Genome-wide mapping of the DNA modification uracil and its mechanism of incorporation into DNA

Abstract

The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. In this study, we developed a novel, specific method for mapping positions and local content of modified DNA nucleobases through combination of in vitro base excision with massively parallel DNA sequencing. We applied this method, named Excision-seq, to map uracil in Escherichia coli and Saccharomyces cerevisiae, and discovered significant variation in uracil content wherein uracil is excluded from the earliest and latest-replicating regions of the genome. Widely used antimetabolite chemotherapies increase uracil levels in DNA, and thus identification of factors that influence the content of uracil in DNA may have implications for understanding uracil-mediated chromosomal instability. We manipulated nucleotide biosynthesis in S. cerevisiae to determine whether genomic uracil content variation was affected. Removal of dCMP deaminase (Dcd1), which synthesizes dTTP continuously throughout the cell cycle, resulted in uracil incorporation into DNA sooner after the firing of replication origins due to rapid dTTP pool depletion. When the activity of ribonucleotide reductase, which is required for the synthesis of all dNTPs, was increased throughout the cell cycle, dUTP and dTTP pool equilibration occurred and uracil depletion at replication origins was markedly reduced. These data indicate that uracil availability and the dUTP:dTTP ratio govern uracil incorporation into the genome; therefore, manipulating nucleotide biosynthesis to increase the dUTP pool in human cells may improve the efficacy of clinical chemotherapies. Secondary repair mechanisms such as mismatch repair and translesion synthesis have been purported to recognize and act on genomic uracil. We deleted components of mismatch repair, which primarily recognizes U:G base pairs created from cytosine deamination, and translesion synthesis, which uses an error-prone translesion polymerase to replicate past some DNA modifications. Using Excision-seq, we determined that neither repair process played a significant role in establishing the variation in genomic uracil content in S. cerevisiae. In conclusion, we improved the ability to detect modified nucleobases with cognate repair enzymes genome-wide using the broadly applicable Excision-seq method. We discovered a previously unknown bias in genomic uracil content, in which some regions of the genome were protected from uracil incorporation. We proposed that this protection is a result of the temporal regulation of nucleotide biosynthesis and the intracellular dUTP:dTTP ratio, and therefore suggest that clinical chemotherapeutic drugs like 5-fluorouracil be combined with nucleotide metabolism-altering drugs that favor uracil incorporation in order to improve overall treatment efficacy and patient prognosis.