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Sequencing uracil in DNA at single-nucleotide resolution

Jiang, L.; Yin, J.; Qian, M.; Rong, S.; Chen, K.; Zhao, C.; Tan, Y.; Guo, J.; Chen, H.; Gao, S.; Liu, T.; Liu, Y.; Shen, B.; Yang, J.; Zhang, Y.; Meng, F.-L.; Hu, J.; Ma, H.; Chen, Y.-H.

2021-06-15 biochemistry
10.1101/2021.06.14.448445 bioRxiv
Show abstract

As an aberrant base in DNA, uracil is generated by dUMP misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution or specificity. Here, we present a UNG-independent Single-Nucleotide resolution Uracil Sequencing (SNU-seq) method utilizing the UdgX protein which specifically excises the uracil and forms a covalent bond with the resulting deoxyribose. SNU-seq was validated on synthetic DNA and applied to mammalian genomes. We found that the uracil content of pemetrexed-treated cells fluctuated along with DNA replication timing. We also accurately detected uracil introduced through cytosine deamination by the cytosine base editor (nCas9-APOBEC) and verified uracil occurrence in "WRC" motif within Activation-Induced Cytidine Deaminase (AID) hotspot regions in CSR-activated UNG -/- B cells.

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