent information regulator Sir4 and yKu. Intriguingly, we find that both Ebp2 and Rrs1 interact with the C-terminal domain of Sir4, and that conditional inactivation of either ebp2 or rrs1 interferes with both the clustering and silencing of yeast telomeres, while telomere tethering to the nuclear periphery remains intact. Importantly, expression of an Ebp2-Mps3 fusion protein in the ebp2 mutant suppresses the defect in telomere clustering, but not its defects in growth or ribosome biogenesis. Our results suggest that the ribosome biogenesis factors Ebp2 and Rrs1 cooperate with Mps3 to mediate telomere clustering, but not telomere tethering, by binding Sir4.     

The nuclear matrix: Three-dimensional architecture and protein composition

The structural filament network of the nucleus is prepared while still connected to the cytoskeleton. The relatively gentle procedure removes about 98% of the DNA and at least 86% of the histones. The matrix is bounded by an outer nuclear lamina connected to the cytoskeletal framework, as well as the inner filaments. The filaments range in diameter from 3 to 22 nm, and are organized in a three-dimensional anastomosing network in which nucleoli are enmeshed. The nuclear matrix is separated from the cytoskeletal framework by a double detergent and then partitioned into a chromatin fraction and a matrix fraction by nuclease and high salt. Two-dimensional gel electrophoresis shows that the proteins of the cytoskeleton, chromatin and nuclear matrix are very different. A major protein found in all fractions cofocuses with actin. Vimentin is largely associated with the nuclear matrix, probably as a corona external of filaments.     

Mammalian telomeres end in a large duplex loop.

Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat-binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3' overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.     

Mammalian protein SCP1 forms synaptonemal complex-like structures in the absence of meiotic chromosomes

Synaptonemal complexes (SCs) are evolutionary conserved, meiosis-specific structures that play a central role in synapsis of homologous chromosomes, chiasmata distribution, and chromosome segregation. However, it is still for the most part unclear how SCs do assemble during meiotic prophase. Major components of mammalian SCs are the meiosis-specific proteins SCP1, 2, and 3. To investigate the role of SCP1 in SC assembly, we expressed SCP1 in a heterologous system, i.e., in COS-7 cells that normally do not express SC proteins. Notably, under these experimental conditions SCP1 is able to form structures that closely resemble SCs (i.e., polycomplexes). Moreover, we show that mutations that modify the length of the central alpha-helical domain of SCP1 influence the width of polycomplexes. Finally, we demonstrate that deletions of the nonhelical N- or C-termini both affect polycomplex assembly, although in a different manner. We conclude that SCP1 is a primary determinant of SC assembly that plays a key role in synapsis of homologous chromosomes.     

Identification of a meiotic prophase-specific nuclear matrix protein in the rat

We have identified a 110-kDa pI 5.6 phosphoprotein with DNA binding properties in the rat pachytene spermatocyte nuclear matrix. By immunoblotting and indirect immunofluorescence assays using polyclonal antibodies against the 110-kDa protein, we observed that it was germ cell nuclear matrix specific, more prominent in pachytene spermatocytes compared to premeiotic spermatogonia or postmeiotic round spermatids, and present in rat oocytes and in germ cells of mouse and monkey. We propose that this protein could play an important role in the meiotic process.     

Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements.

An enzyme capable of specifically modifying, with a geranylgeranyl isoprenoid, candidate proteins containing a consensus prenylation sequence ending in leucine has been purified from bovine brain. This protein geranylgeranyltransferase (PGGT), isolated using affinity chromatography on an immobilized peptide column, contains two subunits with molecular masses of 48 and 43 kDa, designated alpha and beta, respectively. An antiserum raised to the alpha subunit of the related enzyme, protein farnesyltransferase (PFT), also recognizes this chromatographically identical alpha-subunit of the PGGT by immunoblot analysis. The PGGT and PFT enzymes from bovine brain are shown to be dependent on both Mg2+ and Zn2+ for optimal activity. Demonstration of the Zn2+ dependence of the enzymes requires prolonged incubation or purification in the presence of a chelating agent; we therefore propose that these enzymes be placed into the category of metalloenzymes. Under optimal assay conditions, these enzymes show high specificity toward their prenyl diphosphate substrates, with only a weak competition observed with farnesyl diphosphate in the PGGT reaction or geranylgeranyl diphosphate in the PFT reaction. The two enzymes are differentially sensitive to several detergents tested to determine suitable ones for product stabilization in the reactions. These results confirm previous predictions on the subunit structure of the PGGT and provide an avenue to initiating a molecular analysis of the geranylgeranyl modification of many mammalian proteins.     

SUMO: ligases, isopeptidases and nuclear pores

Small ubiquitin-related modifier (SUMO) proteins are reversibly coupled to numerous intracellular targets and modulate their interactions, localization, activity or stability. Recent advances in the SUMO field have uncovered the first SUMO E3 ligases and point to a complex family of isopeptidases. SUMO has been linked to many different pathways, including nucleocytoplasmic transport. Modifying enzymes and an isopeptidase have been detected at nuclear pore complexes. In addition, studies in yeast suggest a requirement of SUMO conjugation for nuclear protein import, and specific SUMO targets depend on modification for nuclear import or export.     

Genome-wide redistribution of meiotic double-strand breaks in Saccharomyces cerevisiae

Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the Spo11 protein. DSBs are not randomly distributed along chromosomes. To better understand factors that control the distribution of DSBs in budding yeast, we have examined the genome-wide binding and cleavage properties of the Gal4 DNA binding domain (Gal4BD)-Spo11 fusion protein. We found that Gal4BD-Spo11 cleaves only a subset of its binding sites, indicating that the association of Spo11 with chromatin is not sufficient for DSB formation. In centromere-associated regions, the centromere itself prevents DSB cleavage by tethered Gal4BD-Spo11 since its displacement restores targeted DSB formation. In addition, we observed that new DSBs introduced by Gal4BD-Spo11 inhibit surrounding DSB formation over long distances (up to 60 kb), keeping constant the number of DSBs per chromosomal region. Together, these results demonstrate that the targeting of Spo11 to new chromosomal locations leads to both local stimulation and genome-wide redistribution of recombination initiation and that some chromosomal regions are inherently cold regardless of the presence of Spo11.