Sequencing of DNA Lesions Facilitated by Site-Specific Excision via Base Excision Repair DNA Glycosylases Yielding Ligatable Gaps

Abstract

Modifications to nucleotides in the genome can lead to mutations or are involved in regulation of gene expression, and therefore, finding the site of modification is a worthy goal. Robust methods for sequencing modification sites on commercial sequencers have not been developed beyond the epigenetic marks on cytosine. Herein, a method to sequence DNA modification sites was developed that utilizes DNA glycosylases found in the base excision repair pathway to excise the modification. This approach yields a gap at the modification site that is sealed by T4-DNA ligase yielding a product strand missing the modification. Upon sequencing, the modified nucleotide is reported as a deletion mutation, identifying its location. This approach was used to detect a uracil (U) or 8-oxo-7,8-dihydroguanine (OG) in codon 12 of the KRAS gene in synthetic oligodeoxynucleotides. Additionally, an OG modification site was placed in the VEGF promoter in a plasmid and sequenced. This method requires only commercially available materials and can be put into practice on any sequencing platform, allowing this method to have broad potential for finding modifications in DNA.

Oxidative DNA damage is epigenetic by regulating gene transcription via base excision repair

“…Confirmation that the DNA modifications were introduced into the plasmid was achieved using a protocol established in the C.J.B. laboratory, in which the modification was removed by Fpg and APE1 to yield a ligatable gap (40). After ligation of the gap with T4-DNA ligase, Sanger sequencing provided a characteristic nucleotide loss at the modification site to confirm its presence (SI Appendix, Fig.…”

Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine

“…Sequencing OG by conversion to a deletion signature harnesses enzymes of the base excision repair pathway to delete OG from the DNA strand leading to a deletion signature at the OG site by Sanger sequencing (Riedl et al, 2015b). The DNA glycosylase formamidopyrimidine DNA glycosylase (Fpg) removes OG from duplex DNA by its N -glycosylase and AP-lyase activities (Fig.…”

“…1 Step IV), the amplicons are subjected to Sanger sequencing to produce a chromatogram that has two peaks out of register by 1 nucleotide starting at the OG site (Fig. 1 Step V) (Riedl et al, 2015b). The two peaks are observed in the chromatogram because the OG-containing strand is one nucleotide shorter after processing than the complementary strand, and both are amplified during PCR prior to Sanger sequencing.…”

“…The two peaks are observed in the chromatogram because the OG-containing strand is one nucleotide shorter after processing than the complementary strand, and both are amplified during PCR prior to Sanger sequencing. This approach is capable of sequencing one OG or two OGs in the same strand separated by at least five nucleotides (Riedl et al, 2015b), as this is the closest Fpg can recognize and operate efficiently on two substrates (Cunniffe et al, 2007). It is conceivable that more than two OGs could be sequenced via this method, although this concept has yet to be verified.…”

“…Direct sequencing for OG is achievable by SMRT or nanopore sequencing methods on native DNA (Clark et al, 2011; Schibel et al, 2010); however, a drawback to these methods is that the DNA cannot be PCR amplified and retain the OG location during this step. This methods paper outlines three approaches developed in our laboratory for sequencing OG in DNA (Ding et al, 2017; Riedl et al, 2015a; Riedl et al, 2015b). The three methods described allow PCR amplification of the OG-containing DNA.…”