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High-resolution mapping of mitotic DNA synthesis under conditions of replication stress in cultured cells.

ABSTRACT: Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we describe a protocol to identify at genome wide and at high resolution the genomic sites where MiDAS occurs in cells exposed to aphidicolin. We use EdU incorporation to label nascent DNA in mitotic cells, followed by isolation of the EdU-labeled DNA and next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Groelly et al. (2022)¹ and Macheret et al. (2020).².

SUBMITTER: Groelly FJ

PROVIDER: S-EPMC9826876 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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High-resolution mapping of mitotic DNA synthesis under conditions of replication stress in cultured cells.

Groelly Florian J FJ Dagg Rebecca A RA Mailler Jonathan J Halazonetis Thanos D TD Tarsounas Madalena M

STAR protocols 20230103 1

Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we describe a protocol to identify at genome wide and at high resolution the genomic sites where MiDAS occurs in cells exposed to aphidicolin. We use EdU incorporation to label nascent DNA in mitotic cells, followed by isolation of the EdU-labeled DNA and next-generation sequencing. For complete details on the use and execution of this ...[more]

PMID: 36598851

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Project description:Genome-wide Association Studies (GWAS) identify genome variations related to specific phenotypes using Single Nucleotide Polymorphism (SNP) markers. Genotyping platforms like SNP-Array or sequencing-based techniques (GBS) can genotype samples with many SNPs. These approaches may bias tropical maize analyses due to reliance on the temperate line B73 as the reference genome. An alternative is a simulated genome called "Mock," adapted to the population using bioinformatics. Recent studies show SNP-Array, GBS, and Mock yield similar results for population structure, heterotic groups definition, tester selection, and genomic hybrid prediction. However, no studies have examined the results generated by these different genotyping approaches for GWAS. This study aims to test the equivalence among the three genotyping scenarios in identifying significant effect genes in GWAS. To achieve this, maize was used as the model species, where SNP-Array genotyped 360 inbred lines from a public panel via the Affymetrix platform and GBS. The GBS data were used to perform SNP calling using the temperate inbred line B73 as the reference genome (GBS-B73) and a simulated genome "Mock" obtained in-silico (GBS-Mock). The study encompassed four above-ground traits with plants grown under two levels of water supply: well-watered (WW) and water-stressed (WS). In total, 46, 34, and 31 SNP were identified in the SNP-Array, GBS-B73, and GBS-Mock scenarios, respectively, across the two water levels, associated with the evaluated traits following the comparative analysis of each genotyping method individually. Overall, the identified candidate genes varied along the various scenarios but had the same functionality. Regarding SNP-Array and GBS-B73, genes with functional similarity were identified even without coincidence in the physical position of the SNPs. These genes and regions are involved in various processes and responses with applications in plant breeding. In terms of accuracy, the combination of genotyping scenarios compared to those isolated is feasible and recommended, as it increased all traits under both water conditions. In this sense, it is worth highlighting the combination of GBS-B73 and GBS-Mock scenarios, not only due to the increase in the resolution of GWAS results but also the reduction of costs associated with genotyping and the possibility of conducting genomic breeding methods.

Dataset Information

High-resolution mapping of mitotic DNA synthesis under conditions of replication stress in cultured cells.

Publications

High-resolution mapping of mitotic DNA synthesis under conditions of replication stress in cultured cells.

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