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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
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The histone H2A deubiquitinase USP16 interacts with HERC2 and fine-tunes cellular response to DNA damage

Zhuo Zhang1, Huirong Yang2, Hengbin Wang3

  • 1From the Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, Alabama 35294 and.

The Journal of Biological Chemistry
|October 12, 2014
PubMed

Insights

The histone deubiquitinase USP16, interacting with HERC2, regulates ubiquitin signals during DNA repair. USP16 is crucial for terminating these signals after DNA damage repair.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Histone ubiquitination is vital for DNA double-strand break repair by recruiting repair proteins.
  • The dynamic regulation and termination of histone ubiquitination during and after DNA repair remain unclear.

Purpose of the Study:

  • To investigate the role of USP16 in regulating and terminating histone ubiquitination at DNA double-strand breaks.
  • To elucidate the interaction between USP16 and HERC2 in the DNA damage response.

Main Methods:

  • Co-immunoprecipitation to study protein interactions.
  • Western blotting to analyze ubiquitinated H2A levels.
  • Knockdown experiments to assess the function of HERC2 and USP16.
  • In vitro deubiquitination assays.

Main Results:

  • USP16 interacts with HERC2, and this interaction is crucial for fine-tuning and terminating histone ubiquitination signals post-DNA damage.
  • HERC2 knockdown impacts ubiquitinated H2A levels via USP16.
  • USP16 levels increase upon DNA damage in a HERC2-dependent manner, negatively regulating ubiquitin foci.
  • USP16 deubiquitinates H2A at Lys-119 and Lys-15 in vitro.

Conclusions:

  • USP16 is a critical regulator of the DNA damage response, specifically in terminating histone ubiquitination.
  • USP16's interaction with HERC2 is essential for this regulatory function.
  • H2A Lys-15 ubiquitination is identified as a potential target of USP16 in DNA repair processes.
Keywords:
DNA DamageDeubiquitylation (Deubiquitination)HistoneUbiquitin LigaseUbiquitylation (Ubiquitination)

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