Monitoring early S-phase origin firing and replication fork movement by sequencing nascent DNA from synchronized cells

Macheret, Morgane; Halazonetis, Thanos D.

doi:10.1038/s41596-018-0081-y

Cited by 26 publications

(27 citation statements)

References 25 publications

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“…MiDAS permits CFSs to be mapped at high resolution on the human genome Previously, the study of MiDAS was limited to inspection of EdUpositive foci on mitotic chromosomes. 37,44 Using a variant of EdUseq, 52 we mapped the genomic coordinates of MiDAS at high resolution. Depending on the cells studied, the number of MiDAS regions ranged between 36 and 293.…”

Section: Discussionmentioning

confidence: 99%

“…Biotinylation and isolation of EdU-labeled DNA was then performed as described previously. 52,53 Briefly, after fixation, the cells were permeabilized with 0.2% Triton-X in PBS, and EdU was attached to a cleavable biotin linker (Azide-PEG(3 + 3)-S-S-biotin) (Jena Biosciences, Cat. No.…”

Section: Midasmentioning

confidence: 99%

“…Sequencing read alignment and processing Sequencing reads were aligned and processed as described in detail previously. 52 Briefly, the Burrows-Wheeler Aligner software was used to align the reads on the masked human genome assembly (GRCh37/hg19) retaining only the reads with the highest quality score. Custom Perl scripts, all provided previously, 52 were then used to assign the aligned reads to 10 kb genomic bins; the data were normalized, sigma values were calculated and the data visualized, as described previously.…”

Section: Midasmentioning

confidence: 99%

“…52 Briefly, the Burrows-Wheeler Aligner software was used to align the reads on the masked human genome assembly (GRCh37/hg19) retaining only the reads with the highest quality score. Custom Perl scripts, all provided previously, 52 were then used to assign the aligned reads to 10 kb genomic bins; the data were normalized, sigma values were calculated and the data visualized, as described previously. 52 Identification and classification of MiDAS peaks A custom Perl script was used to identify genomic regions with sigma values that were significantly higher than the sigma values of the background.…”

Section: Midasmentioning

confidence: 99%

“…Here, we studied aphidicolin-treated cells using EdUseq 52 to map the genomic regions that remain under-replicated prior to mitotic entry and the genomic sites undergoing MiDAS. Our analysis revealed that the main function of MiDAS is to help complete replication of the genome.…”

Section: Introductionmentioning

confidence: 99%

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High-resolution mapping of mitotic DNA synthesis regions and common fragile sites in the human genome through direct sequencing

et al. 2020

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DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.

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Section: Discussionmentioning

confidence: 99%

Section: Midasmentioning

confidence: 99%