Keeping intracellular DNA untangled: A new role for condensin?

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.

Condensin minimizes topoisomerase II-mediated entanglements of DNA in vivo.

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.

Transcriptional supercoiling boosts topoisomerase II-mediated knotting of intracellular DNA.

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.

Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis.

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.

Intracellular nucleosomes constrain a DNA linking number difference of -1.26 that reconciles the Lk paradox.

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.

Electrophoretic Analysis of the DNA Supercoiling Activity of DNA Gyrase.

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.

DNA knots occur in intracellular chromatin.

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.

DNA Topology and Global Architecture of Point Centromeres.

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.

Prevalence of Helicobacter pylori vacA, cagA, and iceA Genotypes in Cuban Patients with Upper Gastrointestinal Diseases.

Virulence factors of Helicobacter pylori can predict the development of different gastroduodenal diseases. There are scarce reports in Cuba about H. pylori isolates genotyping. The aim of the present investigation was to identify allelic variation of the virulence genes vacA, cagA, and iceA in sixty-eight patients diagnosed as H. pylori positive by culture. In seven out of 68 patients, strains from both gastric regions were obtained and considered independent. DNA was extracted from all the H. pylori strains and evaluated by PCR-genotyping. The vacA s1 allele, cagA gene, and iceA2 allele were the most prevalent (72.0%, 56.0%, and 57.3%, respectively). Alleles from m-region showed a similar frequency as s1a and s1b subtypes. The presence of multiple H. pylori genotypes in a single biopsy and two gastric region specimens were found. Significant statistical association was observed between iceA2 allele and patients with non-peptic ulcer dyspepsia (NUD) (P = 0.037) as well as virulence genotypes (s1, s1m2) and patients over 40 years old (P < 0.05). In conclusion, the results demonstrated a high prevalence of H. pylori virulent genotypes in Cuban patients over 40 years old while iceA2 alleles demonstrated a good specificity in patients with NUD.

Automedicación, ¿beneficio o perjuicio? / Self-medication: benefit or damage?

Se hizo un estudio descriptivo en el Centro Provincial Desarrollo Medicina Tradicional y Natural de Sancti Spíritus dirigido a evaluar la relación riesgo-beneficio, respecto a la automedicación con fármacos naturales. El universo lo conformaron 3060 pacientes que acudieron a la farmacia de esta institución y que recibieron sus servicios en el período de enero a mayo del 2003, para la selección de la muestra se utilizó el método probabilístico aleatorio simple, que correspondió al 20 por ciento de la unidad de análisis total (612 individuos), a los cuales se les realizaron encuestas; los datos se procesaron con el auxilio de una computadora y la ayuda de un paquete estadístico SPSS-PC. Se pudo comprobar que sin la prescripción del facultativo se ocasiona involuntariamente daño al organismo, donde se afecta la farmacocinética del medicamento por desconocimiento en la posología y el tiempo en que debe ser administrado, además de las interacciones que pueden producirse, sin embargo la población la acepta como modalidad terapéutica teniendo en cuenta que es una terapia poco agresiva a la salud humana, proveniente de una fuente renovable de materias primas, sin efectos tóxicos, ni nocivos marcados para el hombre y sin necesidades tecnológicas avanzadas para su producción, todo lo cual favorece en el momento que el médico la indique y se empleé para beneficio del paciente, logrando una mejor calidad de vida en su enfermedad(AU)

A descriptive study was made in the Provincial Center of Development of Traditional and Natural Medicine of Sancti Spíritus, aimed at evaluating the risk-benefit relationship, regarding self-medication with natural drugs. The sample was made up of 3060 patients that went to the pharmacy of this institution and received its services in the period from January to May of 2003. For sample selection the method of simple probabilistic randomization was used that corresponded to 20 percent of the unit of total analysis (612 individuals), to whom surveys were carried out. Data were processed with the help of a computer and an SPSS-PC statistical package. It could be confirmed that without the doctor's prescription, non-desired damage is caused to the body, where the pharmacokinetics of the medication is affected by ignorance in the posology and the time in which it must be administered, besides the interactions that can take place. However, the population accepts it as a therapeutic modality taking into account that it is a not very aggressive therapy to human health, coming from a renewable source of raw material, without marked toxic or noxious effects for man and without advanced technological needs for its elaboration, all of which favors its presciption and use at the moment for the patient's benefit, with a better quality of life being achieved in his/her disease(AU)

Automedicación, ¿beneficio o perjuicio? / Self-medication: benefit or damage?

Se hizo un estudio descriptivo en el Centro Provincial Desarrollo Medicina Tradicional y Natural de Sancti Spíritus dirigido a evaluar la relación riesgo-beneficio, respecto a la automedicación con fármacos naturales. El universo lo conformaron 3060 pacientes que acudieron a la farmacia de esta institución y que recibieron sus servicios en el período de enero a mayo del 2003, para la selección de la muestra se utilizó el método probabilístico aleatorio simple, que correspondió al 20 % de la unidad de análisis total (612 individuos), a los cuales se les realizaron encuestas; los datos se procesaron con el auxilio de una computadora y la ayuda de un paquete estadístico SPSS-PC. Se pudo comprobar que sin la prescripción del facultativo se ocasiona involuntariamente daño al organismo, donde se afecta la farmacocinética del medicamento por desconocimiento en la posología y el tiempo en que debe ser administrado, además de las interacciones que pueden producirse, sin embargo la población la acepta como modalidad terapéutica teniendo en cuenta que es una terapia poco agresiva a la salud humana, proveniente de una fuente renovable de materias primas, sin efectos tóxicos, ni nocivos marcados para el hombre y sin necesidades tecnológicas avanzadas para su producción, todo lo cual favorece en el momento que el médico la indique y se empleé para beneficio del paciente, logrando una mejor calidad de vida en su enfermedad.[AU]

Factor desencadenante y de hiperractividad de la penicilina en las conectivopatias / Penicillin unchaining and hiperactivity factors in collagen diseases

Se plantea que a 6 pacientes de un total de 18 recibidos en nuestro servicio de medicina interna (fajardo A) en forma continuada se les diagnostico a su inicio, conectivopatias lupus eritematoso generalizado (LEG), poliarteritis nudosa (PAN) y dermatomiositis (DM) probocada por el empleo de la penicilina, y que se le desarrollo una forma hiperactiva de la enfermedad con rapido fallecimiento.El promedio de sobrevivencia fue de 0.5 años. El estudio necropsico de todos los fallecidos demostro la marcada extension del grave daño histico. Las subitas manifestaciones clinicas se acompañaron de trastornos humorales significativos y, en ocasiones, peculiares. El estudio clinico permitio observaciones que pudieran ser de interes en el orden patogenico, asi como recomendaciones de utilidad desde el punto de vista profilactivo