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.

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.

Schizont transcriptome variation among clinical isolates and laboratory-adapted clones of the malaria parasite Plasmodium falciparum.

BACKGROUND: Malaria parasites are genetically polymorphic and phenotypically plastic. In studying transcriptome variation among parasites from different infections, it is challenging to overcome potentially confounding technical and biological variation between samples. We investigate variation in the major human parasite Plasmodium falciparum, generating RNA-seq data on multiple independent replicate sample preparations of merozoite-containing intra-erythrocytic schizonts from a panel of clinical isolates and from long-term laboratory-adapted clones, with a goal of robustly identifying differentially expressed genes. RESULTS: Analysis of biological sample replicates shows that increased numbers improve the true discovery rate of differentially expressed genes, and that six independent replicates of each parasite line allowed identification of most differences that could be detected with larger numbers. For highly expressed genes, focusing on the top quartile at schizont stages, there was more power to detect differences. Comparing cultured clinical isolates and laboratory-adapted clones, genes more highly expressed in the laboratory-adapted clones include those encoding an AP2 transcription factor (PF3D7_0420300), a ubiquitin-binding protein and two putative methyl transferases. In contrast, higher expression in clinical isolates was seen for the merozoite surface protein gene dblmsp2, proposed to be a marker of schizonts forming merozoites committed to sexual differentiation. Variable expression was extremely strongly, but not exclusively, associated with genes known to be targeted by Heterochromatin Protein 1. Clinical isolates show variable expression of several known merozoite invasion ligands, as well as other genes for which new RT-qPCR assays validate the quantitation and allow characterisation in samples with more limited material. Expression levels of these genes vary among schizont preparations of different clinical isolates in the first ex vivo cycle in patient erythrocytes, but mean levels are similar to those in continuously cultured clinical isolates. CONCLUSIONS: Analysis of multiple biological sample replicates greatly improves identification of genes variably expressed between different cultured parasite lines. Clinical isolates recently established in culture show differences from long-term adapted clones in transcript levels of particular genes, and are suitable for analyses requiring biological replicates to understand parasite phenotypes and variable expression likely to be relevant in nature.

Chromatin regulates DNA torsional energy via topoisomerase II-mediated relaxation of positive supercoils.

Eukaryotic topoisomerases I (topo I) and II (topo II) relax the positive (+) and negative (-) DNA torsional stress (TS) generated ahead and behind the transcription machinery. It is unknown how this DNA relaxation activity is regulated and whether (+) and (-)TS are reduced at similar rates. Here, we used yeast circular minichromosomes to conduct the first comparative analysis of topo I and topo II activities in relaxing chromatin under (+) and (-)TS. We observed that, while topo I relaxed (+) and (-)TS with similar efficiency, topo II was more proficient and relaxed (+)TS more quickly than (-)TS. Accordingly, we found that the relaxation rate of (+)TS by endogenous topoisomerases largely surpassed that of (-)TS. We propose a model of how distinct conformations of chromatin under (+) and (-)TS may produce this unbalanced relaxation of DNA. We postulate that, while quick relaxation of (+)TS may facilitate the progression of RNA and DNA polymerases, slow relaxation of (-)TS may serve to favor DNA unwinding and other structural transitions at specific regions often required for genomic transactions.

Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage.

By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.

Nucleosomal DNA has topological memory.

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.

Intrinsic multiplication rate variation and plasticity of human blood stage malaria parasites.

Pathogen multiplication rate is theoretically an important determinant of virulence, although often poorly understood and difficult to measure accurately. We show intrinsic asexual blood stage multiplication rate variation of the major human malaria parasite Plasmodium falciparum to be associated with blood-stage infection intensity in patients. A panel of clinical isolates from a highly endemic West African population was analysed repeatedly during five months of continuous laboratory culture, showing a range of exponential multiplication rates at all timepoints tested, mean rates increasing over time. All isolates had different genome sequences, many containing within-isolate diversity that decreased over time in culture, but increases in multiplication rates were not primarily attributable to genomic selection. New mutants, including premature stop codons emerging in a few isolates, did not attain sufficiently high frequencies to substantially affect overall multiplication rates. Significantly, multiplication rate variation among the isolates at each of the assayed culture timepoints robustly correlated with parasite levels seen in patients at clinical presentation, indicating innate parasite control of multiplication rate that contributes to virulence.

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.

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.

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.