Genome-wide mapping of 8-oxo-7,8-dihydro-2′-deoxyguanosine reveals accumulation of oxidatively-generated damage at DNA replication origins within transcribed long genes of mammalian cells

Abstract

Abstract8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is one of the major DNA modifications and a potent pre-mutagenic lesion prone to mispair with 2′-deoxyadenosine (dA). Several thousand residues of 8-oxodG are constitutively generated in the genome of mammalian cells, but their genomic distribution has not yet been fully characterized. Here, by using OxiDIP-Seq, a highly sensitive methodology that uses immuno-precipitation with efficient anti–8-oxodG antibodies combined with high-throughput sequencing, we report the genome-wide distribution of 8-oxodG in human non-tumorigenic epithelial breast cells (MCF10A), and mouse embryonic fibroblasts (MEFs). OxiDIP-Seq revealed sites of 8-oxodG accumulation overlapping with γH2AX ChIP-Seq signals within the gene body of transcribed long genes, particularly at the DNA replication origins contained therein. We propose that the presence of persistent single-stranded DNA, as a consequence of transcription-replication clashes at these sites, determines local vulnerability to DNA oxidation and/or its slow repair. This oxidatively-generated damage, likely in combination with other kinds of lesion, might contribute to the formation of DNA double strand breaks and activation of DNA damage response.

Targeted DNA oxidation by LSD1–SMAD2/3 primes TGF-β1/ EMT genes for activation or repression

“…Immunocytochemistry was performed according to the manufacturer’s instructions (Trevigen Inc.). The anti-8-oxo-dG antibodies (mouse monoclonal 15A3) were validated by confocal microscopy ( 16 ) and chromatin immunoprecipitation (ChIP) ( 17 ). The signal was resistant to RNase A and sensitive to DNase I and NAC.…”

Next-generation DNA damage sequencing

“…As one example, OxiDIP-seq used an 8-oxodG antibody for immunoprecipitation followed by high-throughput sequencing in human non-tumorigenic epithelial breast cells and mouse embryonic fibroblasts. 55,56 The sequencing revealed that 8-oxodG sites accumulated in the transcribed regions of long genes and at DNA replication origins, overlapping with gH2AX ChIP-seq signals and double-strand breaks. Furthermore, a strong reduction of 8-oxodG was observed within promoter regions with high GC content in quiescent (G0) cells without DNA replication.…”

ELIXIR-IT HPC@CINECA: high performance computing resources for the bioinformatics community

“…This computational effort led to further insights about the molecular mechanisms underlying the heterogeneity of the local mutation rate and the understanding of why certain regions seem to be more, while others less, prone to oxidation. A full description of this work can be found in [19].…”