Combination of AID2 and BromoTag expands the utility of degron-based protein knockdowns

Yuki Hatoyama, Moutushi Islam, Adam G. Bond, Ken Ichiro Hayashi, Alessio Ciulli, Masato T. Kanemaki (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

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Abstract

Acute protein knockdown is a powerful approach to dissecting protein function in dynamic cellular processes. We previously reported an improved auxin-inducible degron system, AID2, but recently noted that its ability to induce degradation of some essential replication factors, such as ORC1 and CDC6, was not enough to induce lethality. Here, we present combinational degron technologies to control two proteins or enhance target depletion. For this purpose, we initially compare PROTAC-based degrons, dTAG and BromoTag, with AID2 to reveal their key features and then demonstrate control of cohesin and condensin with AID2 and BromoTag, respectively. We develop a double-degron system with AID2 and BromoTag to enhance target depletion and accelerate depletion kinetics and demonstrate that both ORC1 and CDC6 are pivotal for MCM loading. Finally, we show that co-depletion of ORC1 and CDC6 by the double-degron system completely suppresses DNA replication, and the cells enter mitosis with single-chromatid chromosomes, indicating that DNA replication is uncoupled from cell cycle control. Our combinational degron technologies will expand the application scope for functional analyses.

Original languageEnglish
Pages (from-to)4062-4077
Number of pages16
JournalEMBO Reports
Volume25
Issue number9
Early online date23 Aug 2024
DOIs
Publication statusPublished - 10 Sept 2024

Keywords

  • AID2
  • BromoTag
  • Degron
  • dTAG
  • PROTAC and Protein Degradation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Genetics

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