Topology of ubiquitin chains in the e3 ubiquitin ligase rnf168 chromatosome entourage

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Abstract

Genome stability is critical for normal functioning of cells and depends on accuracy of DNA replication, chromosome segregation, and DNA repair. Cellular defense mechanisms against DNA damage are important for preventing the development of cancer and aging. The E3 ubiquitin ligase RNF168 of the RING superfamily is an essential component of the complex responsible for the ubiquitination of H2A/H2A.X histones near DNA double-strand breaks, which is a key step in attracting repair factors to the injury site. In this study, we unequivocally showed that RNF168 does not have ability to directly distinguish the architecture of polyubiquitin chains, except for tropism of its two ubiquitin-binding domains UDM1/2 to K63 ubiquitin chains. Analysis of the intracellular chromatosomal environment of full-length RNF168 and its domains by ligand-induced bioluminescence resonance energy transfer (BRET) revealed that the C-terminal part of UDM1 is associated with K63 ubiquitin chains; RING and the N-terminal part of UDM2 are sterically close to K63- and K48- ubiquitin chains, while the C-terminal part of UDM1 is colocalized with all possible ubiquitin variants. Our observations together with the available structural data suggest that the C-terminal part of UDM1 binds K63 polyubiquitin chains on linker histone H1; RING and the N-terminal part of UDM2 are located in the central part of the nucleosome and sterically close to H1 and K48-ubiquitinated alternative substrates of RNF168, such as JMJD2A/B demethylases, while the C-terminal part of UDM1 is in the region of an activated ubiquitin residue associated with E2 ubiquitin ligase, engaged by RNF168.

About the authors

A. A Kudriaeva

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: anna.kudriaeva@gmail.com
117997 Moscow, Russia

L. A Yakubova

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

117997 Moscow, Russia

G. A Saratov

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

117997 Moscow, Russia

V. I Vladimirov

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

117997 Moscow, Russia

V. M Lipkin

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

117997 Moscow, Russia

A. A Belogurov

Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

117997 Moscow, Russia

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