TI - DISCUSSION . AB - RAD18 plays a pivotal role in post-replication DNA repair in human cells exposed to UV light . In response to UV irradiation , RAD18 forms nuclear foci , almost all of which colocalize with PCNA , suggesting that RAD18 is recruited to stalled replication forks (6) . In the present study , cells exposed to X-rays formed distinct nuclear foci containing RAD18 , which colocalized with gamma-H2AX foci but not with PCNA (Figure 1A) . These results suggest that in X-ray-irradiated cells , the recruitment of RAD18 to DSBs and its localization into foci occur in a replication -independent manner . RAD18 is also involved in S-phase-specific single strand break repair ( 10 ) . It is recruited to single strand break sites induced by UV-A laser irradiation ( 25 ) . Although X-ray irradiation causes various types of DNA breaks , the IR -induced RAD18 foci that we observed likely represent DSBs based on their colocalization with gamma-H2AX and other early markers of the response to DSBs ( Figure 1B and C ) . In our study , the formation of RAD18 IRIFs in G1 cells was dependent on 53BP1 ( Figure 3A and B ) . Given that RAD18 IRIFs were observed in S - and G2/M-phase cells irrespective of the presence of 53BP1 , the mechanism responsible for the recruitment of RAD18 to DSBs in G1 cells differ from that in S - or G2/M-phase cells . Consistent with the G1-specific and RAD18/53BP1 -dependent mechanism for DSB repair , the physical interaction between these two proteins could be detected efficiently at G1-phase but not at S or G2/M-phase Supplementary Figure 3 ) . NHEJ is thought to work mainly during G1-phase and various proteins including 53BP1 are recruited to DSBs to perform DSBs repair . The G1-specific binding of RAD18 with 53BP1 and putative G1-specific chromatin structure might contribute to the G1-specific dependency on 53BP1 for RAD18 IRIF . The binding of full-length 53BP1 and RAD18 was observed only when cells were irradiated with IR (Figure 4A) , whereas bacterially-expressed 53BP1 KBD domain specifically bound to purified RAD18 in vitro ( Figure 4B and C ) . We speculate that the apparent discrepancy between in vivo and in vitro experiments arises because in vivo binding of full length of 53BP1 to RAD18 is usually strictly suppressed by other domains of 53BP1 or by 53BP1 -binding proteins unless the suppression is released by IR -induced post-transcriptional modifications or changes in associations with binding partners . To assess the role of RAD18 at different cell -cycle stages , we investigated the formation of nuclear foci by a Rad18 cysteine 207 ( C207F ) mutant . The C207F RAD18 mutant failed to form foci during G1-phase , S-phase or G2/M ( data not shown ) . Because 53BP1 was dispensable for IRIF of RAD18 during S - or G2/M phase , molecules other than 53BP1 will likely interact with RAD18 following completion of G1 . Previous studies have shown that the zinc finger is required for accumulation of hRAD18 at sites of DNA damage ( 25 ) . Additionally , DNA-damage -induced poly-ubiquitin chains are produced on histone H2A and H2AX on damaged chromosomes after irradiation of X-ray . These poly-ubiquitin chains assemble additional DSB regulators ( 26,27 ) . It was also reported that Rad18 binds poly-ubiquitin chains through its zinc finger domains ( 28 ) . Moreover , Surface plasmon resonance analysis ( using GST-fusions of wt or GST-C207F zinc fingers derived from hRAD18 ) revealed that the zinc finger is indeed important for binding to ubiquitin ( 29 ) . Therefore , we conclude that the zinc finger domain of RAD18 interacts with multiple targets , possibly including the poly-ubiquitin chains on histone H2A and H2AX , during different stages of the cell cycle . In the early response to IR , local changes in chromatin structure are required for the initial recruitment of 53BP1 to DSBs . The initial recruitment of 53BP1 to DSBs was proposed to involve the indirect sensing of DSBs by 53BP1 through its interaction with the methylated Lys 79 residue of histone H3 and Lys 20 of histone H4 in the higher-order chromatin structure ( 20,30 ) . After 53BP1 is recruited , its retention at the chromatin surrounding the break requires MDC1 . Histone H2AX PHOSphorylation mediated by ATM and MDC1 , which is also required for the retention of 53BP1 , is thought to be extended from the site of breakage ( 31 ) . Therefore , the initial recruitment of 53BP1 to DSBs and the subsequent accumulation of 53BP1 at these sites are mechanically dissimilar processes . This study demonstrates that RAD18 monoubiquitinates fragments of 53BP1 in vitro and that RAD18-RAD6 complex plays a role in retaining 53BP1 near DSBs ( Figure 6A and B ) . The extent of 53BP1 retention correlates well with its monoubiquitylation in cells expressing wt , C207F or DR6 mutant RAD18 (Figure 6A) . Moreover , the introduction of K1268R mutation into human 53BP1 dramatically decreased the efficiency of monoubiquitylation of 53BP1 in vitro ( Fig 5C ) . Additionally , the introduction of K1253R mutation into mouse 53BP1 decreased the efficiency of its foci formation ( Fig 6D ) . These results suggest very strongly that 53BP1 monoubiquitylation and nuclear retention at DSBs are closely related . A fragment of 53BP1 was efficiently monoubiquitinated by RAD18-RAD6 complex in vitro , however , monoubiquitination of endogenous 53BP1 could not be detected in vivo . After IR-irradiation , most of 53BP1 appear to be poly-ubiquitinated and degradated by the ubiquitin-proteasome system ( 32 ) . Moreover , this ubiquitin-proteasome system is required for IR -induced 53BP1 foci formation at DSBs ( 33 ) . So the whole mechanism of 53BP1 foci formation remains