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1.
Nat Commun ; 15(1): 4526, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38806488

ABSTRACT

One elusive aspect of the chromosome architecture is how it constrains the DNA topology. Nucleosomes stabilise negative DNA supercoils by restraining a DNA linking number difference (∆Lk) of about -1.26. However, whether this capacity is uniform across the genome is unknown. Here, we calculate the ∆Lk restrained by over 4000 nucleosomes in yeast cells. To achieve this, we insert each nucleosome in a circular minichromosome and perform Topo-seq, a high-throughput procedure to inspect the topology of circular DNA libraries in one gel electrophoresis. We show that nucleosomes inherently restrain distinct ∆Lk values depending on their genomic origin. Nucleosome DNA topologies differ at gene bodies (∆Lk = -1.29), intergenic regions (∆Lk = -1.23), rDNA genes (∆Lk = -1.24) and telomeric regions (∆Lk = -1.07). Nucleosomes near the transcription start and termination sites also exhibit singular DNA topologies. Our findings demonstrate that nucleosome DNA topology is imprinted by its native chromatin context and persists when the nucleosome is relocated.


Subject(s)
DNA, Fungal , Nucleosomes , Saccharomyces cerevisiae , Nucleosomes/metabolism , Nucleosomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , DNA, Fungal/genetics , DNA, Fungal/metabolism , Nucleic Acid Conformation , Chromatin/genetics , Chromatin/metabolism , Telomere/genetics , Telomere/metabolism , DNA/genetics , DNA/chemistry
2.
EMBO J ; 42(3): e111913, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36533296

ABSTRACT

Condensin, an SMC (structural maintenance of chromosomes) protein complex, extrudes DNA loops using an ATP-dependent mechanism that remains to be elucidated. Here, we show how condensin activity alters the topology of the interacting DNA. High condensin concentrations restrain positive DNA supercoils. However, in experimental conditions of DNA loop extrusion, condensin restrains negative supercoils. Namely, following ATP-mediated loading onto DNA, each condensin complex constrains a DNA linking number difference (∆Lk) of -0.4. This ∆Lk increases to -0.8 during ATP binding and resets to -0.4 upon ATP hydrolysis. These changes in DNA topology do not involve DNA unwinding, do not spread outside the condensin-DNA complex and can occur in the absence of the condensin subunit Ycg1. These findings indicate that during ATP binding, a short DNA domain delimited by condensin is pinched into a negatively supercoiled loop. We propose that this loop is the feeding segment of DNA that is subsequently merged to enlarge an extruding loop. Such a "pinch and merge" mechanism implies that two DNA-binding sites produce the feeding loop, while a third site, plausibly involving Ycg1, might anchor the extruding loop.


Subject(s)
Chromosomes , DNA, Superhelical , DNA/metabolism , Adenosine Triphosphate/metabolism , Cell Cycle Proteins/metabolism
3.
Bioessays ; 44(1): e2100187, 2022 01.
Article in English | MEDLINE | ID: mdl-34761394

ABSTRACT

The DNA-passage activity of topoisomerase II accidentally produces DNA knots and interlinks within and between chromatin fibers. Fortunately, these unwanted DNA entanglements are actively removed by some mechanism. Here we present an outline on DNA knot formation and discuss recent studies that have investigated how intracellular DNA knots are removed. First, although topoisomerase II is able to minimize DNA entanglements in vitro to below equilibrium values, it is unclear whether such capacity performs equally in vivo in chromatinized DNA. Second, DNA supercoiling could bias topoisomerase II to untangle the DNA. However, experimental evidence indicates that transcriptional supercoiling of intracellular DNA boosts knot formation. Last, cohesin and condensin could tighten DNA entanglements via DNA loop extrusion (LE) and force their dissolution by topoisomerase II. Recent observations indicate that condensin activity promotes the removal of DNA knots during interphase and mitosis. This activity might facilitate the spatial organization and dynamics of chromatin.


Subject(s)
Adenosine Triphosphatases , Multiprotein Complexes , Cell Cycle Proteins , Chromatin , DNA , DNA-Binding Proteins/genetics , Multiprotein Complexes/genetics
4.
EMBO J ; 40(1): e105393, 2021 01 04.
Article in English | MEDLINE | ID: mdl-33155682

ABSTRACT

The juxtaposition of intracellular DNA segments, together with the DNA-passage activity of topoisomerase II, leads to the formation of DNA knots and interlinks, which jeopardize chromatin structure and gene expression. Recent studies in budding yeast have shown that some mechanism minimizes the knotting probability of intracellular DNA. Here, we tested whether this is achieved via the intrinsic capacity of topoisomerase II for simplifying the equilibrium topology of DNA; or whether it is mediated by SMC (structural maintenance of chromosomes) protein complexes like condensin or cohesin, whose capacity to extrude DNA loops could enforce dissolution of DNA knots by topoisomerase II. We show that the low knotting probability of DNA does not depend on the simplification capacity of topoisomerase II nor on the activities of cohesin or Smc5/6 complexes. However, inactivation of condensin increases the occurrence of DNA knots throughout the cell cycle. These results suggest an in vivo role for the DNA loop extrusion activity of condensin and may explain why condensin disruption produces a variety of alterations in interphase chromatin, in addition to persistent sister chromatid interlinks in mitotic chromatin.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Multiprotein Complexes/metabolism , Cell Cycle/physiology , Chromatids/metabolism , Chromatin/metabolism , Saccharomyces cerevisiae/metabolism , Cohesins
5.
Nucleic Acids Res ; 47(13): 6946-6955, 2019 07 26.
Article in English | MEDLINE | ID: mdl-31165864

