Structural And Functional Research Of RING Domain Of RNF168

DNA double-strand break (DSB) is one of the most lethal types of DNA lesions. Failure to repair DSBs contributes to genome instability and numerous human diseases, including cancer and immunodeficiency. To eliminate the hazardous effects of DSBs on genome integrity, cells have evolved elaborate protein networks of checkpoints that abort progression of cell cycle and promote the onset of DNA damage repair in response to genotoxic stress. Upon DSB, cells signal by initiating a yH2AX-dependent signaling cascade that lead to the assembly of DNA damage mediators and repair proteins. Accordingly, DSB signals are transmitted and amplified through a series of post-translational modifications, and recent studies have highlighted usage of nondegradative ubiquitin chains catalyzed by the E3ubiquitin ligases RNF8and RNF168.The RING finger protein RNF8has an FHA domain at its N terminus, which targets phosphorylated MDC1at DSB foci. Through its RING-dependent interaction with E2Ubc13, RNF8conjugates ubiquitin adducts onto H2A-type histones and allows recruitment of the second E3ubiquitin ligase-RNF168to further amplify the signals. The current working model suggests that RNF168, in concert with Ubc13, extends and spreads the RNF8-primed ubiquitin signals and represents the primary Lys63-ubiquitylating activity at DSBs important for assembly of downstream components, including tumor suppressors BRCA1and53BP1. While recent reports have identified a number of negative regulators of the RNF8/RNF168-dependent ubiquitin signaling pathway, mechanistically how the RNF168-Ubcl3complex amplifies ubiquitin signals has remained obscure.To examine roles of RNF168repair, we solved the crystal structure of the RNF168RING domain (residues1-111). We found that the core RING domain of RNF168adopted a typical RING finger fold comprising one central a helix, two antiparallelβ strands and two long loops that are stabilized by the coordination of two zinc ions. Intriguingly, by comparing the structure of RNF168(1-111) to the complex structures of TRAF6-Ubc13, CHIP-Ubc13-MMS2and RNF8-Ubc13-MMS2, we identified a number of structural determinants on RNF168(1-111) that may preclude its binding to Ubc13. Our in vitro and in vivo data suggest that the RING domains of RNF8and RNF168may operate in different modes in the ubiquitin dependent DNA damage signaling pathway. In eukaryotes, PCNA (proliferating cell nuclear antigen) is an important cofactor of replicative DNA polymerases that encircles DNA as a trimer. It orchestrates several replication-linked processes by providing a molecular platform that controls dynamic recruitment of crucial players to the replication fork. Remarkably, a plethora of PCNA-associated proteins interact with a particular face of PCNA, they share a conserved motif called PIP-box. RING finger protein TRAIP is an E3ubiquitin ligase, it contains a PIP-box sequence at its C-terminus, suggesting it’s a new binding partner of PCNA. MTH2is a member of the MutT-related proteins that hydrolyzes8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), it was found to bind to PCNA, but intriguingly, this interaction was abolished by PCNA acetylation. MTH2depletion reduced the half-life of PCNA in a proteasome-dependent manner, suggesting that interaction with MTH2stabilises PCNA. We verify the interaction between PCNA and TRAIP, PCNA and MTH2in vitro for further research.