Background

DNA, as a highly dynamic molecule, is constantly exposed to mutation events (Lindahl, 1993). Among DNA mutant factors, reactive oxygen species (ROS) are the most prominent source of DNA modifications that may trigger processes such as neurodegeneration (Kim et al., 2015), ageing (Beckman and Ames, 1998), and cancer (Klaunig and Kamendulis, 2004). The 2'-deoxyguanosine (dG) is the most ROS-targeted nucleobase due to its lower oxidation potential (Steenken and Jovanovic, 1997; Baik et al., 2001). The best characterized product of dG oxidation induced by ROS is 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG; Figure 1A) (Baik et al., 2001; Cooke et al., 2003; Evans and Cooke, 2004; van Loon et al., 2010). This altered base is chemically generated by C8 oxidation and subsequent addition of a hydrogen atom on the N7 in the imidazole ring of deoxyguanosine.

The 8-oxodG is considered a premutagenic DNA lesion that may bond 2′-deoxyadenosine, thus causing a dC:dG to dA:dT premutagenic transversion (Shibutani et al., 1991; Maga et al., 2007; Batra et al., 2010; Koga et al., 2013; Boiteux et al., 2017). Human cells can survive this mutational phenomenon through the base excision repair (BER), a multi-layer defence machinery (Lindahl, 1990; Lindahl and Barnes, 2000).

The OGG1 glycosylase/AP(apurinic/apyrimidinic) lyase initiates the BER pathway, specifically recognizing and removing the 8-oxodGs from the sugar backbone. The OGG1 activity creates an abasic site, which is subsequently incised by either the OGG1 intrinsic AP lyase activity, or by the AP endonuclease 1, APE1. The lyases’ activity generates a single strand DNA (ssDNA) break. Then, the short patch BER carries on with the gap filling, mediated by the DNA polymerase beta, while the long patch BER generates a 5′ overhanging flap that is removed by FEN1 (flap structure–specific endonuclease 1). Finally, the DNA ligase I (short patch) or DNA ligase III (long patch) complete the repair process by fixing the nicked strand (Frosina et al., 1996; Fortini et al., 1999; van Loon et al., 2010).

Several next-generation sequencing (NGS)–based strategies have recently been developed by different laboratories to provide a high-resolution mapping of 8-oxodG in human and mouse genomes (Ding et al., 2017; Poetsch et al., 2018; Wu et al., 2018; Amente et al., 2019; Liu et al., 2019, 2019; Cao et al., 2020; Fang and Zou, 2020; Poetsch, 2020). The previous study developed a highly sensitive methodology named OxiDIP-Seq to isolate and map oxidized DNA fragments in mammalian cells (Amente et al., 2019; Gorini et al., 2020; Scala et al., 2022). OxiDIP-Seq combines the immuno-precipitation of single-stranded DNA through specific anti-8-oxodG antibodies with NGS, with a resolution of approximately 200–300 bp (Amente et al., 2019; Gorini et al., 2020; Scala et al., 2022).

Briefly, with this methodology, genomic DNA containing 8-oxodG residues were first extracted and then fragmented by sonication. The DNA fragments were then denatured at 95 °C to expose the 8-oxodG on the single-stranded DNA. Then, the single-stranded DNA containing 8-oxodG residues was immunoprecipitated using a specific antibody targeting 8-oxodG. The so formed immunocomplexes were then pulled down through specific magnetic beads. The eluted DNA was enriched in fragments containing 8-oxodGs that can be analyzed by qPCR and/or sequenced by high-throughput sequencing (Figure 1B). In this protocol, crucial steps were carried out in low light conditions and in the presence of free radical scavengers to preserve the oxidized state of DNA extracted from cells and to prevent the introduction of possible new nonspecific 8-oxodGs during DNA handling.

Figure 1. OxiDIP-Seq technique. (A) ROS oxidation of 2′-deoxyguanosine generates 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodG). (B) Schematic protocol of the OxiDIP-Seq technique.

Sequencing was performed adopting the SBS (sequencing by synthesis) chemistry and using the Illumina HiSeq 2000 platform. Reads with an average length of 50 bp were obtained. Raw sequenced data were collected in a FASTQ file and subjected to further analyses.

With the OxiDIP-Seq approach, it has been demonstrated that in the genome of normal human cells 42% of the identified 8-oxodG peaks map within gene loci, specifically in the promoter and gene body regions (Amente et al., 2019). Moreover, it has been revealed that 8-oxodG regions show a specific G4-enrichment and that there is a complex relationship between 8-oxodG and guanine–cytosine (GC) content. In particular, the promoter regions with high (>47%) GC content display low levels of 8-oxodG (Gorini et al., 2020). This suggests that other mechanisms, such as the epigenetic regulation of transcription and replication, may be involved in the accumulation of 8-oxodG (Amente et al., 2019; Gorini et al., 2020). Moreover, a set of oxidized enhancers in human epithelial cells have been recently identified and characterized, which could be classified as super-enhancers. Specifically, it has been demonstrated that these oxidized enhancers are associated with bidirectional-transcribed enhancer RNAs and DNA damage response activation. Additionally, it has been revealed that the oxidized enhancer is physically associated with promoter regions in specific CTCF-mediated chromatin loops (Scala et al., 2022).

In conclusion, the OxiDIP-Seq technique allowed us to demonstrate that 8-oxodG accumulation in enhancers–promoters regions occur in a transcription-dependent manner, providing novel mechanistic insights on the intrinsic fragility of chromatin loops containing oxidized enhancers–promoters interactions (Scala et al., 2022).