Topoisomerase II binds nucleosome-free DNA and acts redundantly with topoisomerase I to enhance recruitment of RNA Pol II in budding yeast.

DNA topoisomerases are believed to promote transcription by removing excessive DNA supercoils produced during elongation. However, it is unclear how topoisomerases in eukaryotes are recruited and function in the transcription pathway in the context of nucleosomes. To address this problem we present high-resolution genome-wide maps of one of the major eukaryotic topoisomerases, Topoisomerase II (Top2) and nucleosomes in the budding yeast, Saccharomyces cerevisiae. Our data indicate that at promoters Top2 binds primarily to DNA that is nucleosome-free. However, although nucleosome loss enables Top2 occupancy, the opposite is not the case and the loss of Top2 has little effect on nucleosome density. We also find that Top2 is involved in transcription. Not only is Top2 enriched at highly transcribed genes, but Top2 is required redundantly with Top1 for optimal recruitment of RNA polymerase II at their promoters. These findings and the examination of candidate-activated genes suggest that nucleosome loss induced by nucleosome remodeling factors during gene activation enables Top2 binding, which in turn acts redundantly with Top1 to enhance recruitment of RNA polymerase II.

RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli.

The superhelicity of the chromosome, which is controlled by DNA topoisomerases, modulates global gene expression. Investigations of transcriptional responses to the modulation of gyrase function have identified two types of topoisomerase-mediated transcriptional responses: (i) steady-state changes elicited by a mutation in gyrase, such as the D82G mutation in GyrA, and (ii) dynamic changes elicited by the inhibition of gyrase. We hypothesize that the steady-state effects are due to the changes in biochemical properties of gyrase, whereas the dynamic effects are due to an imbalance between supercoiling and relaxation activities, which appears to be influenced by the RecA activity. Herein, we present biochemical evidence for hypothesized mechanisms. GyrA D82G gyrase exhibits a reduced supercoiling activity. The RecA protein can influence the balance between supercoiling and relaxation activities either by interfering with the activity of DNA gyrase or by facilitating the relaxation reaction. RecA has no effect on the supercoiling activity of gyrase but stimulates the relaxation activity of topoisomerase I. This stimulation is specific and requires formation of an active RecA filament. These results suggest that the functional interaction between RecA and topoisomerase I is responsible for RecA-mediated modulation of the relaxation-dependent transcriptional activity of the Escherichia coli chromosome.

Analysis of pleiotropic transcriptional profiles: a case study of DNA gyrase inhibition.

Genetic and environmental perturbations often result in complex transcriptional responses involving multiple genes and regulons. In order to understand the nature of a response, one has to account for the contribution of the downstream effects to the formation of a response. Such analysis can be carried out within a statistical framework in which the individual effects are independently collected and then combined within a linear model. Here, we modeled the contribution of DNA replication, supercoiling, and repair to the transcriptional response of inhibition of the Escherichia coli gyrase. By representing the gyrase inhibition as a true pleiotropic phenomenon, we were able to demonstrate that: (1) DNA replication is required for the formation of spatial transcriptional domains; (2) the transcriptional response to the gyrase inhibition is coordinated between at least two modules involved in DNA maintenance, relaxation and damage response; (3) the genes whose transcriptional response to the gyrase inhibition does not depend on the main relaxation activity of the cell can be classified on the basis of a GC excess in their upstream and coding sequences; and (4) relaxation by topoisomerase I dominates the transcriptional response, followed by the effects of replication and RecA. We functionally tested the effect of the interaction between relaxation and repair activities, and found support for the model derived from the microarray data. We conclude that modeling compound transcriptional profiles as a combination of downstream transcriptional effects allows for a more realistic, accurate, and meaningful representation of the transcriptional activity of a genome.

Spatial patterns of transcriptional activity in the chromosome of Escherichia coli.

BACKGROUND: Although genes on the chromosome are organized in a fixed order, the spatial correlations in transcription have not been systematically evaluated. We used a combination of genomic and signal processing techniques to investigate the properties of transcription in the genome of Escherichia coli K12 as a function of the position of genes on the chromosome. RESULTS: Spectral analysis of transcriptional series revealed the existence of statistically significant patterns in the spatial series of transcriptional activity. These patterns could be classified into three categories: short-range, of up to 16 kilobases (kb); medium-range, over 100-125 kb; and long-range, over 600-800 kb. We show that the significant similarities in gene activities extend beyond the length of an operon and that local patterns of coexpression are dependent on DNA supercoiling. Unlike short-range patterns, the formation of medium and long-range transcriptional patterns does not strictly depend on the level of DNA supercoiling. The long-range patterns appear to correlate with the patterns of distribution of DNA gyrase on the bacterial chromosome. CONCLUSIONS: Localization of structural components in the transcriptional signal revealed an asymmetry in the distribution of transcriptional patterns along the bacterial chromosome. The demonstration that spatial patterns of transcription could be modulated pharmacologically and genetically, along with the identification of molecular correlates of transcriptional patterns, offer for the first time strong evidence of physiologically determined higher-order organization of transcription in the bacterial chromosome.