Biomedical subjects

P Pasero

Publications and source records attributed to P Pasero.

14 recordsLinked to original sources

PubMed2001

Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains.

Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.

Blotting, Southern↗

PubMed2000

Think global, act local--how to regulate S phase from individual replication origins.

All eukaryotes use similar proteins to licence replication origins but, paradoxically, origin DNA is much less conserved. Specific binding sites for these proteins have now been identified on fission yeast and Drosophila chromosomes, suggesting that the DNA-binding activity of the origin recognition complex has diverged to recruit conserved initiation factors on polymorphic replication origins. Once formed, competent origins are activated by cyclin- and Dbf4-dependent kinases. The latter have been shown to control S phase in several organisms but, in contrast to cyclin-dependent kinases, seem regulated at the level of individual origins. Global and local regulations generate specific patterns of DNA replication that help establish epigenetic chromosome states.

Animals↗

PubMed1999

A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.

Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs.

CDC28 Protein Kinase, S cerevisiae↗

PubMed1999

In vitro DNA replication in yeast nuclear extracts.

Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase. In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G(1). In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating system. Semiconservative DNA replication is monitored by substitution of newly synthesized DNA with bromodeoxyuridine 5'-triphosphate (BrdUTP) and density gradient analysis. In addition, neutral-neutral two-dimensional gel analyses and, in the case of nuclei, detection of newly synthesized DNA in replication foci by DIG-dUTP incorporation can be used to monitor replication.

Cell Nucleus↗

PubMed1999

Short DNA fragments without sequence similarity are initiation sites for replication in the chromosome of the yeast Yarrowia lipolytica.

We have previously shown that both a centromere (CEN) and a replication origin are necessary for plasmid maintenance in the yeast Yarrowia lipolytica (). Because of this requirement, only a small number of centromere-proximal replication origins have been isolated from Yarrowia. We used a CEN-based plasmid to obtain noncentromeric origins, and several new fragments, some unique and some repetitive sequences, were isolated. Some of them were analyzed by two-dimensional gel electrophoresis and correspond to actual sites of initiation (ORI) on the chromosome. We observed that a 125-bp fragment is sufficient for a functional ORI on plasmid, and that chromosomal origins moved to ectopic sites on the chromosome continue to act as initiation sites. These Yarrowia origins share an 8-bp motif, which is not essential for origin function on plasmids. The Yarrowia origins do not display any obvious common structural features, like bent DNA or DNA unwinding elements, generally present at or near eukaryotic replication origins. Y. lipolytica origins thus share features of those in the unicellular Saccharomyces cerevisiae and in multicellular eukaryotes: they are discrete and short genetic elements without sequence similarity.

Centromere↗

PubMed1999

Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts.

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

Animals↗

PubMed1998

Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template.

We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [alpha-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis. We demonstrate dependence on DNA pol delta and the pol alpha/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions. In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p. Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2.

Adenosine Triphosphatases↗

PubMed1998

New systems for replicating DNA in vitro.

Current paradigms for the regulation of genomic DNA replication in eukaryotes are derived primarily from cell fusion experiments, yeast genetics, and from in vitro assays in Xenopus egg extracts. Initially, many aspects seemed irreconcilably different among the various organisms and model systems. In the past year, however, divergent approaches have arrived at a consensus on how the cell cycle regulates the initiation of DNA replication. All major players appear to be conserved from yeast to vertebrates, yet the important challenge of reconstituting eukaryotic replication from purified components remains. Three novel in vitro assays that replicate nuclear templates bring us closer to this goal.

Animals↗

PubMed1997

ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.

We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.

Cell Cycle↗

PubMed1996

Common DNA structural features exhibited by eukaryotic ribosomal gene promoters.

Nucleotide sequences of DNA regions containing eukaryotic ribosomal promoters were analysed using strategies designed to reveal sequence-directed structural features. DNA curvature, duplex stability and pattern of twist angle variation were studied by computer modelling. Although ribosomal promoters are known to lack sequence homology (unless very closely related species are considered), investigation of these structural characteristics uncovered striking homologies in all the taxonomic groups examined so far. This wide conservation of DNA structures, while DNA sequence is not conserved, suggests that the determined structures are fundamental for ribosomal promoter function. Moreover, this result agrees well with the recent observations showing that RNA polymerase I transcription factors have not evolved as intensively as previously suspected.

Animals↗

PubMed1995

Promoter structure and intron-exon organization of a scorpion alpha-toxin gene.

