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REPOSITORIES: ENA
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Project description:Multiple replication abnormalities cause cells lacking BRCA2 to enter mitosis with under-replicated DNA and to activate mitotic DNA synthesis (MiDAS). However, the precise position of these MiDAS sites, as well as their origin, remains unknown. Here we labelled mitotic nascent DNA and performed high-throughput sequencing to identify at high-resolution the sites where MiDAS occurs in the absence of BRCA2. This approach revealed 150 genomic loci affected by MiDAS, which map within regions replicating during early S-phase and are therefore distinct from the aphidicolin-induced common fragile sites. Moreover, these sites largely localise near early firing origins and within genes transcribed in early S, suggesting that they stem from transcription-replication conflicts (TCRs). Inhibiting transcription with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) during early S-phase abrogates MiDAS. Strikingly, MiDAS sites co-localise with genomic loci where R-loops form in unchallenged conditions, suggesting that R-loop accumulation caused by BRCA2 inactivation leads to DNA lesion which are repaired by MiDAS. RAD52 is required in this process, as its abrogation in BRCA2-deficient cells reduces the rate of MiDAS and causes DNA damage accumulation in G1. Furthermore, MiDAS sites triggered by BRCA2 inactivation are hotspots for genomic rearrangement in BRCA2-mutated breast tumours. These results indicate that BRCA2 acts in early S-phase to protect TRC- and R-loop-induced DNA lesions, thereby preventing them from becoming a source of genomic instability and tumorigenesis.
2022-08-01 | GSE196163 | GEO
Project description:DNA replication stress is an established driver of cancer-associated chromosomal rearrangements. Replication stress perturbs the duplication of late-replicating loci and activates a mitotic DNA repair pathway (termed MiDAS) for completion of replication. We here investigated RAD51-independent MiDAS.
2022-06-27 | GSE196278 | GEO
Project description:Genomic DNA from pools of H. pylori strain G27 Clones as indicated (pools of 300 (300p) or insertions in specific mapped genes) were amplifed using the MATT method to label DNA adjacent to the site of transposon insertion with the primer pairs indicated. The left side of the transposon was labeled in the Cy3 channel (Primer S) and the right side of the transposon was labeled in the Cy5 channel (Primer N). Keywords: reference_design
2005-08-26 | GSE3197 | GEO
Project description:Investigation of detection limits and the influence of DNA extraction and primer choice on the observed microbial communities in drinking water samples using 16S rRNA gene amplicon sequencing.
| PRJEB26658 | ENA
Project description:Human PDCD4 wild-type (wt) promoter fragments were amplified from U2OS genomic DNA and X-box deletion mutants (mut) were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies). DNA probes for affinity purification with the same sequences were obtained by PCR using a biotinylated primer for labeling the 3' end (Thermo Fisher Scientific). DNA affinity purification with nuclear extracts from Nutlin-3a treated U2OS cells.
2024-08-08 | PXD020932 | Pride