Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins

Viduth K. Chaugule (Lead / Corresponding author), Connor Arkinson, Rachel Toth, Helen Walden (Lead / Corresponding author)

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Citations (Scopus)

Abstract

In higher eukaryotes, DNA damage repair response pathways are orchestrated by several molecular signals including ubiquitination. In particular the repair of DNA interstrand crosslinks, toxic to transcription and replication processes, involve the activation of the Fanconi anemia repair pathway. At the heart of this pathway lies the monoubiquitination of FANCD2 and FANCI proteins, which triggers the recruitment of DNA repair factors. A major road block in our understanding of this fundamental repair pathway arises from the challenge with generating sufficient quantities of site-specifically monoubiquitinated FANCD2 and FANCI proteins to enable mechanistic and molecular studies. Current in vitro methods rely on the purification of a large (~ 0.8 MDa), multiprotein E3 complex that can only partially monoubiquitinate a FANCD2-FANCI-DNA complex. In this chapter, we describe detailed protocols for the preparation of homogeneously and natively monoubiquitinated FANCD2 and FANCI proteins in isolation. The method relies on the use of a minimal E3 module and an engineered E2 variant that together drive site-specific ubiquitination of the isolated substrates, without the requirement of DNA cofactors. Using the enzymatic approach, we also demonstrate how added functionalities such as a fluorescently labeled ubiquitin can be conjugated on the FANCD2 and FANCI substrates, thus enabling multiple downstream applications.

Original languageEnglish
Title of host publicationMethods in Enzymology
EditorsMark Hochstrasser
Place of PublicationCambridge, MA
PublisherAcademic Press Inc.
Chapter4
Pages73-104
Number of pages32
Volume618
ISBN (Print)9780128163597
DOIs
Publication statusPublished - 2019

Publication series

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

Keywords

  • DNA repair
  • Fanconi anemia
  • RING E3
  • Ubiquitin E2
  • Ubiquitination
  • USP1

Fingerprint Dive into the research topics of 'Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins'. Together they form a unique fingerprint.

  • Cite this

    Chaugule, V. K., Arkinson, C., Toth, R., & Walden, H. (2019). Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins. In M. Hochstrasser (Ed.), Methods in Enzymology (Vol. 618, pp. 73-104). (Methods in Enzymology; Vol. 618). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2018.12.021