RNF168 plays a critical role in the initiation of the DNA damage response to double-strand breaks (DSBs). In order to gain a better understanding of the process, a new study describes the crystal structure of RNF168 and delves into its binding to ubiquitylated proteins. According to scientists at Osaka University and The University of Tokyo, to activate the DNA repair machinery, DSBs stimulate ubiquitylation, and RNF168 binds to polyubiquitylated proteins and accumulates at the damaged DNA as part of the repair process.
Ubiquitin will then further bind to itself through one of its seven lysine amino acid residues to form a ubiquitin chain. RNF168 binds with high specificity to ubiquitin chains linked at lysine 63. Because the binding of RNF168 to lysine 63 chains is imperative for the recruitment of other DNA repair proteins, the scientists investigated the molecular interactions that assure this binding. In particular, they were interested in two RNF168 domains appropriately named ubiquitin-dependent DSB recruitment module (UDM)1 and UDM2.
In a study published in Nature Communications last week, crystal structures were made of each RNA168 domain bound to a lysine 63 chain two ubiquitin molecules long. The structures revealed that the RNF168-lysine 63 chain interaction is stabilized by many unique hydrogen bonds and hydrophobic interactions not seen in other ubiquitin lysine chains. Furthermore, UDM1 and UDM2 each folded as single α-helices to bind to the distal and proximal ubiquitins, but the specificity for lysine 63 chains by RNF168 depended on the interaction with the distal ubiquitin.
Using the crystal structures, the researchers could identify which regions in each UDM bound to the two ubiquitin molecules: LRM1 and UMI in UDM1, and UAD and MIU2 in UDM2. However, the conditions for binding were different. For UDM1, the specificity to lysine 63 chains depended on the distance and orientation between LRM1 and UMI while RNF168 was bound to ubiquitin. On the other hand, for UDM2, it was the space between the two ubiquitin molecules while RNF168 was bound to ubiquitin. Subsequent mutant experiments revealed that UDM2 binding was essential for RNF168 recruitment, while UDM1 was auxiliary.
This study suggests that the interaction of UDM2 with ubiquitylated and polyubiquitylated targets mainly contributes to the RNF168 recruitment.