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Abstract
Ubiquitin adducts surrounding DNA double-strand breaks (DSBs) have emerged as molecular platforms important for the assembly of DNA damage mediator and repair proteins. Central to these chromatin modifications lies the E2 UBC13, which has been implicated in a bipartite role in priming and amplifying lys63-linked ubiquitin chains on histone molecules through coupling with the E3 RNF8 and RNF168. However, unlike the RNF8-UBC13 holoenyzme, exactly how RNF168 work in concert with UBC13 remains obscure. To provide a structural perspective for the RNF168-UBC13 complex, we solved the crystal structure of the RNF168 RING domain. Interestingly, while the RNF168 RING adopts a typical RING finger fold with two zinc ions coordinated by several conserved cystine and histine residues arranged in a C3HC4 “cross-brace” manner, structural superimposition of RNF168 RING with other UBC13-binding E3 ubiquitin ligases revealed substantial differences at its corresponding UBC13-binding interface. Consistently, and in stark contrast to that between RNF8 and UBC13, RNF168 did not stably associate with UBC13 in vitro or in vivo. Moreover, domain-swapping experiments indicated that the RNF8 and RNF168 RING domains are not functionally interchangeable. We propose that RNF8 and RNF168 operate in different modes with their cognate E2 UBC13 at DSBs.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank the staff at beamline BL17U1 of the Shanghai Synchrotron Radiation Facility for assistance with data collection, and Dr. Grant Stewart for the RIDDLE cells. This work was supported by the Chinese Ministry of Science and Technology (Project Nos. 2009CB825502 and 2012CB917202 to J.Z.), the Ministry of Education of China (Ph.D. Programs Foundation Project No. 20113402110033 to J.Z.), the National Natural Science Foundation of China (Project Nos. 31171241 and 30970576 to J.Z.), the Chinese Academy of Sciences (“100 Talents Program” to J.Z.) and the Research Grant Council Hong Kong (General Research Fund Project No. 767811 to M.S.Y.H.).