Complex Genomic Patterns of Abasic Sites in Mammalian DNA Revealed by High-Resolution SSiNGLe-AP Method
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PROVIDER: PRJNA789172 | ENA |
REPOSITORIES: ENA
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Project description:DNA damage plays critical role in biology and disease, however, precise understanding of how different types of DNA damage affect cellular functions is far from clear. A major underlying reason for this is paucity of high-resolution methods that can map locations of different types of DNA damage in complex genomes such as those of mammals. Here, we present development and validation of SSiNGLe-AP method that can map a common type of DNA damage, abasic (AP) sites, genome-wide and with high-resolution. We applied this method to six different tissues of mice of different ages and human cancer cell lines. We found non-random distribution of AP sites in mammalian genome that exhibits dynamic enrichment at specific genomic locations, and is significantly influenced by gene expression, age and especially by tissue-type. Overall, these results suggest that we are at the very beginning of understanding the true complexities of genomic patterns of DNA damage.
2022-09-15 | GSE190955 | GEO
Project description:Abasic (AP) sites are one of the most common forms of DNA damage which can lead to polymerase stalling, strand breaks and mutations. We developed snA-seq, a mapping method that reveals the location of abasic sites at base-resolution. Using synthetic DNA, we show that high selectivity for AP DNA is achieved. We use this method to explore the distribution of thymine modifications in the Leishmania major genome, by converting these into abasic sites using a glycosylase enzyme. We also apply snAP-seq to the human genome to study the distribution of endogenous AP sites, in both APE1 knockdown and control cells.
2019-06-12 | E-MTAB-7152 | biostudies-arrayexpress
Project description:The AP-seq provides a genome-wide method for detecting abasic sites among the human and mouse genomes Purpose: Abasic sites are one of the most frequent DNA damage among the genome , which often carries harmful consequences for the genome stability and the normal cell functions. So it is necessary to develop a whole genome abasic sites (AP sites) profiling method to detect all the AP sites among the genome, which we called the AP-seq. Methods: The human and mouse genome are processed with enzymatic fragmentation, damage repair, biotin-dU labeling, and pull-down steps. After the sequencing library preparation, the FASTQ files are produced by the Illumina HiSeq XTen platform. To guarantee the reproducibility, each case of the experiment is present with two biological replicates. Results: The reproducibility of our two biological replicates shows a high correlation which larger than 0.95. And the overlap AP site peaks between the HEK293T and MCF-7 cell line are over 50%, which show a great similarity between the different cell lines. Totally, we identified more than 2000 and 1000 abasic site peaks in human, and mouse cell lines separately. Moreover, the distribution of these AP sites prefer to locate in the intergenic regions and introns.
2020-12-01 | GSE125254 | GEO
Project description:We developed a novel method, AP-seq, capable of mapping apurinic sites and 8-oxo-7,8-dihydroguanine bases at approximately 250bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of apurinic sites varies widely across the genome, with hot spots acquiring many times more damage than cold spots. The experiment was performed in triplicates on HepG2 cells, 30 min after X-ray treatment (6Gy). AP-Seq is an approach that specifically enriches AP-sites via a biotin-labelled aldehyde-reactive probe under pH neutral conditions, which has been well established for the specific detection of AP-sites since its development by Kubo et al. in 1992. Under neutral conditions (pH7), 1h at 37ºC, the probe is highly specific for the aldehydes occurring at AP-sites. After biotin labelling of genomic DNA, DNA is sonicated into ~250 bp fragments, enriched, in vitro repaired (PreCR, NEB), and used for Illumina short read sequencing (125 bp, paired end, HiSeq2000). To assess oxidative damage as the sum of AP-sites and 8-oxoG, we applied recombinant OGG1 in vitro to the extracted DNA (OGG1-AP-seq). Under the conditions chosen, any remaining 8-oxoG is excised in a largely sequence-independent fashion after DNA extraction to result in a set of secondary AP-sites and to a lesser extent the associated beta-elimination product.
2018-10-22 | GSE121005 | GEO
Project description:Understanding the genomic locations of DNA modifications is pivotal for deciphering their roles in gene regulation, mutagenesis, and pathology. The affinity probe-based enrichment of lesion-containing DNA represents a key strategy for sequencing DNA modifications. Existing methods are limited in the specificity of labeling and enrichment of abasic (AP) sites, a prevalent DNA modification and repair intermediate. Herein, we devise a novel approach, termed dual chemical labeling-assisted sequencing (DCL-seq), for mapping AP sites. DCL-seq features two designer compounds for enriching and mapping AP sites specifically at single-nucleotide resolution. For proof of principle, we mapped AP sites in mitochondrial DNA (mtDNA) from cultured human cells under different biological conditions. Also, we demonstrated the broader applicability of the method in sequencing additional DNA modifications in mtDNA, such as N7-methyl-2'-deoxyguanosine and N3-methyl-2'-deoxyadenosine, when coupled with a lesion-specific repair enzyme. Together, DCL-seq holds the promise to sequence multiple DNA modifications in various biological samples.
2022-09-24 | GSE213585 | GEO
Project description:Genome-wide maps of apurinic sites (AP-sites) and 8-oxoG derived secondary AP-sites
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Project description:This phase I trial is studying the best dose of 3-AP and the side effects of giving 3-AP together with gemcitabine in treating patients with advanced solid tumors or lymphoma. Drugs used in chemotherapy, such as 3-AP and gemcitabine (GEM), work in different ways to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. 3-AP may help gemcitabine kill more cancer cells by making the cells more sensitive to the drug. 3-AP may also stop the growth of tumor cells by blocking some of the enzymes needed for cell growth.
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Project description:The polarization and activation of macrophages are controlled synergistically by transcription factors such as NF-κB and AP-1 transcription factor members. Surprisingly, little is known about the role of the AP-1 member, Fra-1, during macrophage responses. To determine the full spectrum of Fra-1 binding sites in macrophages, chromatin immunoprecipitation sequencing analysis was performed in macrophages pooling down for the transcription factor Fra-1.
2021-06-25 | GSE178865 | GEO
Project description:Promiscuous labeling enzymes, such as APEX2 or TurboID, are commonly used in in situ biotinylation studies of subcellular proteomes or protein-protein interactions. Although the conventional approach of enriching biotinylated proteins is widely implemented, in-depth identification of specific biotinylation sites remains challenging, and current approaches are technically demanding with low yields. A novel method to systematically identify specific biotinylation sites for LC-MS analysis followed by proximity labeling showed excellent performance compared with that of related approaches in terms of identification depth with high enrichment power. The systematic identification of biotinylation sites enabled a simpler and more efficient experimental design to identify subcellular localized proteins within membranous organelles. Applying this method to the processing body (PB), a non-membranous organelle, successfully allowed unbiased identification of PB core proteins, including novel candidates. We anticipate that our newly developed method will replace the conventional method for identifying biotinylated proteins labeled by promiscuous labeling enzymes.
2024-05-22 | PXD047979 | Pride