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Structural basis for sequence context-independent single-stranded DNA cytosine deamination by the bacterial toxin SsdA
Open accessPreprint — published as 10.1038/s41467-025-63943-9 · Paper page
Abstract
DNA deaminase toxins are involved in interbacterial antagonism and the generation of genetic diversity in surviving bacterial populations. These enzymes have also been adopted as genome engineering tools. The single-stranded (ss)DNA deaminase SsdA represents the bacterial deaminase toxin family-2 (BaDTF2) and it deaminates ssDNA cytosines with little sequence context dependence, which contrasts with the AID/APOBEC family of sequence-selective ssDNA cytosine deaminases. Here we report the crystal structure of SsdA in complex with a ssDNA substrate. The structure reveals a unique mode of substrate binding, in which a cluster of aromatic residues of SsdA engages ssDNA in a V-shaped conformation sharply bent across the target cytosine. The bases 5’ or 3’ to the target cytosine are stacked linearly and make few sequence-specific protein contacts, thus explaining the broad substrate selectivity of SsdA. Unexpectedly, SsdA contains a β-amino acid isoaspartate, which is important for enzymatic activity and may contribute to the stability of SsdA as a toxin. Structure- function studies helped to design SsdA mutants active in human cells, which could lead to future applications in genome engineering.
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Metadata
- Authors
- Yin, L., Shi, K., Chen, Y., Harris, R. S., & Aihara, H.
- Affiliations
- University of Minnesota (US)
- The University of Texas at San Antonio Health Science Center (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Aihara, Hideki
- Venue
- Nature Communications
- Publisher
- openRxiv
- Published
- 2024-09-08
- Volume
- 16
- Issue
- 1
- Pages
- 8841-8841
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC12494967
- OpenAlex
- W4414793805
- Cited by
- 3
- References
- 1
- Citations by year
- 2026: 2 · 2025: 1
- FWCI
- 0.90
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- cc-by-nc-nd
- www.biorxiv.org/about/FAQ#license (vor)
- Locations
- Nature Communications (publishedVersion, cc-by-nc-nd)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (publishedVersion, cc-by-nc-nd)
- PubMed Central (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- U.S. Department of Health and Human Services (R35-GM118047, P01-CA234228)
- Cancer Prevention and Research Institute of Texas (CPRIT RR220053, RR220053)
- National Institutes of Health (GM124165, NIGMS P30 GM124165)
- National Cancer Institute (CA234228, P01CA234228)
- National Institute of General Medical Sciences (GM118047, GM124165, NIGMS R35-GM118047, P30GM133893, R35GM118047, P30GM124165)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- crystal structure, substrate binding, π-π stacking, genome editing, base editing, APOBEC, AID
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