TI - . AB - The histone variant H2AX is instantly phosphorylaTED at chromatin regions flanking DSBs , and the number of PHOSphorylated H2AX ( gamma-H2AX ) foci in cells reflects the number of DSBs ( 17 ) . To assess whether RAD18 forms nuclear foci at the chromatin flanking DSB sites , we examined the localization of RAD18 and gamma-H2AX foci in cells exposed to IR . In the absence of X-ray exposure , RAD18 showed diffuse nuclear localization ( Figure 1B , upper panel ) . After X-ray exposure , however , RAD18 formed nuclear foci that largely colocalized with gamma-H2AX foci ( Figure 1B , center panel ) . Exposure to bleomycin , another DSB -inducing agent , also caused RAD18 to form nuclear foci that colocalized with gamma-H2AX foci ( Figure 1B , lower panel ) . Taken together , these results indicate that RAD18 forms nuclear foci at DSBs . In response to DSB formation , various proteins are recruited to the regions flanking the breaks to form IRIFs containing NBS1 , PHOSphorylated ATM , BRCA1 and 53BP1 ( 13,14,18 ) . To determine if RAD18 is recruited to IRIFs , we examined whether nuclear RAD18 foci colocalized with IRIF component proteins . In X-irradiated cells , RAD18 foci largely colocalized with IRIF component proteins including NBS1 , pATM , BRCA1 and 53BP1 (Figure 1C) . As shown in Figure 1D , RAD18 foci appeared within 15 min after X-ray exposure and reached a plateau at 30 min ( ~90% the cells contained RAD18 foci ) . Thirty minutes after irradiation , the number of cells containing RAD18 foci began to gradually decrease ; after 24 h , the number of cells with nuclear RAD18 foci had returned to the background level ( ~10% ) ( Figure 1D ) . The kinetics with which RAD18 was recruited to nuclear foci following irradiation was similar to those for 53BP1 and gamma-H2AX , known components of IRIF . These results indicate that RAD18 is a component of IRIFs .