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Mapping Replication Fork Pause Sites in S. cerevisiae

ABSTRACT: Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. We alse mapped pause sites using a second replication fork component, Rrm3-13MYC and got similar results.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE16218 | GEO | 2009/06/15

SECONDARY ACCESSION(S): PRJNA117157

REPOSITORIES: GEO

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Mapping Replication Fork Pause Sites in S. cerevisiae

Project description:Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome wide approach to identify 96 sites of very high DNA polymerase binding in wild type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared to wild type cells as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are the first class of natural pause sites that are not exacerbated in rrm3 cells. We alse mapped pause sites using a second replication fork component, Rrm3-13MYC and got similar results. Genomic input (labelled with Cy3) and IP'ed DNA (labelled with Cy5) using a MYC Ab of either DNA Pol2-13MYC or Rrm3-MYC from asynchronously grown S. cerevisiae cells in rich media were hybridized to whole-genome PCR-based arrays containing ORF and intergenic regions of the entire genome (Ivery et al 2001). At least three biological replication and one technical replicate (dye swap) were performed. Log2 transformed median normalized ratios (IP/IN) were averaged for each experiment and significant peaks of either DNA Pol2 or Rrm3 association were identified

2009-06-15 | E-GEOD-16218 | biostudies-arrayexpress

Rad53-mediated regulation of Rrm3 and Pif1 DNA helicases contributes to prevention of aberrant fork transitions under replication stress.

Project description:Replication stress activates the Mec1ATR and Rad53 kinases. Rad53 phosphorylates nuclear pores to counteract gene gating, thus preventing aberrant transitions at forks approaching transcribed genes. Here, we show that Rrm3 and Pif1, DNA helicases assisting fork progression across pausing sites, are detrimental in rad53 mutants experiencing replication stress. Rrm3 and Pif1 ablations rescue cell lethality, chromosome fragmentation, replisome-fork dissociation, fork reversal, and processing in rad53 cells. Through phosphorylation, Rad53 regulates Rrm3 and Pif1; phospho-mimicking rrm3 mutants ameliorate rad53 phenotypes following replication stress without affecting replication across pausing elements under normal conditions. Hence, the Mec1-Rad53 axis protects fork stability by regulating nuclear pores and DNA helicases. We propose that following replication stress, forks stall in an asymmetric conformation by inhibiting Rrm3 and Pif1, thus impeding lagging strand extension and preventing fork reversal; conversely, under unperturbed conditions, the peculiar conformation of forks encountering pausing sites would depend on active Rrm3 and Pif1. BrdU incorporation profiles by ssDNA-BrdU IP on chip have been generated as described (Katou et al., 2003). Protein binding profiles by ChIP-chip analysis were generated as described (Bermejo et al., 2009). Labeled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.

2015-09-24 | E-GEOD-68214 | biostudies-arrayexpress

Rad53-mediated regulation of Rrm3 and Pif1 DNA helicases contributes to prevention of aberrant fork transitions under replication stress.

2015-09-24 | GSE68214 | GEO

Fork Pausing Complex Engages Topoisomerases at the Replisome

Project description:Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) acts as a ‘molecular brake’ and promotes fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C-terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a ‘sTOP’ mechanism (‘slowing down with TOPoisomerases I-II’), which we show also contributes to protecting cells from topoisomerase-blocking agents.

2019-11-07 | GSE140034 | GEO

VEGF promotes RNAPII pausing release through ETS1 to stimulate angiogenesis

Project description:RNA polymerase II (RNAPII) pausing release is a recently recognized checkpoint for transcriptional regulation. The biological roles of RNAPII pausing release and the mechanisms that by which extracellular signals control it are incompletely understood. Here we identify a novel mechanism by which VEGF stimulates RNAPII pausing-release through acetylation of ETS1, a master endothelial cell transcriptional regulator. In endothelial cells (ECs), ETS1 uniquely bound transcribed gene promoters and stimulated their expression by broadly increasing RNA polymerase II (RNAPII) pause release. VEGF enhanced ETS1 chromatin occupancy. Furthermore, VEGF increased ETS1 acetylation, enhancing its binding by BRD4 and thereby stimulating RNAPII pause release. This ETS1-mediated transduction of VEGF signaling to increase RNAPII pausing release was essential for EC angiogenic responses in vitro and in vivo. Together, our results define a new angiogenic pathway in which VEGF enhances ETS1-Brd4 interaction to broadly promote RNAPII pause release and drive angiogenesis.

2016-12-31 | GSE93030 | GEO

GAGA Factor maintains promoters in nucleosome-free conformation and allows promoter-proximal pausing [MNase]

Project description:Promoter-proximal pausing of RNA polymerase II (Pol II) is a widespread in higher eukaryotes. Previous studies have shown that GAF is enriched at paused genes, but the role of GAF in pausing has not been well characterized on a genome-wide level. To investigate the role of GAF in pausing, we RNAi-depleted GAF from Drosophila S2 cells, and examined the effects on promoter-proximal polymerase. We confirmed the importance of GAF for pausing on the classic pause model gene Hsp70. To determine the dependence of pausing on GAF genome-wide, we assayed the levels of transcriptionally-engaged polymerase genome-wide using GRO-seq in control and GAF-RNAi cells. We found that promoter-proximal polymerase was significantly reduced on a subset of paused genes with GAF-bound promoters. There is a dramatic change in nucleosome distribution at genes with reduction in pausing upon GAF depletion and intergenic GAF binding sites in GAF knock-down, suggesting that GAF allows the establishment of pausing at these genes by directing nucleosome displacement off of the promoter. In addition, the insulator factor BEAF, BEAF-interacting protein Chriz, and transcription M1BP enrichment on unaffected genes suggests that redundant transcription factors or insulators protect other GAF-bound paused genes from GAF knock-down effects. Three biological replicates of MNase digested chromatin from LacZ-RNAi and GAGA factor-RNAi cells.

2015-04-04 | E-GEOD-58956 | biostudies-arrayexpress

Conserved DNA sequence features underlie pervasive RNA polymerase pausing

Project description:This study reveals widespread Pol II pausing at single-nucleotide resolution across the human genome with the majority of Pol II pauses occur outside of promoter-proximal gene regions primarily along the gene-body of transcribed genes. We show DNA sequence properties underlying widespread transcriptional pausing including a new pause motif. The study indicates pervasive sequence-induced transcriptional pausing in human cells.

2021-03-23 | GSE162857 | GEO

Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-Seq

Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-seq data for Pol II and additional factors controlling pause release in mouse ES cells.

2010-04-02 | E-GEOD-20530 | biostudies-arrayexpress

Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-chip

Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-chip data for Pol II and additional factors controlling pause release in mouse ES cells.

2010-04-02 | E-GEOD-20529 | biostudies-arrayexpress

Paused RNA polymerase II inhibits new transcriptional initiation

Project description:RNA polymerase II (Pol II) pauses downstream of the transcription initiation site before beginning productive elongation. This pause is a key component of metazoan gene expression regulation. Some promoters have a strong disposition for Pol II pausing and often mediate faster, more synchronous changes in expression. This requires multiple rounds of transcription and thus cannot rely solely on pause release. But it is unclear how pausing affects the initiation of new transcripts during consecutive rounds of transcription. Using our recently developed ChIP-nexus method, we find that Pol II pausing inhibits new initiation. We propose that paused Pol II helps prevent new initiation between transcription bursts, which may reduce noise.

2017-04-17 | GSE85741 | GEO

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