Carboxymethylcytosine is a natural base modification and a handle for bacteriophage DNA hypermodification

Bacteriophages possess a wide array of DNA modifications, with many acting as molecular camouflage to evade host immune defenses. Sequence databases contain numerous bacteriophage enzymes of unknown function, with some potentially involved in yet to be identified DNA modifications. Here we report the discovery of a DNA cytosine C5-carboxymethyltransferase (CmoX) in Synechococcus phage S-B43, which catalyzes the formation of a 5-carboxymethylcytosine (5cxmC), previously reported as an unnatural DNA modification formed by an engineered cytosine methyltransferase. The carboxy-S-adenosyl-L-methionine (Cx-SAM) cofactor required by CmoX is provided by a phage-encoded Cx-SAM synthase (CmoA), a homolog of the bacterial CmoA involved in tRNA modification. A crystal structure of CmoX in complex with Cx-SAM revealed the basis for its substrate selectivity, involving a key Arg residue interacting with the substrate carboxy group. In addition, we characterize a phage-encoded ATP-dependent amide ligase, CmoY that catalyzes the formation of 5cxmC-glycine amide. CmoA is present in many bacteriophage genomes, typically alongside CmoX and homologs of CmoY, suggesting that 5cxmC modification is a widespread naturally occurring DNA modification serving as a handle for further hypermodifications in bacteriophages. Our study underscores the ability of bacteriophages to repurpose RNA modification enzymes to expand their repertoire of DNA modifications.

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Publication Details

Journal
Nature Communications
Published
2025-12-07
DOI
https://doi.org/10.1038/s41467-025-66999-9
Primary Topic
RNA modifications and cancer
Type
article
Field-Weighted Citation Impact
0.00

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article

Carboxymethylcytosine is a natural base modification and a handle for bacteriophage DNA hypermodification

Yiling Hu, Haoyu Ma, Chuyuan Zhang, Jason Tan et al.
Nature Communications
RNA modifications and cancer
article

Carboxymethylcytosine is a natural base modification and a handle for bacteriophage DNA hypermodification

Yiling Hu, Haoyu Ma, Chuyuan Zhang, Jason Tan, Kai-Liang Ma, Lifu Song, Lin Guang Luo, Suwen Zhao, Min Wang, Lin Zhang, Liang Zhang, Yan Zhang, Yifeng Wei, Yang Tong, Yiwei Chen, Qiaoyu Yang, Yantao Liang
article en

Abstract

Bacteriophages possess a wide array of DNA modifications, with many acting as molecular camouflage to evade host immune defenses. Sequence databases contain numerous bacteriophage enzymes of unknown function, with some potentially involved in yet to be identified DNA modifications. Here we report the discovery of a DNA cytosine C5-carboxymethyltransferase (CmoX) in Synechococcus phage S-B43, which catalyzes the formation of a 5-carboxymethylcytosine (5cxmC), previously reported as an unnatural DNA modification formed by an engineered cytosine methyltransferase. The carboxy-S-adenosyl-L-methionine (Cx-SAM) cofactor required by CmoX is provided by a phage-encoded Cx-SAM synthase (CmoA), a homolog of the bacterial CmoA involved in tRNA modification. A crystal structure of CmoX in complex with Cx-SAM revealed the basis for its substrate selectivity, involving a key Arg residue interacting with the substrate carboxy group. In addition, we characterize a phage-encoded ATP-dependent amide ligase, CmoY that catalyzes the formation of 5cxmC-glycine amide. CmoA is present in many bacteriophage genomes, typically alongside CmoX and homologs of CmoY, suggesting that 5cxmC modification is a widespread naturally occurring DNA modification serving as a handle for further hypermodifications in bacteriophages. Our study underscores the ability of bacteriophages to repurpose RNA modification enzymes to expand their repertoire of DNA modifications.

Nature CommunicationsVol. 17(1)
Agency for Science, Technology and Research (SG), Tianjin University of Science and Technology (CN), Tianjin University (CN), Shanghai Jiao Tong University (CN), Chinese Academy of Sciences (CN), ShanghaiTech University (CN), Shanghai Advanced Research Institute (CN), Tianjin Institute of Industrial Biotechnology (CN), Ocean University of China (CN)
Agency for Science, Technology and Research, National Natural Science Foundation of China, Ocean University of China, Tianjin University, Shanghai Jiao Tong University, School of Life Sciences and Biotechnology Division of Life Sciences, Korea University, National Cancer Institute
Openalex Percentile: Top 65%
RNA modifications and cancer
DOIOpen Access PDFOpenAlex
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