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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
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The BAH domain of BAF180 is required for PCNA ubiquitination

Atsuko Niimi1, Suzanna R Hopkins2, Jessica A Downs2

  • 1Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.

Mutation Research
|June 29, 2015
PubMed

Insights

BAF180 promotes PCNA ubiquitination, a key step in DNA repair, independent of the PBAF complex. Its bromo-adjacent homology domains are sufficient to enhance this process following UV damage.

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Monoubiquitination of proliferating cell nuclear antigen (PCNA) is crucial for post-replication DNA repair (PRR).
  • BAF180, a subunit of the PBAF chromatin remodeling complex, influences PCNA ubiquitination and DNA fork progression after damage.
  • The precise mechanism by which BAF180 affects PCNA ubiquitination remains largely unknown.

Purpose of the Study:

  • To elucidate the mechanism by which BAF180 influences PCNA ubiquitination.
  • To determine if the chromatin remodeling activity of the PBAF complex is required for BAF180's effect on PCNA.
  • To identify the specific domains of BAF180 responsible for promoting PCNA ubiquitination.

Main Methods:

  • Expression of exogenous BAF180 and its deletion mutants in human cells.
  • UV irradiation to induce DNA damage and trigger PRR.
  • Analysis of PCNA ubiquitination levels via Western blotting.
  • Assessment of BAF180's ability to integrate into the PBAF complex.

Main Results:

  • Exogenous BAF180 expression enhances PCNA ubiquitination during S-phase post-UV irradiation, with sustained levels.
  • No correlation was found between BAF180 expression and the protein levels of USP1, a known regulator of PCNA ubiquitination.
  • Deletion analysis revealed that the bromo-adjacent homology (BAH) domains of BAF180 are essential for promoting PCNA ubiquitination.
  • A construct containing only the BAH domains was sufficient to increase ubiquitinated PCNA, even without PBAF complex assembly.

Conclusions:

  • The bromo-adjacent homology (BAH) domains of BAF180 are sufficient to promote PCNA ubiquitination.
  • The chromatin remodeling activity of the PBAF complex is not necessary for BAF180's role in PCNA ubiquitination.
  • BAF180's BAH domains directly facilitate PCNA ubiquitination, independent of its function within the PBAF complex.

Keywords:
BAF180BAH domainChromatin remodelingPCNA ubiquitinationPost replication repair

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