TI - RAD18 interacts with 53BP1 via its zinc-finger domain , which is also required for RAD18 IRIF formation . AB - Next , we sought to identify the region within RAD18 that is required for its recruitment to IRIFs and its interaction with 53BP1 . To determine the region of RAD18 required for IRIF formation , we transfected Rad18-null cells with myc-tagged expression vectors encoding wt or mutant forms of RAD18 . As shown in Figure 4D , localization to nuclear foci post-IR was observed for full-length RAD18 and several of the deletion mutants , but not for RAD18DZ , a mutant lacking the zinc-finger domain and its flanking regions or C207F . These findings indicate that the zinc-finger domain of RAD18 is required for IRIF formation . Next , we tested whether RAD18 forms nuclear foci by using its zinc-finger domain to interact with 53BP1 . C terminal myc-tagged wt and C207F RAD18 were expressed in COS-7 cells and examined for their ability to interact with GST-53BP1-CDeltaBRCT in pull-down assays . As shown in Figure 4E , wt RAD18 was efficiently retrieved from COS-7 whole cell extracts by GST-53BP1-CDeltaBRCT beads . In contrast , RAD18C207F was not recovered by GST-53BP1-CDeltaBRCT , indicating that 53BP1 interacts with RAD18 via the RAD18 zinc-finger domain . We next tested whether RAD18C207F , which does not interact with 53BP1 , could also monoubiquitinate the KBD fragment . As shown in Figure 5B , RAD18C207F exhibited no ubiquitination activity towards the KBD fragment . In contrast with 53BP1 , PCNA was efficiently monoubiquitinated by RAD18C207F in vitro ( data not shown ) . Therefore , RAD18C207F retains ubiquitin ligase activity towards PCNA but not the 53BP1 KBD fragment . Most likely , the failure of RAD18C207F to ubiquitinate the KBD fragment is due to the inability of RAD18C207F and 53BP1 to interact . Therefore , we conclude that wt RAD18 functions as an E3 enzyme for the monoubiquitination of 53BP1 in vitro . Finally , to identify the site in 53BP1 that is monoubiquitinated by RAD18 , we introduced point mutations into the putative monoubiquitination sites in KBD and used the purified fragments as SUBstrates for in vitro ubiquitination assays with RAD18 (Figure 5C) . The efficiency of monoubiquitination was remarkably reduced in the mutant K1268R . Taken together , we conclude that Lys 1268 of 53BP1 is specifically monoubiquitinated by RAD18 in vitro , although we could not detect monoubiquitinated form of endogenous 53BP1 in vivo ( data not shown ) .