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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
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Differential regulation of Rad18 through Rad6-dependent mono- and polyubiquitination

Shiho Miyase1, Satoshi Tateishi, Kenji Watanabe

  • 1Institute of Molecular Embryology and Genetics, Kumamoto University Kumamoto 860-8556, Japan.

The Journal of Biological Chemistry
|October 29, 2004
PubMed

Insights

Rad18 protein exists in nonubiquitinated and monoubiquitinated forms, with self-association critical for its modification. Differential ubiquitination regulates Rad18 nuclear levels, impacting DNA repair.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Rad18 protein plays a key role in postreplication DNA repair.
  • Its function is primarily linked to the monoubiquitination of proliferating cell nuclear antigen (PCNA).

Purpose of the Study:

  • To investigate the different forms of Rad18 protein in human cells.
  • To elucidate the regulatory mechanisms controlling Rad18 levels and localization.

Main Methods:

  • Western blotting to detect Rad18 protein forms.
  • Mass spectrometry for identification of ubiquitinated Rad18.
  • In vitro ubiquitination assays with purified proteins and E1/E2 enzymes.
  • Site-directed mutagenesis to study Rad18 self-association.
  • Cellular localization studies and proteasome inhibition experiments.

Main Results:

  • Rad18 was detected as 75 kDa (nonubiquitinated) and 85 kDa (monoubiquitinated) forms.
  • Rad18 undergoes autoubiquitination, forming multiple ubiquitinated species in vitro.
  • Self-association, mediated by the zinc finger domain, is essential for Rad18 monoubiquitination.
  • Monoubiquitinated Rad18 localizes to the cytoplasm, while nonubiquitinated Rad18 is nuclear.
  • Rad18 is polyubiquitinated and degraded by proteasomes, a process enhanced by proteasome inhibitors.

Conclusions:

  • Rad18 exists in distinct ubiquitinated states that influence its cellular localization.
  • Self-association is crucial for Rad18 monoubiquitination.
  • Both mono- and polyubiquitination differentially regulate nuclear Rad18 levels, impacting DNA repair pathways.

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