Bisulfite-Free Sequencing of 5-Hydroxymethylcytosine with APOBEC-Coupled Epigenetic Sequencing (ACE-Seq)

Wang, Tong; Luo, Meiqi; Berríos, Kiara N.; Schutsky, Emily K; Wu, Hao; Kohli, Rahul M.

doi:10.1007/978-1-0716-0876-0_27

Cited by 8 publications

(8 citation statements)

References 20 publications

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“…The single-stranded glucosylated DNA was then deaminated in 50 mM bis-tris (pH 6.0), 0.1% Triton X-100 (APOBEC Reaction Buffer, EM-seq component E7134AA, NEB), and 20 μg of bovine serum albumin (NEB) using 0.2 μg of APOBEC3A (EM-seq component E7133AA, NEB), in a total volume of 10 μl. DNA was deaminated by APOPBEC3A at 4°C for 10 min, then ramping from 4° to 50°C over 2 hours followed by 50°C for 10 min as previously reported ( 46 ). First-strand synthesis, second-strand synthesis for adapter tagging, and PCR were completed as described above for PBAT.…”

Section: Methodsmentioning

confidence: 99%

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

et al. 2022

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TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

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Section: Methodsmentioning

confidence: 99%

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

et al. 2022

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Section: Methodsmentioning

confidence: 99%

“…Bacterial expression of A3A and eA3A constructs has been previously described in detail. 33 Purified MBP-A3A-His, MBP-eA3A-His or isolated A3A were dialyzed overnight in 50 mM Tris-Cl (pH 7.5), 50 mM NaCl, 10% glycerol, 0.5 mM DTT and 0.01% Tween-20 and the concentrations of proteins were determined using a BSA standard curve.…”

Section: Methodsmentioning

confidence: 99%

The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

Barka

Berríos

Bailer

et al. 2021

Preprint

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Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, off-target mutagenesis by A3A has been readily detected in both cellular DNA and RNA, which has been shown to promote oncogenesis. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA, but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G) which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

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Section: Materials and Methodsmentioning

confidence: 99%

“…The A3A expression construct (Addgene #109231) has been previously described and can be used to purify A3A as a fusion protein (MBP-A3A-His) that can be further processed to generate the isolated A3A domain. , For eA3A (A3A-N57G), the N57G mutation was introduced via Q5 site-directed mutagenesis (New England Biolabs, NEB). The bacterial expression of A3A and eA3A constructs has been previously described in detail . Purified MBP-A3A-His, MBP-eA3A-His, or isolated A3A was dialyzed overnight in 50 mM Tris–Cl (pH 7.5), 50 mM NaCl, 10% glycerol, 0.5 mM DTT, and 0.01% Tween-20, and the concentrations of proteins were determined using a BSA standard curve.…”

Section: Materials and Methodsmentioning

confidence: 99%

The Base-Editing Enzyme APOBEC3A Catalyzes Cytosine Deamination in RNA with Low Proficiency and High Selectivity

et al. 2022

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Human APOBEC3A (A3A) is a nucleic acid-modifying enzyme that belongs to the cytidine deaminase family. Canonically, A3A catalyzes the deamination of cytosine into uracil in single-stranded DNA, an activity that makes A3A both a critical antiviral defense factor and a useful tool for targeted genome editing. However, mutagenesis by A3A has also been readily detected in both cellular DNA and RNA, activities that have been implicated in cancer. Given the importance of substrate discrimination for the physiological, pathological, and biotechnological activities of A3A, here we explore the mechanistic basis for its preferential targeting of DNA over RNA. Using a chimeric substrate containing a target ribocytidine within an otherwise DNA backbone, we demonstrate that a single hydroxyl at the sugar of the target base acts as a major selectivity determinant for deamination. To assess the contribution of bases neighboring the target cytosine, we show that overall RNA deamination is greatly reduced relative to that of DNA but can be observed when ideal features are present, such as preferred sequence context and secondary structure. A strong dependence on idealized substrate features can also be observed with a mutant of A3A (eA3A, N57G), which has been employed for genome editing due to altered selectivity for DNA over RNA. Altogether, our work reveals a relationship between the overall decreased reactivity of A3A and increased substrate selectivity, and our results hold implications both for characterizing off-target mutagenesis and for engineering optimized DNA deaminases for base-editing technologies.

show abstract

Bisulfite-Free Sequencing of 5-Hydroxymethylcytosine with APOBEC-Coupled Epigenetic Sequencing (ACE-Seq)

Cited by 8 publications

References 20 publications

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function

The base-editing enzyme APOBEC3A catalyzes cytosine deamination in RNA with low proficiency and high selectivity

The Base-Editing Enzyme APOBEC3A Catalyzes Cytosine Deamination in RNA with Low Proficiency and High Selectivity

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