RESUMO
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.
Assuntos
Cromossomos , DNA Super-Helicoidal , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismoRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Complexos Multiproteicos/metabolismo , Ciclo Celular/fisiologia , Cromátides/metabolismo , Cromatina/metabolismo , Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
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.
Assuntos
Adenosina Trifosfatases , Complexos Multiproteicos , Proteínas de Ciclo Celular , Cromatina , DNA , Proteínas de Ligação a DNA/genética , Complexos Multiproteicos/genéticaRESUMO
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.
Assuntos
DNA/química , Eletroforese em Gel Bidimensional , Conformação de Ácido Nucleico , DNA Super-Helicoidal/química , Reprodutibilidade dos TestesRESUMO
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.
Assuntos
DNA Topoisomerases Tipo II/fisiologia , DNA Super-Helicoidal/metabolismo , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae/fisiologia , Transcrição Gênica/genética , Cromatina/ultraestrutura , DNA Topoisomerases Tipo I/metabolismo , DNA Fúngico/metabolismo , Humanos , Nucleossomos/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
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 â¼0.025 in domains of â¼20 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.
Assuntos
Cromatina/química , DNA Fúngico/química , DNA Super-Helicoidal/química , Conformação de Ácido Nucleico , Divisão Celular/genética , Cromatina/genética , Cromatina/metabolismo , Replicação do DNA/genética , DNA Topoisomerases Tipo II/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Modelos Moleculares , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
DNA Girase/metabolismo , DNA Super-Helicoidal/metabolismo , Eletroforese em Gel de Ágar/métodos , Animais , Bovinos , Humanos , Especificidade por SubstratoRESUMO
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.