ABSTRACT

Recent studies have revealed that the DNA cross-inversion mechanism of topoisomerase II (topo II) not only removes DNA supercoils and DNA replication intertwines, but also produces small amounts of DNA knots within the clusters of nucleosomes that conform to eukaryotic chromatin. Here, we examine how transcriptional supercoiling of intracellular DNA affects the occurrence of these knots. We show that although (-) supercoiling does not change the basal DNA knotting probability, (+) supercoiling of DNA generated in front of the transcribing complexes increases DNA knot formation over 25-fold. The increase of topo II-mediated DNA knotting occurs both upon accumulation of (+) supercoiling in topoisomerase-deficient cells and during normal transcriptional supercoiling of DNA in TOP1 TOP2 cells. We also show that the high knotting probability (Pkn ≥ 0.5) of (+) supercoiled DNA reflects a 5-fold volume compaction of the nucleosomal fibers in vivo. Our findings indicate that topo II-mediated DNA knotting could be inherent to transcriptional supercoiling of DNA and other chromatin condensation processes and establish, therefore, a new crucial role of topoisomerase II in resetting the knotting-unknotting homeostasis of DNA during chromatin dynamics.


Subject(s)
DNA Topoisomerases, Type II/physiology , DNA, Superhelical/metabolism , Nucleic Acid Conformation , Saccharomyces cerevisiae Proteins/physiology , Transcription, Genetic/genetics , Chromatin/ultrastructure , DNA Topoisomerases, Type I/metabolism , DNA, Fungal/metabolism , Humans , Nucleosomes/metabolism , Saccharomyces cerevisiae/metabolism
6.
Nucleic Acids Res ; 47(5): e29, 2019 03 18.
Article in English | MEDLINE | ID: mdl-30649468

ABSTRACT

The characterization of knots formed in duplex DNA has proved useful to infer biophysical properties and the spatial trajectory of DNA, both in free solution and across its macromolecular interactions. Since knotting, like supercoiling, makes DNA molecules more compact, DNA knot probability and knot complexity can be assessed by the electrophoretic velocity of nicked DNA circles. However, the chirality of the DNA knots has to be determined by visualizing the sign of their DNA crossings by means of electron microscopy. This procedure, which requires purifying the knotted DNA molecules and coating them with protein, is semi-quantitative and it is impracticable in biological samples that contain little amount of knotted DNA forms. Here, we took advantage of an earlier observation that the two chiral forms of a trefoil knot acquire slightly different electrophoretic velocity when the DNA is supercoiled. We introduced a second gel dimension to reveal these chiral forms in DNA mixtures that are largely unknotted. The result is a high-resolution 2D-gel electrophoresis procedure that quantitatively discerns the fractions of positive- and negative-noded trefoil knots formed in vitro and in vivo systems. This development in DNA knot analysis may uncover valuable information toward disclosing the architecture of DNA ensembles.


Subject(s)
DNA/chemistry , Electrophoresis, Gel, Two-Dimensional , Nucleic Acid Conformation , DNA, Superhelical/chemistry , Reproducibility of Results
7.
Nat Commun ; 9(1): 3989, 2018 09 28.
Article in English | MEDLINE | ID: mdl-30266901

ABSTRACT

The interplay between chromatin structure and DNA topology is a fundamental, yet elusive, regulator of genome activities. A paradigmatic case is the "linking number paradox" of nucleosomal DNA, which refers to the incongruence between the near two left-handed superhelical turns of DNA around the histone octamer and the DNA linking number difference (∆Lk) stabilized by individual nucleosomes, which has been experimentally estimated to be about -1.0. Here, we analyze the DNA topology of a library of mononucleosomes inserted into small circular minichromosomes to determine the average ∆Lk restrained by individual nucleosomes in vivo. Our results indicate that most nucleosomes stabilize about -1.26 units of ∆Lk. This value balances the twist (∆Tw ≈ + 0.2) and writhe (∆Wr ≈ -1.5) deformations of nucleosomal DNA in terms of the equation ∆Lk = ∆Tw + ∆Wr. Our finding reconciles the existing discrepancy between theoretical and observed measurement of the ΔLk constrained by nucleosomes.


Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA, Fungal/metabolism , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Base Sequence , Chromosomes, Fungal/genetics , DNA, Circular/genetics , DNA, Circular/metabolism , DNA, Fungal/genetics , Nucleosomes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
8.
Methods Mol Biol ; 1805: 291-300, 2018.
Article in English | MEDLINE | ID: mdl-29971724

ABSTRACT

Most bacterial cells have a motor enzyme termed DNA gyrase, which is a type-2 topoisomerase that reduces the linking number (Lk) of DNA. The supercoiling energy generated by gyrase is essential to maintain the bacterial chromosome architecture and regulate its DNA transactions. This chapter describes the use of agarose-gel electrophoresis to detect the unconstrained supercoiling of DNA generated by gyrase or other gyrase-like activities. Particular emphasis is made on the preparation of a relaxed plasmid as initial DNA substrate, on the distinction of constrained and unconstrained DNA supercoils, and on the measurement of the DNA supercoiling density achieved by gyrase activity.


Subject(s)
DNA Gyrase/metabolism , DNA, Superhelical/metabolism , Electrophoresis, Agar Gel/methods , Animals , Cattle , Humans , Substrate Specificity
9.
Nucleic Acids Res ; 46(2): 650-660, 2018 01 25.
Article in English | MEDLINE | ID: mdl-29149297

ABSTRACT

In vivo DNA molecules are narrowly folded within chromatin fibers and self-interacting chromatin domains. Therefore, intra-molecular DNA entanglements (knots) might occur via DNA strand passage activity of topoisomerase II. Here, we assessed the presence of such DNA knots in a variety of yeast circular minichromosomes. We found that small steady state fractions of DNA knots are common in intracellular chromatin. These knots occur irrespective of DNA replication and cell proliferation, though their abundance is reduced during DNA transcription. We found also that in vivo DNA knotting probability does not scale proportionately with chromatin length: it reaches a value of ∼0.025 in domains of ∼20 nucleosomes but tends to level off in longer chromatin fibers. These figures suggest that, while high flexibility of nucleosomal fibers and clustering of nearby nucleosomes facilitate DNA knotting locally, some mechanism minimizes the scaling of DNA knot formation throughout intracellular chromatin. We postulate that regulation of topoisomerase II activity and the fractal architecture of chromatin might be crucial to prevent a potentially massive and harmful self-entanglement of DNA molecules in vivo.


Subject(s)
Chromatin/chemistry , DNA, Fungal/chemistry , DNA, Superhelical/chemistry , Nucleic Acid Conformation , Cell Division/genetics , Chromatin/genetics , Chromatin/metabolism , DNA Replication/genetics , DNA Topoisomerases, Type II/metabolism , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Models, Molecular , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
10.
Cell Rep ; 13(4): 667-677, 2015 Oct 27.
Article in English | MEDLINE | ID: mdl-26489472

ABSTRACT

DNA is wrapped in a left-handed fashion around histone octasomes containing the centromeric histone H3 variant CENP-A. However, DNA topology studies have suggested that DNA is wrapped in a right-handed manner around the CENP-A nucleosome that occupies the yeast point centromere. Here, we determine the DNA linking number difference (ΔLk) stabilized by the yeast centromere and the contribution of the centromere determining elements (CDEI, CDEII, and CDEIII). We show that the intrinsic architecture of the yeast centromere stabilizes +0.6 units of ΔLk. This topology depends on the integrity of CDEII and CDEIII, but it is independent of cbf1 binding to CDEI and of the variable length of CDEII. These findings suggest that the interaction of the CBF3 complex with CDEIII and a distal CDEII segment configures a right-handed DNA loop that excludes CDEI. This loop is then occupied by a CENP-A histone complex, which does not have to be inherently right-handed.


Subject(s)
Centromere/metabolism , Saccharomyces cerevisiae/genetics , DNA, Fungal/genetics , Nucleosomes/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
11.
Proc Biol Sci ; 280(1771): 20131945, 2013 Nov 22.
Article in English | MEDLINE | ID: mdl-24068360

ABSTRACT

Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primates and Carnivora to better understand the dynamics of mammalian recombination. Our results suggest a phylogenetic component in recombination rates (RRs), which appears to be directional, strongly punctuated and subject to selection. Species that diversified earlier in the evolutionary tree have lower RRs than those from more derived phylogenetic branches. Furthermore, chromosome-specific recombination maps in distantly related taxa show that crossover interference is especially weak in the species with highest RRs detected thus far, the tiger. This is the first example of a mammalian species exhibiting such low levels of crossover interference, highlighting the uniqueness of this species and its relevance for the study of the mechanisms controlling crossover formation, distribution and resolution.


Subject(s)
Biological Evolution , Crossing Over, Genetic/genetics , Genetic Variation , Mammals/genetics , Phylogeny , Recombination, Genetic/genetics , Animals , Basal Metabolism , Bayes Theorem , Body Size , Body Temperature , Crossing Over, Genetic/physiology , Fluorescent Antibody Technique , Humans , Likelihood Functions , Male , Models, Genetic , Species Specificity , Testis/metabolism
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