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Transposase N-terminal phosphorylation and asymmetric transposon ends inhibit piggyBac transposition in mammalian cells
Open accessPreprint — published as 10.1093/nar/gkac1191
Abstract
Mechanistic regulation of DNA transposon systems in mammalian cells remains poorly understood. Using modeling, biochemical, and cell-based assays, we sought to extend the recent cryoEM structural insight into the piggyBac transpososome to evaluate the previously unexplained role of the transposase N-terminus, the need for asymmetric transposon ends, and the complexity of transposase tetramer formation for transposition in mammalian cells. We found that N-terminal phosphorylation by casein kinase II inhibits transposase-DNA interaction and designed deletion of this phosphorylated domain releases inhibition thereby enhancing activity. We also found that the N-terminal domain promotes transposase dimerization in the absence of transposon DNA. N-terminal deletion enables transposition of symmetric transposon ends that was previously not achievable with piggyBac . The complex transposase tetramer needed for transposition of asymmetric transposon ends can be overcome via appending a second transposase C-terminal domain in combination with symmetric transposon ends overcoming the negative regulation by asymmetric ends. Our results demonstrate that N-terminal transposase phosphorylation and the requirement for asymmetric transposon ends both negatively regulate piggyBac transposons in mammalian cells. These novel insights into mechanism and structure of the piggyBac transposase expand its potential use for genomic applications.
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Metadata
- Authors
- Luo, W., Hickman, A. B., Genzor, P., Ghirlando, R., Furman, C. M., Menshikh, A., Haase, A., Dyda, F., & Wilson, M. H.
- Affiliations
- Vanderbilt University Medical Center (US)
- National Institutes of Health (US)
- United States Department of Veterans Affairs (US)
- Corresponding author
- Dyda, Fred
- Wilson, Matthew H.
- ORCID
- Venue
- Nucleic Acids Research
- Publisher
- openRxiv
- Published
- 2022-09-26
- Volume
- 50
- Issue
- 22
- Pages
- 13128-13142
- ISSN
- 0305-1048 (ISSN-L)
- PMC
- PMC9825180
- OpenAlex
- W4312081968
- Cited by
- 13
- References
- 1
- Citations by year
- 2026: 4 · 2025: 6 · 2023: 3
- FWCI
- 0.73
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- public-domain
- Locations
- Nucleic Acids Research (publishedVersion, public-domain)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- U.S. Department of Veterans Affairs (I01 BX004258)
- National Institutes of Health (DK036153-16, DK114809)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK093660, P30DK114809, DK114809, DK093660, ZIADK036153, ZIADK075111)
- National Institute of Biomedical Imaging and Bioengineering (R01EB033676)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Animal Genetics and Reproduction
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Keywords
- piggyBac transposon, casein kinase 2, transposition regulation, protein redesign, mammalian cells
- MeSH terms
- Cell Line, DNA Transposable Elements, Humans, Phosphorylation, Transposases
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