Topoisomerase 1 dependent R-loop deficiency drives accelerated replication and genomic instability
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE168738 | GEO | 2022/09/08
REPOSITORIES: GEO
Similar Datasets
Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis
2007-08-01 | GSE8305 | GEO
Project description:DNA topological stress inhibits DNA replication fork (RF) progression and contributes to DNA replication stress. In Saccharomyces cerevisiae we demonstrate that centromeric DNA and the rDNA array are especially vulnerable to DNA topological stress during replication. The activity of the SMC complexes cohesin and condensin are linked to both the generation and repair of DNA topological stress linked damage in these regions. At cohesin enriched centromeres cohesin activity causes the accumulation of DNA damage, RF rotation and precatenation, confirming that cohesin dependent DNA topological stress impacts on normal replication progression. In contrast, at the rDNA cohesin and condensin activity inhibit the repair of damage caused by DNA topological stress. We propose that as well as generally acting to ensure faithful genetic inheritance, SMCs can disrupt genome stability by trapping DNA topological stress.
2020-03-03 | GSE131558 | GEO
Project description:To ensure efficient genome duplication, cells have evolved a multitude of factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in individuals with microcephalic dwarfism. We demonstrate that DONSON is a component of the replisome that stabilises forks during normal genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent ,signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability
2017-03-28 | MSV000080704 | MassIVE
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
Project description:Top1 and Top2-mediated replication fork integrity
| PRJNA101273 | ENA
Project description:DNA topoisomerases solve topological problems during chromosome metabolism. We investigated where and when Top1 and Top2 are recruited on replicating chromosomes and how their inactivation affects fork integrity and DNA damage checkpoint activation. We show that, in the context of replicating chromatin, Top1 and Top2 act within a 600 bp region spanning the moving forks. Top2 exhibits additional S-phase clusters at specific intergenic loci, mostly containing promoters. TOP1 ablation does not affect fork progression and stability and does not cause activation of the Rad53 checkpoint kinase. top2 mutants accumulate sister chromatid junctions in S phase without affecting fork progression and activate Rad53 at the M/G1 transition. top1 top2 double mutants exhibit fork block and processing, and phosphorylation of Rad53 and γH2A in S phase. The exonuclease Exo1 influences fork processing and DNA damage checkpoint activation in top1 top2 mutants. Our data are consistent with a coordinated action of Top1 and Top2 in counteracting the accumulation of torsional stress and sister chromatid entanglement at replication forks, thus preventing the diffusion of topological changes along large chromosomal regions. A failure in resolving fork-related topological constrains during S phase may therefore result in abnormal chromosome transitions, DNA damage checkpoint activation and chromosome breakage during segregation. Keywords: ChIP-chip analysis S. cerevisiae chromosomes IIIâV, and chromosome VI highdensity oligonucleotide microarrays were provided by Affymetrix Custom Express Service (SC3456a520015F, P/N 520015; rikDACF, P/N 510636, respectively). Sequence and position of oligonucleotides on the microarrays are available from Affymetrix. ChIP was carried out as previously described (Katou et al. 2003; Katou et al. 2006): we disrupted 1.5 x 108 cells byMulti-beads shocker (MB400U, Yasui Kikai) using glass beads. Anti-HA monoclonal antibody HA.11 (16B12) (CRP Inc.) and anti-Flag monoclonal antibody M2 (Sigma-Aldrich) were used for chromatin immunoprecipitation. ChIPed DNA was purified and amplified by random priming as described (Katou et al. 2003): a total of 10 μg of amplified DNA was digested with DNaseI to a mean size of 100 bp, purified, and the fragments were end-labelled with biotin-N6-ddATP23. Hybridization, washing, staining and scanning were performed according to the manufacturerâs instruction (Affymetrix). Primary data analyses were carried out using the Affymetrix microarray Suite version 5.0 software to obtain hybridization intensity, fold change value, change P-value and detection P-value for each locus. For the discrimination of positive and negative signals for the binding, we compared ChIPed fraction with supernatant fraction by using three criteria. First, the reliability of strength of signal was judged by detection P-value of each locus (P â¥0.025). Second, reliability of binding ratio was judged by change P-value (P ⥠0.025). Third, clusters consisting of at least three contiguous loci that filled the above Bermejo et al. 26 two criteria were selected, because it was known that a single site of proteinâDNA interaction will result in immunoprecipitation of DNA fragments that hybridized not only to the locus of the actual binding site but also to its neighbours. For the analyses of BrdU incorporation, cells were fixed by ice-cold buffer containing 0.1% azide, and then total DNA from 3 x108 cells was purified. DNA was sheared to 300 bp by sonication, denatured, and mixed with 2μg anti-BrdU monoclonal antibody (2B1D5F5H4E2; MBL). Antibody-bound and unbound fractions were subsequently purified, amplified, labelled and hybridized to the DNA chip.
2010-06-05 | E-GEOD-8305 | biostudies-arrayexpress
Project description:To ensure efficient genome duplication, cells have evolved a multitude of factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in individuals with microcephalic dwarfism. We demonstrate that DONSON is a component of the replisome that stabilises forks during normal genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent ,signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability
2017-02-10 | PXD005690 | Pride
Project description:Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate - a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc) - is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase, TDP1, which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3’ phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. We find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.
2020-03-13 | PXD017239 | Pride
Project description:R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, we have mapped RNA:DNA hybrids, replication stress markers and DNA double strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids were found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR was only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also cumulated at TTS, leading to persistent checkpoint activation, spreading of -H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.
2020-06-20 | GSE108172 | GEO
Project description:Topoisomerase I (Top1) is a key enzyme acting at the interface between DNA replication, transcription and mRNA maturation. Here, we show that Top1 suppresses genomic instability in mammalian cells by preventing conflicts between transcription and DNA replication. Using DNA combing and ChIP-on-chip, we found that Top1-deficient cells accumulate stalled replication forks and chromosome breaks in S phase and that breaks occur preferentially at gene-rich regions of the genome. Strikingly, these phenotypes were suppressed by preventing the formation of RNA-DNA hybrids (R-loops) during transcription. Moreover, these defects could be mimicked by depletion of the splicing factor ASF/SF2, which interacts functionally with Top1. Taken together, these data indicate that Top1 prevents replication fork collapse by suppressing the formation of R-loops in an ASF/SF2-dependent manner. We propose that interference between replication and transcription represents a major source of spontaneous replication stress, which could drive genomic instability during early stages of tumorigenesis. Bed files contain the gamma-H2AX enrichment sites described in the paper
2009-08-08 | GSE17552 | GEO