Project description:This SuperSeries is composed of the following subset Series: GSE18240: Saccharomyces cerevisiae cells: control vs positive supercoiling accumulation after 0, 30 and 120 min GSE18241: S. cerevisiae cells: control vs positive supercoiling accumulation in absence of telomere silencing after 0 and 120 min GSE18605: Saccharomyces cerevisiae cells: effect of Top2 depletion without accumulation of positive superhelical stress Refer to individual Series
Project description:Positive DNA helical stress accumulates in vivo by the unbalanced relaxation of positive and negative DNA supercoils in M-NM-^Ttop1, top2ts, pGPD:TopA yeast cells. The resulting overwinding of DNA greatly diminishes overall RNA synthesis. Here we show that whereas most genes reduce their transcript levels by several fold, genes situated at less than 100 kb from the chromosomal ends (near 15% of the genome) are gradually unaffected. This positional effect denotes that chromosomal ends are topologically open, thus precluding the accumulation of DNA helical stress in telomere-proximal regions. The progressive escape from the transcription stall observed in all the chromosome extremities indicates also that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, suffice to confine DNA helical tension along eukaryotic chromatin. Keywords: Time course of positive helical tension accumulation in absence of telomere silencing. Two-condition experiment: delta top1 delta sir3-TOPA vs delta top1 delta sir3 top2-ts-TOPA. 2 time points: 0min, 120min. 3 biological replicates per condition and time point, independently grown in leu- selective media and harvested.
Project description:Positive DNA helical stress accumulates in vivo by the unbalanced relaxation of positive and negative DNA supercoils in Δtop1, top2ts, pGPD:TopA yeast cells. The resulting overwinding of DNA greatly diminishes overall RNA synthesis. Here we show that whereas most genes reduce their transcript levels by several fold, genes situated at less than 100 kb from the chromosomal ends (near 15% of the genome) are gradually unaffected. This positional effect denotes that chromosomal ends are topologically open, thus precluding the accumulation of DNA helical stress in telomere-proximal regions. The progressive escape from the transcription stall observed in all the chromosome extremities indicates also that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, suffice to confine DNA helical tension along eukaryotic chromatin. Keywords: Time course of positive helical tension accumulation in absence of telomere silencing.
Project description:In this project, we follow the temporal proteome dynamics during heat stress response in a model organism Saccharomyces cerevisiae. Using label-free quantification we determine changes in the yeast proteome at different times after mild temperature shift, from 30˚C to 37˚C. The time points measured were 0 min (30˚C, no stress) and then at 10 min, 30 min, 60 min, 120 min and 240 min after cells were transferred to 37 °C. For each time point, four biological replicates were collected for the haploid wild type (BY4742, Mat ALPHA, his3Δ1; leu2Δ0; lys2Δ0; ura3Δ0; YJL088w::kanMX4) strain and a haploid strain with a chaperone Ssb1p deletion (ssb1Δ).
Project description:Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, replication-transcription encounters, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach that can be applied in any organism to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.
Project description:Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three- dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.
Project description:Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three-dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and enriched between convergently-oriented genes, consistent with the ?twin-domain? model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, replication-transcription encounters, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach that can be applied in any organism to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.
Project description:Supercoiling impacts DNA replication, transcription, protein binding to DNA, and the three- dimensional organization of chromosomes. However, there are currently no methods to directly interrogate or map positive supercoils, so their distribution in genomes remains unknown. Here, we describe a method, GapR-seq, based on the chromatin immunoprecipitation of GapR, a bacterial protein that preferentially recognizes overtwisted DNA, for generating high-resolution maps of positive supercoiling. Applying this method to E. coli and S. cerevisiae, we find that positive supercoiling is widespread, associated with transcription, and particularly enriched between convergently-oriented genes, consistent with the “twin-domain” model of supercoiling. In yeast, we also find positive supercoils associated with centromeres, cohesin binding sites, autonomously replicating sites, and the borders of R-loops (DNA-RNA hybrids). Our results suggest that GapR-seq is a powerful approach, likely applicable in any organism, to investigate aspects of chromosome structure and organization not accessible by Hi-C or other existing methods.