Covalent binding of uracil DNA glycosylase UdgX to abasic DNA upon uracil excision

Ahn, Woo-Chan; Aroli, S.; Kim, Jin-Hahn; Moon, Jeong Hee; Lee, Ga Seal; Sang, Pau Biak; Oh, Byung‐Ha; Varshney, Umesh; Woo, Eui-Jeon

doi:10.1038/s41589-019-0289-3

Cited by 36 publications

(34 citation statements)

References 45 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%

“…Recently, it has been reported that a novel DNA glycosylase from Mycobacterium smegmatis, named UdgX, could specifically recognize and excise uracil in DNA and form an irreversible covalent bond with the deoxyribose (Figure 1a). 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 .…”

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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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supporting

confidence: 92%

mentioning

confidence: 99%

Sequencing uracil in DNA at single-nucleotide resolution

Jiang

Yin

Qian

et al. 2021

Preprint

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“…This discrepancy between the activities of polyHis- and FLAG-tagged proteins made us wonder whether the manner of purification of the polyHis-UdgX creates a partially inactive protein. In particular, we wondered whether the imidazole used during its purification replaces one or more histidine residues involved in catalysis or the binding of the Fe–S cluster ( 34 , 35 ). To test this, the FLAG-tagged UdgX was preincubated with increasing concentrations of imidazole and then used in the covalent binding assay.…”

Section: Resultsmentioning

confidence: 99%

“…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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“…30,31 UdgX from UDG family 4 forms a stable covalent link to the deoxyribose ring with a histidine residue. 32,33 We envision that introducing mutations in these enzymes that block the repair process may convert them into useful enTRAP-seq tools for targeting a battery of DNA damages.…”

mentioning

confidence: 99%

Genome-Wide Mapping of Oxidative DNA Damage via Engineering of 8-Oxoguanine DNA Glycosylase

Fang

Zou

2019

Biochemistry

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The occurrence of 8-oxo-7,8-dihydroguanine (OG) in the genome, as one of the major DNA oxidative damages, has been implicated in an array of biological processes, ranging from mutagenesis to transcriptional regulation. Genome-wide mapping of oxidative damages could shed light on the underlying cellular mechanism. In the present study, we engineered the hOGG1 enzyme, a primary 8-oxoguanine DNA glycosylase, into a guanine oxidation-profiling tool. Our method, called enTRAP-seq, successfully identified more than 1400 guanine oxidation sites in the mouse embryonic fibroblast genome. These OG peaks were enriched in open chromatin regions and regulatory elements, including promoters, 5′ untranslated regions, and CpG islands. Collectively, we present a simple and generalizable approach for the genome-wide profiling of DNA damages with high sensitivity and specificity.

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Covalent binding of uracil DNA glycosylase UdgX to abasic DNA upon uracil excision

Cited by 36 publications

References 45 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

Genome-Wide Mapping of Oxidative DNA Damage via Engineering of 8-Oxoguanine DNA Glycosylase

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