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Methods Enzymol. 2019;618:1-27. doi: 10.1016/bs.mie.2019.01.003. Epub 2019 Feb 26.

Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage.

Author information

1
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, United States.
2
Schulich Faculty of Chemistry, Technion-Israel Institute of Technology, Haifa, Israel.
3
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, United States. Electronic address: cwolberg@jhmi.edu.

Abstract

Posttranslational modifications of histone proteins regulate all biological processes requiring access to DNA. Monoubiquitination of histone H2B is a mark of actively transcribed genes in all eukaryotes that also plays a role in DNA replication and repair. Solution and structural studies of the mechanism by which histone ubiquitination modulates these processes depend on the ability to generate homogeneous preparations of nucleosomes containing ubiquitin conjugated to a specific lysine residue. We describe here methods for generating milligram quantities of histone H2B with ubiquitin (Ub) conjugated to Lys 120 via either a nonhydrolyzable, dichloroacetone linkage or a cleavable isopeptide bond. H2B-Ub with an isopeptide linkage is generated by a combination of intein-fusion protein derivatization and native chemical ligation, yielding a fully native ubiquitinated lysine that can be cleaved by Ub isopeptidases. We also describe how to reconstitute nucleosomes containing ubiquitinated H2B.

KEYWORDS:

Chromatin; Histone modifications; Intein; Ubiquitin

PMID:
30850047
DOI:
10.1016/bs.mie.2019.01.003

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