Division of Molecular Cell Engineering • Kanemaki Group

A new genetics of human cells for the study of DNA transactions

Faculty




Research Summary

We established the auxin-inducible degron (AID) technology, by which the expression of a protein of interest can be rapidly controlled by the addition of a plant hormone, auxin. By combining the CRISPR–Cas-based genome-editing technology with the AID system, we can now make human conditional mutants, in which a protein of interest can be depleted in a half-life of less than 30 min. By applying the AID technology, we wish to understand how genomic DNA is maintained in human cells. In particular, we are focusing on the relationship between DNA replication and other DNA transactions.

The auxin-inducible degron (AID) technology
(A) Schematic illustration of the AID system. TIR1 derived from a plant forms an SCF-TIR1 E3 ubiquitin ligase with the endogenous factors when expressed in human cells. Auxin promotes the interaction between TIR1 and AID for poly-ubiqutylation of AID. Therefore, AID-fused proteins are degraded by the proteasome.
(B) TIR1 and GFP-AID fused with a nuclear localization signal were expressed in human 293 cells. After addition of auxin, GFP was detected by a time-lapse microscopy.
(C) Quantification of GFP signal. Most of GFP was degraded by 60 min.

Publications

Natsume, T., Kiyomitsu, T., Saga, Y., and Kanemaki, M.T. (2016). Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Reports 15, 210-218.

Nishimura, K., and Kanemaki, M.T. (2014). Rapid depletion of budding yeast proteins via the fusion of an auxin-inducible degron (AID). Curr Protoc Cell Biol 64, 20.9.1-20.9.16.

Nishimura, K., Ishiai, M., Horikawa, K., Fukagawa, T., Takata, M., Takisawa, H., and Kanemaki, M.T. (2012). Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks. Molecular Cell 47, 511-522.


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