Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA

Datta, Madhurima; Aroli, S.; Karmakar, Kapudeep; Dutta, Somnath; Chakravortty, Dipshikha; Varshney, Umesh

doi:10.1016/j.bbrc.2019.08.005

Cited by 11 publications

(12 citation statements)

References 27 publications

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“…The gel shift results showed UdgX quantitatively reacted with uracil only (Figure 1b), in line with previous findings 26 . There are conflict reports on the reactivity of UdgX with uracil in double-stranded DNA (dsDNA) [24][25]27 , our results showed that UdgX could only efficiently crosslink with uracil on singlestranded DNA (ssDNA) but not those on dsDNA (Figure 1c), which was consistent with a recent report 28 . We then further optimized the UdgX reaction conditions to maximize the efficiency and minimize possible side reactions (Figure S2b, S2c).…”

supporting

confidence: 92%

“…This UdgX-DNA covalent complex was exceedingly stable even in harsh treatments such as SDS, NaOH and heat [24][25][26] . UdgX has been successfully used to visualize uracil in situ 27,28 . Based on these findings, we develop a method named 'SNU-seq' (Single-Nucleotide resolution Uracil sequencing) by combining the unique property of UdgX and the Damage-seq strategy 29 of high-fidelity DNA polymerase stalling at the protein-DNA adducts to pinpoint uracil at single base resolution.…”

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confidence: 99%

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Sequencing uracil in DNA at single-nucleotide resolution

Jiang

Yin

Qian

et al. 2021

Preprint

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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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confidence: 92%

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confidence: 99%

Sequencing uracil in DNA at single-nucleotide resolution

Jiang

Yin

Qian

et al. 2021

Preprint

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“…It does not excise normal DNA bases and its covalent link with abasic sites is stable under harsh conditions used for denaturing gels-boiling of samples in the presence of formamide under strong alkaline conditions and electrophoresis in 8 M urea gels ( 32 ). This protein has been expressed in Escherichia coli ( 32 , 33 ), purified to homogeneity and the structure of its covalent complex with ssDNA substrate has been reported ( 34 , 35 ). We have now expressed this protein in mammalian cells and used it to detect uracils in the cellular genome using immunohistochemistry.…”

Section: Introductionmentioning

confidence: 99%

Visualization of uracils created by APOBEC3A using UdgX shows colocalization with RPA at stalled replication forks

Stewart

Schauer

Bhagwat

2020

Nucleic Acids Research

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The AID/APOBEC enzymes deaminate cytosines in single-stranded DNA (ssDNA) and play key roles in innate and adaptive immunity. The resulting uracils cause mutations and strand breaks that inactivate viruses and diversify antibody repertoire. Mutational evidence suggests that two members of this family, APOBEC3A (A3A) and APOBEC3B, deaminate cytosines in the lagging-strand template during replication. To obtain direct evidence for the presence of these uracils, we engineered a protein that covalently links to DNA at uracils, UdgX, for mammalian expression and immunohistochemistry. We show that UdgX strongly prefers uracils in ssDNA over those in U•G or U:A pairs, and localizes to nuclei in a dispersed form. When A3A is expressed in these cells, UdgX tends to form foci. The treatment of cells with cisplatin, which blocks replication, causes a significant increase in UdgX foci. Furthermore, this protein- and hence the uracils created by A3A- colocalize with replication protein A (RPA), but not with A3A. Using purified proteins, we confirm that RPA inhibits A3A by binding ssDNA, but despite its overexpression following cisplatin treatment, RPA is unable to fully protect ssDNA created by cisplatin adducts. This suggests that cisplatin treatment of cells expressing APOBEC3A should cause accumulation of APOBEC signature mutations.

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“… 22 Moreover, several different approaches were also designed for in situ detection of uracil–DNA in the cellular context using fluorescence microscopies. 8 , 25 , 26 Nonetheless, the mapping of the distribution of uracil at genome-wide still remains a challenge besides impeding an in-depth view of global uracil events. dU-seq, 23 UPD-seq, 24 and U-DNA-Seq 8 were also developed to obtain the local information regarding the dU.…”

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confidence: 99%

Base-Resolution Analysis of Deoxyuridine at the Genome Scale Based on the Artificial Incorporation Modified Nucleobase

et al. 2021

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Deamination of cytosine and dUMP misincorporation have been found to be capable of producing uracil in the genome. This study presents the AI-seq (artificial incorporation modified nucleobase for sequencing), a “base substitution”, which not only is capable of profiling uracil at single-nucleotide resolution and showing its centromeric enrichment but could also reveal that the identified uracil sites are derived from cytosine deamination. All the results indicate the potential biological significance of uracil as the epigenetic modification.

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Development of mCherry tagged UdgX as a highly sensitive molecular probe for specific detection of uracils in DNA

Cited by 11 publications

References 27 publications

Sequencing uracil in DNA at single-nucleotide resolution

Sequencing uracil in DNA at single-nucleotide resolution

Visualization of uracils created by APOBEC3A using UdgX shows colocalization with RPA at stalled replication forks

Base-Resolution Analysis of Deoxyuridine at the Genome Scale Based on the Artificial Incorporation Modified Nucleobase

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