Semisynthesis of ubiquitinated histone H2B with a native or nonhydrolyzable linkage

Michael Morgan, Muhammad Jbara, Ashraf Brik, Cynthia Wolberger

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

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.

Original languageEnglish
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages1-27
Number of pages27
DOIs
StatePublished - 2019
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume618
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Chromatin
  • Histone modifications
  • Intein
  • Ubiquitin

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