Abstract
As an aberrant base in DNA, uracil is generated by either deoxyuridine (dU) misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Genome-wide profiles of uracil are important for study of these processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution, specificity, and/or sensitivity. Here, we developed a UdgX cross-linking and polymerase stalling sequencing ("Ucaps-seq") method to detect dU at single-nucleotide resolution. First, the specificity of Ucaps-seq was confirmed on synthetic DNA. Then the effectiveness of the approach was verified on two genomes from different sources. Ucaps-seq not only identified the enrichment of dU at dT sites in pemetrexed-treated cancer cells with globally elevated uracil but also detected dU at dC sites within the "WRC" motif in activated B cells which have increased dU in specific regions. Finally, Ucaps-seq was utilized to detect dU introduced by the cytosine base editor (nCas9-APOBEC) and identified a novel off-target site in cellular context. In conclusion, Ucaps-seq is a powerful tool with many potential applications, especially in evaluation of base editing fidelity.
Highlights
Uracil is a pyrimidine base that possesses similar chemical structure to thymine and forms base-pairing with adenine as well
Since most DNA polymerases are unable to distinguish thymine and uracil, they occasionally incorporate dU instead of deoxythymidine into DNA, especially when the synthesis of thymidine is disturbed.[1−3] On the other hand, uracil can be generated through cytosine deamination catalyzed by the AID/APOBEC family proteins or by spontaneous hydrolysis,[2] leading to C to T conversion
Uracil can be efficiently excised from deoxyribose by uracil-DNA Nglycosylase (UNG),[2,4] generating an apyrimidinic site (AP site) which can be further repaired by the base excision repair (BER) pathway.[5,6]
Summary
Uracil is a pyrimidine base that possesses similar chemical structure to thymine and forms base-pairing with adenine as well. Uracil can be efficiently excised from deoxyribose by UNG,[2,4] generating an apyrimidinic site (AP site) which can be further repaired by the base excision repair (BER) pathway.[5,6] the steady-state frequency of uracil in mammalian genomes is very low (∼10−7 per nucleotide),[7] it plays critical roles in diverse biological processes.[2] If uracil is continually incorporated into DNA, hyperactive BER may lead to DNA breaks and even cell death This so-called “thymine-less cell death” has been exploited in several chemotherapeutic agents.[8,9] In addition, two essential processes during B cell maturation, somatic hypermutation (SHM) and class switch recombination (CSR), are initiated by AID-catalyzed cytosine deamination in immunoglobin genes.[10−12] dysregulation of APOBEC can accumulate undesired C-to-T mutations in genome as well, which might facilitate the progression of specific cancer subtypes.[13] cytosine deaminationmediated C-to-T conversion has been applied in the genomeediting tool CBE (cytosine base editor) that holds great potential for the treatment of genetic diseases.[14,15] there is a growing demand for mapping uracil in the whole genome. It was utilized to trace the editing events of cytosine base editors and identified a novel off-target site in vivo, suggesting that Ucaps-seq is a potential tool for assessing base editor fidelity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
