DNA topoisomerase and supercoil accumulation across yeast genome
Ontology highlight
ABSTRACT: DNA topoisomerases assist DNA replication & transcription events by controlling supercoiling alterations. We investigated supercoil distribution across the yeast genome and compared with the accumulation of RNA pol2 and DNA topoisomerases particularly in S-phase. Our data indicate that Top2 along with Hmo1 maintain negative supercoil at gene boundaries by stabilizing alternative DNA structures. To understand how DNA superhelical tension accumulates across the genome we have adopted previously described method [Naughton C et al., 2013] to budding yeast where a biotin molecule was attached to TMP via a linker (bTMP). The Chip on chip analysis for proteins was carried out as described (Bermejo R et al., 2009). For RNA-DNA hybrids DRIP-chip is carried out as described previously (Chan YA et al., 2014). Supercoiled regions are then compared with RNA pol2 (RPB3-chip), DNA Topoisomerase (Top1-chip) & RNA-DNA hybrid (DRIP-chip).
Project description:DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability. BrdU and proteins ChIP-chip analyses analysis were carried out as described (Bermejo et al., 2009). Labelled probes were hybridized to Affymetrix S.cerevisiae Tiling 1.0 (P/N 900645) arrays and processed with TAS software.
Project description:Human acute myeloid leukemia (AML) KG1a cells or mouse BM cells (mouse bone morrow cells were employed for each ChIP assay. The ChIP procedure was performed according to a previously described protocol (Lee et al., 2006; Ying et al., 2017), using anti-ATF4 antibody (Abcam)
Project description:We have performed a genome wide investigation for the binding locations of the transcriptional co-repressor proteins Ssn6, Tup11 and Tup12 in the fission yeast Schizosaccharomyces pombe. We have used a ChIP protocol described previously (Robyr et al, 2003) with microarrays containing ORF and IGR fragments representing the complete fission yeast genome (Wiren et al, 2005). Keywords: ChIP-CHIP
Project description:We have performed a genome wide investigation for the binding locations of the transcriptional co-repressor proteins Ssn6, Tup11 and Tup12 in the fission yeast Schizosaccharomyces pombe. We have used a ChIP protocol described previously (Robyr et al, 2003) with microarrays containing ORF and IGR fragments representing the complete fission yeast genome (Wiren et al, 2005). Keywords: ChIP-CHIP
Project description:We have performed a genome wide investigation for the binding locations of the transcriptional co-repressor proteins Ssn6, Tup11 and Tup12 in the fission yeast Schizosaccharomyces pombe. We have used a ChIP protocol described previously (Robyr et al, 2003) with microarrays containing ORF and IGR fragments representing the complete fission yeast genome (Wiren et al, 2005). Keywords: ChIP-CHIP
Project description:High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must somehow manage these high levels of torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with Topoisomerases 1 (TOP1) and 2A (TOP2A) that was confirmed in vitro and in vivo. Beyond recruiting topoisomerases, MYC directly stimulates their activities in vitro and in vivo. We identify a MYC-nucleated “topoisome” complex that unites and increases the levels of both both TOP1 and TOP2, as well as their activities at promoters and enhancers. Whether TOP2A or TOP2B is included in the topoisome, is dictated by the presence of c-MYC or MYCN, respectively. Thus, in vivo and in vitro, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.
Project description:High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must somehow manage these high levels of torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with Topoisomerases 1 (TOP1) and 2A (TOP2A) that was confirmed in vitro and in vivo. Beyond recruiting topoisomerases, MYC directly stimulates their activities in vitro and in vivo. We identify a MYC-nucleated “topoisome” complex that unites and increases the levels of both both TOP1 and TOP2, as well as their activities at promoters and enhancers. Whether TOP2A or TOP2B is included in the topoisome, is dictated by the presence of c-MYC or MYCN, respectively. Thus, in vivo and in vitro, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.
Project description:High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must somehow manage these high levels of torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with Topoisomerases 1 (TOP1) and 2A (TOP2A) that was confirmed in vitro and in vivo. Beyond recruiting topoisomerases, MYC directly stimulates their activities in vitro and in vivo. We identify a MYC-nucleated “topoisome” complex that unites and increases the levels of both both TOP1 and TOP2, as well as their activities at promoters and enhancers. Whether TOP2A or TOP2B is included in the topoisome, is dictated by the presence of c-MYC or MYCN, respectively. Thus, in vivo and in vitro, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.