The Androctonus australis scorpion venom contains alpha-toxins for which the complementary DNAs have been cloned [Bougis et al. (1989) J. Biol. Chem. 264, 19259-19265], targeting with high affinity the voltage-sensitive sodium channel. From a genomic library made of this species of scorpion, we have cloned and characterized the gene encoding the toxin AaH I'. The gene transcriptional unit is 793 base pairs long, and the gene has a single intron of 425 base pairs located near the end of the signal peptide of the toxin precursor. The transcription initiation site was determined by primer extension and corresponded to the nucleotide sequence AACAA. Upstream, a promoter region has been identified with positive acting sequence elements at consensus positions, such as a CCAAT box and a TATA box. In addition, putative elements for binding the transcriptional factors MAT-alpha 2, Pit-1, and IEF1 are also present. Analysis of DNA curvature by computer modeling revealed a strong bending centered around the transcription initiation site of the gene. The bending angle (61 degrees) estimated experimentally using polyacrylamide gel electrophoresis correlates well with the value predicted by computer modeling (66 degrees). Other minor deflections of the helix axis cooperate for an overall curvature of nearly 90 degrees, which is significantly stronger than similar structures already reported in eukaryotic cells. It is worth noting that the grooves relative to the CCAAT box and the TATA box lie along the inside of the DNA curve. This observation is in agreement with the previously reported correlation between DNA bending and promoter function.

Amino Acid Sequence↗

PubMed1993

Long-range organization and sequence-directed curvature of Xenopus laevis satellite 1 DNA.

We have investigated the long-range organization and the intrinsic curvature of satellite 1 DNA, an unusual tandemly-repeated DNA family of Xenopus laevis presenting sequence homologies to SINEs. PFGE was used in combination with frequent-cutter restriction enzymes not likely to cut within satellite 1 DNA and revealed that almost all the repeating units are tandemly organized to form large arrays (200 kb to 2 Mb) that are marked by restriction length polymorphism and contain intra-array domains of sequence variation. Besides that, we have analysed the secondary structure of satellite 1 DNA by computer modelling. Theoretical maps of curvature obtained from three independent models of DNA bending (the dinucleotide wedge model of Trifonov, the junction model of Crothers and the model of de Santis) showed that satellite 1 DNA is intrinsically curved and these results were confirmed experimentally by polyacrylamide gel electrophoresis. Moreover, we observed that this bending element is highly conserved among all the members of the satellite 1 DNA family that are accessible to analysis. A potential genetic role for satellite 1 DNA based on this unusual structural feature is discussed.

Animals↗

PubMed1993

Size variation of rDNA clusters in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The higher-order organization of rRNA genes was investigated in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. We used pulsed-field gel electrophoresis (PFGE) in combination with frequent cutter endonucleases having no recognition sites within rDNA repeating units to characterize tandem arrays of ribosomal genes in these two species. Large variations in rDNA cluster length were detected in various S. cerevisiae and S. pombe strains commonly used as PFGE molecular weight markers. This wide range of variability implies that the sizes currently assessed for chromosomes bearing rRNA genes in these organisms are unreliable since they may vary within strains by several hundreds of kilobase pairs, depending on the size of the tandem arrays of rRNA genes. Consequently, there is now a lack of reliable PFGE size standards between 1.6 Mb and 4.5 Mb, even when established yeast strains with calibrated chromosomes are used.

Chromosomes, Fungal↗

PubMed1992

Molecular dissection of a specific nuclear domain: the chromatin region of the ribosomal gene cluster in Xenopus laevis.

Molecular dissection of the nuclear domain corresponding to the ribosomal chromatin cluster was investigated. The experimental scheme was based on the ability of restriction enzymes to digest the whole genome without affecting this region (several megabases in length). Such a strategy involved the judicious choice of restriction enzymes, which is possible in Xenopus laevis, where the rDNA sequence is known and the repeated units are organized into one unique cluster. SalI, XhoI, and EcoRV digestion produced frequent cutting of the genome leaving the ribosomal cluster intact. Isolation of the rDNA cluster was confirmed by separation of the digested DNA by pulsed-field electrophoresis. When applied to purified nuclei, this approach allowed the isolation of the ribosomal chromatin cluster under very mild conditions: no cleavages (either enzymatic or mechanical) were detectable. Since the purification scheme depends only on the DNA sequence outside of the rDNA cluster, it permits the obtention of this domain in different functional states. Electron microscopic analysis demonstrated that the domain organization is substantially preserved and maintains its looped organization (the size and the full number of loops were preserved). This purification scheme provides a powerful tool for studying the structure-function relationships within the ribosomal nuclear domain.

Animals↗