to be elusive . We speculate that both mono - and poly-ubiquitinaiton of 53BP1 function cooperatively in the initial recuruitment and retention of 53BP1 IRIF at DSBs . Thus , we could not identify the monoubiquitination form of 53BP1 in vivo , distinguishing from poly-ubiquitinated form of 53BP1 . Actually the poly-ubiquitination and degradation of 53BP1 were observed in Rad18-null cells as well as in wt cells ( data not shown ) . Salt-resistant 53BP1 nuclear foci persisted for longer periods post-irradiation in wt cells than in Rad18-null cells . Consistent with this finding , our FRAP results showed that the association of 53BP1 with chromatin at DSBs was less stable in Rad18-null cells than in control cells . Moreover , the number of cells with IR -induced foci of GFP-53BP1 harboring K1268R ( or K1253R in mouse 53BP1 ) mutation in wt cells decreased to the level in Rad18-null cells. ( Figure 6D and E ) . Since IR -induced 53BP1 focus formation was observed even in Rad18-null cells , we infer that the initial recruitment of 53BP1 to chromatin is RAD18 - independent whereas RAD18-RAD6 -dependent modification of 53BP1 is important for the retention of 53BP1 at DSBs . However , we cannot entirely exclude the possibility that RAD18 ubiquitinates unidentified SUBstrates , which indirectly influence the dynamics of 53BP1 at DSBs . We attribute the decreased retention of the 53BP1 foci in Rad18-null mouse cells to the loss of an interaction between 53BP1 and RAD18 and to decreased modification of 53BP1 . Even in wt mouse cells , the 53BP1 IRIFs gradually disappeared . The dissociation of 53BP1 from the foci has two possible causes . First , since gamma-H2AX is required for the retention of 53BP1 at DSBs , gamma-H2AX dePHOSphorylation by protein phosphatase 2A ( 34 ) may result in the dissociation of 53BP1 from DSBs . Second , since RAD18 foci appeared after IR exposure and disappeared with a time course almost identical to that of the 53BP1 foci ( Figure 1D ) , the dissociation of RAD18 from 53BP1 may decrease the retention of both proteins at DSBs . The dissociation of RAD18 from 53BP1 may increase the accessibility of an unidentified protein to 53BP1 , resulting in the dislocation of 53BP1 from DSBs . The modification of 53BP1 at lysine 1268 may promote interactions with the chromatin surrounding DSBs . Alternatively , since 53BP1 is a mediator of DSB signaling , the modification of 53BP1 at DSB sites may recruit putative adaptor proteins containing ubiquitin-binding domains to the DSBs . Appropriate localization of DNA repair proteins is crucial for their function. 53BP1 plays a role in a subset of DSB repair events ( 24,35,36 ) . In this study , we demonstrated that 53BP1 IRIFs are stabilized at DSBs through the interaction of RAD18 with 53BP1 and through modification of 53BP1 . In addition , putative modifications of RAD18 , including monoubiqitination , phosphorylaTION or dePHOSphorylation may facilitate the interaction between RAD18 and 53BP1 in response to IR . We also found that a Rad18-null mutation caused inefficient DSB repair in MEFs even after 24 h of X-ray irradiation (Figure 8A) . In addition , the Rad18-null cells were significantly more sensitive to X-rays than the wt cells under the DNA-PKcs-inactive condition . Therefore , we conclude that RAD18 is involved in a subset of DSB repair pathways by promoting stable retention of 53BP1 foci at DSBs . Since the defective DSB repair of RAD18-null cells was most evident in G1-synchronized cells ( especially in the presence of a DNA-PKcs inhibitor ) we speculate that RAD18 plays a role in DSB repair via the non-homologous end joining ( NHEJ ) pathway . This idea is supported by the fact that the recombinant KBD protein stimulates double-stranded DNA end-joining activity of DNA ligase IV/Xrcc4 complex in vitro ( 19 ) , that 53BP1 mutants unable to accumulate at IRIFs cannot correct the DSB repair defect of 53BP1-null cells ( 36 ) , and that 53BP1 is functionally involved in XRCC4 -dependent NHEJ ( 37 ) . Additionally , the most recent reports show that 53BP1 promotes NHEJ at telomere-breakage sites through tethering and increasing mobility of the DNA ends , and 53BP1 also promotes NHEJ for V(D) J recombination ( 38,39 ) . Taken together , we assume that the 53BP1 -directed NHEJ pathway is promoted by RAD18 -directed modification of 53BP1 and/or interaction between RAD18 and 53BP1 . We performed additional assays to detect differences in levels of DSBs in Rad18-wt and -null cells by using pulse-field gel electrophoresis ( PFGE ) . PFGE did not reveal differences between Rad18-wt and -null cells . It should be noted however that PFGE , Comet and related assays are inherently insensitive techniques which only detect large global changes in the integrity of genomic DNA . For example , 53BP1 is a bona-fide mediator of DSB signaling , yet other workers have shown that there is no difference between the PFGE pattern of genomic DNA derived from 53BP1-wt and -null following X-ray irradiation ( 40 ) . Therefore , it is most likely that differences in levels of DSBs between Rad18-wt and -null cells are below the sensitivity levels of conventional assays . Alternatively , it is possible that RAD18 and 53BP1 -mediated mechanisms represent partially redundant or minor back-up pathways of DSB repair . In summary , we identified 53BP1 as a SUBstrate for RAD18 E3 monoubiquitination activity in vitro , and we demonstrated that modification of 53BP1 at lysine 1268 has a functional role in promoting the retention of 53BP1 at DSB sites and that RAD18 is involved in DSB repair . These results increase our understanding of the role of RAD18 in the cellular response to DSBs .