Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA

Chemical modifications in DNA have profound influenceson the structuresand functions of DNA. Uracil, a naturally occurring DNA modification,can originate from the deamination of cytosine or arise from misincorporationof dUTP into DNA during DNA replication. Uracil in DNA will imperilgenomic stability due to their potential in producing detrimentalmutations. An in-depth understanding of the functions of uracil modificationrequires the accurate determination of its site as well as contentin genomes. Herein, we characterized that a new member of the uracil-DNAglycosylase (UDG) family enzyme (UdgX-H109S) could selectively cleaveboth uracil-containing single-stranded DNA (ssDNA) and double-strandedDNA (dsDNA). Based on this unique property of UdgX-H109S, we developedan enzymatic cleavage-mediated extension stalling (ECES) method forthe locus-specific detection and quantification of uracil in genomicDNA. In the ECES method, UdgX-H109S specifically recognizes and cleavesthe N-glycosidic bond of uracil from dsDNA and generatesan apurinic/apyrimidinic (AP) site, which could be broken by APE1to form a one-nucleotide gap. The specific cleavage by UdgX-H109Sis then evaluated and quantified by qPCR. With the developed ECESapproach, we demonstrated that the level of uracil at position Chr4:50566961in genomic DNA of breast cancer tissues was significantly decreased.Collectively, the ECES method has been proved to be accurate and reproduciblein the locus-specific quantification of uracil in genomic DNA frombiological and clinical samples.

Automated summary

A new member of the uracil-DNA glycosylase family enzyme (UdgX-H109S) was characterized to selectively cleave uracil-containing single-stranded and double-stranded DNA. Based on this, an enzymatic cleavage-mediated extension stalling (ECES) method was developed for locus-specific detection and quantification of uracil in genomic DNA. The ECES method was proven to be accurate and reproducible in the quantification of uracil in genomic DNA from biological and clinical samples, with a significant decrease in uracil level observed in breast cancer tissues.