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Topology of Ubiquitin Chains in the Chromatosomal Environment of the E3 Ubiquitin Ligase RNF168

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Abstract

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

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Abbreviations

DSB:

double-strand DNA breaks

HR:

homologous recombination

LR:

leucine-arginine

NanoBRET:

bioluminescence resonance energy transfer-based assay that uses NanoLuc® Luciferase

SPR:

surface plasmon resonance

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Funding

This work was financially supported by the Russian Science Foundation (grant no. 21-74-10154).

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Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997, Moscow, Russia

    Anna A. Kudriaeva, Lyudmila A. Yakubova, George A. Saratov, Vasiliy I. Vladimirov, Valeriy M. Lipkin & Alexey A. Belogurov Jr.

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  1. Anna A. Kudriaeva
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  2. Lyudmila A. Yakubova
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  4. Vasiliy I. Vladimirov
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  5. Valeriy M. Lipkin
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  6. Alexey A. Belogurov Jr.
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Contributions

A.A.K. and A.A.B. concept and supervision of the study; A.A.K., L.A.Ya., G.A.S. and V.I.V. conducting experiments; A.A.K., V.M.L. and A.A.B. discussing results of the study; A.A.K. and A.A.B. writing text of the paper; A.A.K. and A.A.B. editing text of the paper.

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Correspondence to Anna A. Kudriaeva.

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The authors declare no conflict of interests in financial or any other sphere. This paper does not describe any studies involving human participant or animals performed by any of the authors.

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Kudriaeva, A.A., Yakubova, L.A., Saratov, G.A. et al. Topology of Ubiquitin Chains in the Chromatosomal Environment of the E3 Ubiquitin Ligase RNF168. Biochemistry Moscow 88, 2063–2072 (2023). https://doi.org/10.1134/S000629792312009X

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