Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins

Chaugule, V. K., Arkinson, C., Toth, R. and Walden, H. (2019) Enzymatic preparation of monoubiquitinated FANCD2 and FANCI proteins. In: Hochstrasser, M. (ed.) Ubiquitin and Ubiquitin-like Protein Modifiers. Series: Methods in Enzymology, 618 (618). Elsevier, pp. 73-104. ISBN 9780128163597 (doi:10.1016/bs.mie.2018.12.021)

Full text not currently available from Enlighten.

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.

Item Type:Book Sections
Glasgow Author(s) Enlighten ID:Chaugule, Dr Viduth and Arkinson, Connor and Walden, Professor Helen
Authors: Chaugule, V. K., Arkinson, C., Toth, R., and Walden, H.
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Methods in Enzymology
Publisher:Elsevier
ISSN:0076-6879
ISSN (Online):1557-7988
ISBN:9780128163597
Published Online:11 February 2019

University Staff: Request a correction | Enlighten Editors: Update this record