Direct enzymatic sequencing of 5-methylcytosine at single-base resolution

Wang, Tong; Fowler, Johanna M.; Loo, Christian E.; Luo, Meiqi; Schutsky, Emily K; Berríos, Kiara N.; DeNizio, Jamie E.; Dvorak, Ashley; Downey, Nick; Montermoso, Saira; Pingul, Bianca; Nasrallah, MacLean P.; Gosal, Walraj S.; Wu, Hao; Kohli, Rahul M.

doi:10.1038/s41589-023-01318-1

Cited by 17 publications

(4 citation statements)

References 44 publications

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“…In 2023, Wang et al. presented another enzyme‐only method for direct and selective DNA methylation sequencing (DM‐seq) [86] . In DM‐seq workflow, a deaminase‐resistant adapter was first synthesized, with all Cs replaced with 5pyC (5‐propynylcytosine).…”

Section: Chemical and Enzyme‐mediated Chemical Reactions On Cytosine ...mentioning

confidence: 99%

Chemical and Enzyme‐Mediated Chemical Reactions for Studying Nucleic Acids and Their Modifications

Li,

Cheng,

Liu

2024

ChemBioChem

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Nucleic acids are genetic information‐carrying molecules inside cells. Apart from basic nucleotide building blocks, there exist various naturally occurring chemical modifications on nucleobase and ribose moieties, which greatly increase the encoding complexity of nuclei acids, contribute to the alteration of nucleic acid structures, and play versatile regulation roles in gene expression. To study the functions of certain nucleic acids in various biological contexts, robust tools to specifically label and identify these macromolecules and their modifications, and to illuminate their structures are highly necessary. In this review, we summarize recent technique advances of using chemical and enzyme‐mediated chemical reactions to study nucleic acids and their modifications and structures. By highlighting the chemical principles of these techniques, we aim to present a perspective on the advancement of the field as well as to offer insights into developing specific chemical reactions and precise enzyme catalysis utilized for nucleic acids and their modifications.

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Section: Chemical and Enzyme‐mediated Chemical Reactions On Cytosine ...mentioning

confidence: 99%

Chemical and Enzyme‐Mediated Chemical Reactions for Studying Nucleic Acids and Their Modifications

Li,

Cheng,

Liu

2024

ChemBioChem

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“…Since the discoveries of DNA methylation in the genome, enormous efforts have been unceasingly devoted to precisely interpret the functions of DNA methylation. Thanks to the invention of bisulfite conversion and enzyme-based methods − together with high-throughput sequencing and nanopore sequencing techniques, , the genome-wide mapping of 5mC sites in the cellular genome at single-base resolution was successfully achieved. 5mC sequencing uncovered the distributions and dynamic changes of 5mC in the genome and connected them with corresponding phenotypes, which greatly promoted the understanding of the functions of 5mC .…”

Section: Introductionmentioning

confidence: 99%

Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond

Zhu,

Hao,

Liu

2023

JACS Au

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DNA methylation plays a pivotal role in various biological processes and is highly related to multiple diseases. The exact functions of DNA methylation are still puzzling due to its uneven distribution, dynamic conversion, and complex interactions with other substances. Current methods such as chemical-and enzyme-based sequencing techniques have enabled us to pinpoint DNA methylation at single-base resolution, which necessitated the manipulation of DNA methylation at comparable resolution to precisely illustrate the correlations and causal relationships between the functions of DNA methylation and its spatiotemporal patterns. Here a perspective on the past, recent process, and future of precise DNA methylation tools is provided. Specifically, genome-wide and site-specific manipulation of DNA methylation methods is discussed, with an emphasis on their principles, limitations, applications, and future developmental directions.

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“…We further demonstrated 5cxmC results from the mutant taking on the neomorphic ability to use the sparse natural bacterial metabolite carboxy- S -adenosyl- l -methionine (CxSAM) . This novel CpG DNA carboxymethyltransferase (CxMTase) has opened new biotechnological applications. − Direct methylation sequencing (DM-Seq) uses a combination of the CxMTase with a DNA deaminase that can discriminate between cytosine modification states in the first all-enzymatic, base-resolution sequencing approach for localizing only 5mC without confounding by 5hmC …”

Section: Introductionmentioning

confidence: 99%

Revealing Drivers for Carboxy-S-adenosyl-l-methionine Use by Neomorphic Variants of a DNA Methyltransferase

et al. 2023

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Methylation of DNA plays a key role in diverse biological processes spanning from bacteria to mammals. DNA methyltransferases (MTases) typically employ S-adenosyl-l-methionine (SAM) as a critical cosubstrate and the relevant methyl donor for modification of the C5 position of cytosine. Recently, work on the CpG-specific bacterial MTase, M.MpeI, has shown that a single N374K point mutation can confer the enzyme with the neomorphic ability to use the sparse, naturally occurring metabolite carboxy-S-adenosyl-l-methionine (CxSAM) in order to generate the unnatural DNA modification, 5-carboxymethylcytosine (5cxmC). Here, we aimed to investigate the mechanistic basis for this DNA carboxymethyltransferase (CxMTase) activity by employing a combination of computational modeling and in vitro characterization. Modeling of substrate interactions with the enzyme variant allowed us to identify a favorable salt bridge between CxSAM and N374K that helps to rationalize selectivity of the CxMTase. Unexpectedly, we also discovered a potential role for a key active site E45 residue that makes a bidentate interaction with the ribosyl sugar of CxSAM, located on the opposite face of the CxMTase active site. Prompted by these modeling results, we further explored the space-opening E45D mutation and found that the E45D/N374K double mutant in fact inverts selectivity, preferring CxSAM over SAM in biochemical assays. These findings provide new insight into CxMTase active site architecture and may offer broader utility given the numerous opportunities offered by using SAM analogs for selective molecular labeling in concert with nucleic acid or even protein-modifying MTases.

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Direct enzymatic sequencing of 5-methylcytosine at single-base resolution

Cited by 17 publications

References 44 publications

Chemical and Enzyme‐Mediated Chemical Reactions for Studying Nucleic Acids and Their Modifications

Chemical and Enzyme‐Mediated Chemical Reactions for Studying Nucleic Acids and Their Modifications

Reshaping the Landscape of the Genome: Toolkits for Precise DNA Methylation Manipulation and Beyond

Revealing Drivers for Carboxy-S-adenosyl-l-methionine Use by Neomorphic Variants of a DNA Methyltransferase

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