. _:b2303167 . _:b350803847 . _:b350804103 "2"^^ . _:b350803793 . _:b2303164 . _:b350804102 "2"^^ . _:b350803933 . _:b2303203 . . _:b2303206 "of DNA-PKCS phosphorylation sites is also known to interfere with DSB repair, clustered phosphorylation of DNA-PKCS is likely to play an important role during NHEJ (Chan et al., 2002; Ding et al., 2003; Chen et al., 2005; Cui et al., >>2005<<). To test whether kinase activity and clustered phosphorylation of DNA-PKCS are important for its recruitment to DSB sites, we generated two V3 cell lines that stably expressed YFP-tagged mutant forms of DNA-PKCS." . _:b350804343 . _:b350804097 "2"^^ . _:b350804422 . _:b350804096 "2"^^ . _:b350804305 . _:b350803627 . _:b350804099 "2"^^ . _:b350804098 "2"^^ . . _:b350804090 . _:b350804109 "2"^^ . . _:b350804108 "2"^^ . _:b2303208 . . _:b2303213 . _:b350804111 "2"^^ . _:b350804271 . . _:b350804110 "2"^^ . . _:b350804105 "2"^^ . _:b350804158 . _:b350804104 "2"^^ . _:b350803762 . _:b350804009 . . _:b2303209 . . _:b350804107 "2"^^ . _:b350803746 . . _:b350803680 . _:b2303205 . . _:b350804106 "2"^^ . _:b350804286 . _:b350804117 "2"^^ . _:b2303171 "the use of microscope-coupled lasers to introduce DNA damage in specified regions of the cell nucleus to address questions concerning the sequential recruitment of different DSB repair enzymes to those damage sites (Kim et al., >>2005<<; Bekker-Jensen et al., 2006)." . _:b350804048 . _:b350804116 "2"^^ . . . _:b350804119 "2"^^ . _:b350804118 "2"^^ . _:b350803700 . _:b350804113 "2"^^ . _:b350804287 . . _:b350803917 . _:b350804112 "2"^^ . _:b2303217 . _:b350804115 "2"^^ . _:b2303228 "Recent studies have demonstrated that conserved C-terminal sequence motifs in Ku80 have the ability to interact with DNA-PKCS (Falck et al., >>2005<<). However, these in vitro studies do not form direct evidence for Ku80's ability to recruit DNA-PKCS to DSB sites. Some experiments have even suggested that DNA-PKCS is able to bind to DNA ends in the absence of Ku70/80 (Hammarsten and" . _:b350804114 "2"^^ . . . _:b350804125 "2"^^ . . _:b350804099 . _:b350803848 . _:b350804124 "2"^^ . _:b350803645 . . _:b350804127 "2"^^ . _:b350804126 "2"^^ . . . _:b350804121 "2"^^ . . . _:b350804120 "2"^^ . _:b350803892 . _:b350804123 "2"^^ . . _:b350804122 "2"^^ . . . _:b350804133 "2"^^ . _:b350804263 . . _:b350804132 "2"^^ . _:b350804135 "2"^^ . . _:b350804134 "2"^^ . . . _:b350804122 . _:b350804129 "2"^^ . . . . _:b2303219 . _:b350804128 "2"^^ . . . _:b350804131 "2"^^ . _:b2303181 . . _:b350804130 "2"^^ . _:b350803872 . _:b350803873 . _:b350804141 "2"^^ . _:b350803874 . _:b350803875 . _:b2303183 . _:b350803876 . _:b350803877 . _:b350804140 "2"^^ . _:b350803878 . _:b350803879 . . _:b350803880 . . _:b350804143 "2"^^ . . _:b350803881 . _:b350803781 . _:b350803882 . . _:b350803883 . _:b350803884 . _:b350804376 . _:b350803885 . _:b350804240 . _:b350804142 "2"^^ . _:b350803886 . _:b350803887 . _:b350803888 . _:b350803889 . _:b350804137 "2"^^ . . _:b350803890 . _:b350803891 . _:b350803892 . _:b350803893 . _:b350804136 "2"^^ . . _:b350803894 . . _:b350803895 . _:b350803896 . _:b350803897 . _:b350804139 "2"^^ . _:b350803714 . _:b350803898 . _:b350803899 . _:b350803900 . _:b350803901 . _:b2303206 . _:b350804138 "2"^^ . _:b350803902 . _:b350803903 . _:b350803840 . _:b350803841 . _:b350804149 "2"^^ . _:b350803842 . _:b350803843 . _:b350803844 . _:b350803845 . _:b350804148 "2"^^ . _:b350803846 . _:b350803847 . _:b350803848 . _:b350803849 . _:b350804151 "2"^^ . _:b350803850 . _:b350803851 . _:b350803852 . _:b350804177 . _:b350803853 . _:b2303159 "Such phosphorylation-induced alterations are important during DSB repair in vivo because mutations in the phosphorylation cluster cause severely increased radiation sensitivity and decreased DNA repair (Chan et al., >>2002<<; Ding et al., 2003; Soubeyrand et al., 2003)." . _:b350804150 "2"^^ . _:b350803854 . _:b350803855 . _:b350803856 . _:b350804196 . _:b350803934 . _:b350803857 . _:b350804145 "2"^^ . _:b350803858 . _:b350803859 . _:b350803860 . _:b350803861 . _:b350804144 "2"^^ . _:b350803862 . _:b350803863 . _:b350804176 . _:b350803864 . _:b350803865 . _:b350804147 "2"^^ . _:b350803866 . _:b350803867 . _:b350803868 . _:b350803869 . _:b350804146 "2"^^ . _:b350803870 . _:b350803871 . _:b2303198 . _:b350803936 . _:b350803937 . _:b350804157 "2"^^ . _:b350803938 . _:b2303148 "introduction" . _:b350803939 . . _:b350803940 . _:b350803941 . _:b350804156 "2"^^ . _:b350803942 . _:b350803943 . _:b350803944 . _:b350803945 . _:b350804159 "2"^^ . _:b350803946 . _:b350803947 . _:b350803687 . _:b350803948 . _:b350803949 . _:b350804158 "2"^^ . _:b350803950 . _:b350804057 . _:b350803951 . _:b350803952 . _:b350803953 . _:b350804153 "2"^^ . . _:b350803651 . _:b350803954 . . _:b350803945 . _:b350804114 . _:b350803955 . _:b350803956 . _:b350803957 . _:b2303220 . _:b350804152 "2"^^ . _:b350803958 . _:b350803959 . . _:b350803960 . _:b350803961 . _:b2303229 . _:b350804155 "2"^^ . . _:b350803962 . . _:b350803963 . _:b350803964 . . _:b350804154 "2"^^ . _:b350803965 . _:b350803966 . _:b2303165 "have shown that the presence of DNA-PKCS at DNA ends interferes with efficient ligation, most likely caused by the large dimensions of the protein molecule (Calsou et al., 1999; Weterings et al., 2003; Block et al., 2004; Cui et al., >>2005<<). This inhibition of ligation can be relieved by DNA-PKCS autophosphorylation, indicating that autophosphorylation induces a conformational change in the DNA-PKCS molecule that liberates DNA ends (Weterings et al., 2003; Block et al.," . _:b350803998 . _:b350803967 . _:b350803904 . _:b350803905 . _:b350804165 "2"^^ . _:b350803906 . _:b350803907 . _:b350803908 . _:b350803638 . _:b350803728 . _:b350803909 . _:b350804374 . _:b350804164 "2"^^ . _:b350803910 . _:b350803911 . . _:b350803912 . . _:b350803913 . _:b350804167 "2"^^ . . _:b350803914 . _:b350803915 . _:b350804184 . _:b350803916 . _:b350803917 . _:b350804166 "2"^^ . _:b350803918 . _:b350803919 . _:b350803920 . _:b350803921 . _:b350804161 "2"^^ . _:b350803867 . _:b350803806 . _:b350803922 . _:b350803923 . _:b350803924 . _:b350803925 . _:b350804160 "2"^^ . _:b350803926 . _:b350803927 . _:b350803928 . _:b350803929 . _:b2303222 "We chose to use a 365-nm laser system, which is known for its ability to generate DNA damage in a controlled fashion (Lan et al., >>2004<<, 2005). The type and amount of DNA damage that is introduced by this laser can be controlled by adjusting the laser output intensity and the pulse frequency (Lan et al., 2005). A previous study has demonstrated the recruitment of DSB" . _:b350804163 "2"^^ . _:b350803930 . _:b350803931 . _:b350803932 . . _:b350803933 . _:b350804162 "2"^^ . _:b350803934 . _:b350803935 . _:b2303230 . _:b350803744 . _:b350803745 . _:b2303169 . _:b350804173 "2"^^ . _:b350803777 . _:b350803746 . _:b350803747 . _:b350803748 . _:b350803978 . _:b350804172 "2"^^ . _:b350803749 . . _:b350803750 . _:b350803751 . _:b350804152 . _:b350803752 . _:b350804131 . _:b350803753 . _:b350804175 "2"^^ . _:b350803754 . . _:b350803755 . _:b350804018 . _:b350803756 . _:b350803757 . _:b350804174 "2"^^ . _:b350803758 . _:b350803759 . _:b350803760 . _:b350803761 . _:b350804169 "2"^^ . _:b350803762 . _:b350804098 . _:b350803763 . _:b350803764 . _:b350803765 . _:b350804168 "2"^^ . . . _:b350803766 . _:b350803767 . _:b350803768 . _:b350803769 . _:b350804171 "2"^^ . _:b350803770 . _:b350803771 . _:b350803772 . _:b350803773 . _:b350804170 "2"^^ . _:b350803774 . _:b350803775 . _:b350803712 . _:b2303159 . _:b350803795 . _:b350803713 . _:b350804181 "2"^^ . _:b350803714 . . _:b350803715 . _:b350803716 . . _:b350803717 . _:b350804180 "2"^^ . _:b350803718 . _:b350803719 . _:b350804321 . _:b350803720 . _:b350803721 . _:b350804183 "2"^^ . _:b350803722 . _:b350803723 . _:b350803724 . . _:b350804182 "2"^^ . _:b350803725 . _:b350803726 . _:b350803727 . _:b2303216 . _:b350803728 . _:b350803729 . _:b350804177 "2"^^ . _:b350803730 . _:b350803731 . _:b350803732 . _:b350803733 . _:b350804404 . _:b350804176 "2"^^ . _:b350803734 . _:b350803735 . _:b350803736 . _:b350803737 . _:b350804179 "2"^^ . _:b350803738 . _:b350803739 . _:b350803740 . _:b350803741 . _:b350804178 "2"^^ . _:b350803742 . _:b350803743 . _:b350803881 . _:b2303149 . _:b350803808 . _:b350803809 . _:b350804189 "2"^^ . _:b350803810 . _:b350803811 . _:b350803812 . _:b350803813 . _:b350804188 "2"^^ . _:b350803814 . _:b350803815 . _:b350803816 . _:b350803817 . _:b350804191 "2"^^ . . _:b350804000 . _:b350803818 . _:b350803819 . _:b350803820 . _:b350803821 . _:b350804190 "2"^^ . _:b350803822 . _:b350803823 . _:b350804336 . _:b350803824 . _:b350803825 . _:b350804185 "2"^^ . _:b350803826 . _:b350804065 . _:b350803827 . _:b350803828 . . _:b350803877 . _:b350804184 "2"^^ . _:b350803829 . _:b350803830 . _:b350803831 . _:b350803832 . _:b350803833 . _:b350804187 "2"^^ . _:b350803834 . . _:b350803835 . _:b350803836 . . _:b350804186 "2"^^ . _:b350803837 . _:b350803838 . _:b350803839 . . _:b350803776 . _:b350803777 . _:b350804197 "2"^^ . _:b350803778 . _:b350803779 . _:b350804154 . _:b350803780 . _:b350803781 . _:b350804196 "2"^^ . _:b350803782 . _:b350803783 . . _:b350803784 . _:b350803785 . _:b350804199 "2"^^ . _:b350803986 . _:b350803786 . _:b350803787 . _:b350803788 . _:b350803789 . _:b350804198 "2"^^ . _:b350804103 . _:b350803790 . _:b350803765 . . . _:b350803791 . . _:b350803792 . _:b350803793 . _:b350804193 "2"^^ . . _:b350803794 . _:b350803795 . _:b350803796 . _:b350803797 . _:b350804192 "2"^^ . _:b350803798 . _:b350803799 . . _:b350803800 . _:b350803801 . _:b350804195 "2"^^ . _:b350803802 . _:b350803803 . . _:b350803804 . _:b350803805 . _:b350804194 "2"^^ . _:b350803688 . _:b350803806 . . _:b350803678 . _:b350803807 . _:b350804205 "2"^^ . _:b350804204 "2"^^ . _:b350803625 . _:b350804207 "2"^^ . _:b350803626 . _:b350803627 . _:b350803628 . . _:b350804206 "2"^^ . . _:b350803629 . . _:b350804169 . _:b350803630 . _:b350803631 . _:b350803632 . _:b350803633 . _:b350804201 "2"^^ . _:b350803634 . _:b350803635 . _:b350803636 . . _:b350804200 "2"^^ . _:b350803637 . _:b350803638 . _:b350804349 . _:b350803639 . _:b350803640 . _:b350803641 . _:b350804203 "2"^^ . _:b350803642 . _:b350803643 . _:b350804088 . _:b350803644 . _:b350803771 . _:b350804202 "2"^^ . _:b350803645 . _:b350803646 . _:b350803647 . _:b350804213 "2"^^ . _:b350804212 "2"^^ . _:b2303183 "Anti-pS2056 and -pT2647 polyclonal antibodies and anti-pT2609 and -\u03B3H2AX monoclonal antibodies were generated as described previously (Chan et al., 2002; Chen et al., >>2005<<; Yajima et al., 2006)." . _:b350804397 . . _:b350804008 . . _:b350804215 "2"^^ . _:b350804041 . _:b350804022 . _:b350804214 "2"^^ . _:b350803634 . _:b350804249 . _:b350804209 "2"^^ . . _:b350804359 . _:b350804208 "2"^^ . _:b350804194 . . _:b350804211 "2"^^ . _:b350804210 "2"^^ . . _:b350804076 . _:b350804002 . _:b350803680 . _:b350803681 . _:b350804235 . _:b350804221 "2"^^ . _:b350803682 . _:b350803683 . _:b350803684 . _:b350803685 . _:b350804220 "2"^^ . _:b350803686 . _:b2303149 "At least two distinct pathways have evolved that mediate the repair of DSBs: homologous recombination (HR) and nonhomologous end-joining (NHEJ; Critchlow and Jackson, >>1998<<; Kanaar et al., 1998; van Gent et al., 2001; Lieber et al., 2003)." . _:b350803695 . _:b350803687 . _:b350803688 . . _:b350803689 . _:b350804223 "2"^^ . _:b350803690 . _:b350803691 . _:b350803692 . _:b350803693 . _:b350804222 "2"^^ . _:b350803694 . . _:b350804063 . _:b350803695 . _:b350803711 . _:b350803696 . _:b350803697 . _:b350804217 "2"^^ . _:b350803698 . _:b350803699 . _:b350803700 . _:b350803701 . _:b350804216 "2"^^ . _:b350803702 . _:b350803889 . _:b350804042 . _:b350803703 . _:b350803704 . _:b350803705 . _:b350804219 "2"^^ . _:b350803659 . _:b350803706 . _:b350803707 . _:b350803708 . _:b350803709 . _:b350804218 "2"^^ . _:b350803710 . _:b350803711 . _:b350803648 . _:b350803649 . _:b350804229 "2"^^ . _:b350803650 . _:b350803651 . _:b350803652 . _:b350803653 . _:b350804416 . _:b350804228 "2"^^ . _:b350803654 . _:b350804420 . . _:b350803655 . _:b350803656 . _:b350803657 . _:b350804231 "2"^^ . _:b350803735 . _:b350803658 . . _:b350804429 . _:b350803659 . . . _:b350803660 . _:b350803661 . _:b350804230 "2"^^ . _:b350803662 . _:b350803663 . _:b350803664 . . _:b350804225 "2"^^ . _:b350803665 . _:b350803666 . _:b350803667 . _:b350804244 . _:b350803668 . _:b350803707 . _:b350804224 "2"^^ . _:b350804068 . _:b350803669 . _:b350803670 . _:b350803997 . _:b350803671 . _:b350803672 . . _:b350804227 "2"^^ . _:b350803673 . _:b350803674 . _:b350803675 . _:b350803676 . _:b350803677 . _:b350804226 "2"^^ . _:b350803902 . _:b350803678 . _:b350803778 . _:b350803679 . _:b350804237 "2"^^ . _:b350804236 "2"^^ . _:b350803874 . _:b350804239 "2"^^ . _:b350804238 "2"^^ . _:b2303234 . . . _:b350804233 "2"^^ . _:b350804232 "2"^^ . _:b350804012 . _:b350804262 . _:b350804207 . _:b350804235 "2"^^ . _:b350803640 . _:b350804234 "2"^^ . _:b350804231 . . _:b350804245 "2"^^ . . _:b350804244 "2"^^ . _:b350804029 . _:b350804247 "2"^^ . . _:b350804246 "2"^^ . _:b350803941 . _:b350804241 "2"^^ . . _:b350803665 . _:b350804240 "2"^^ . . _:b350804243 "2"^^ . _:b350803766 . . _:b350804242 "2"^^ . _:b350804253 "2"^^ . _:b350804252 "2"^^ . . _:b350804255 "2"^^ . _:b350803866 . _:b350804377 . _:b350804097 . _:b350804092 . . _:b350804254 "2"^^ . _:b350803993 . _:b350804249 "2"^^ . . _:b350804248 "2"^^ . _:b350804251 "2"^^ . _:b350804312 . . _:b350804250 "2"^^ . . _:b350804228 . _:b350804261 "2"^^ . _:b350804260 "2"^^ . . . . _:b350804263 "2"^^ . _:b350803940 . . _:b350804262 "2"^^ . _:b350804257 "2"^^ . _:b350804378 . _:b350804256 "2"^^ . _:b350804259 "2"^^ . _:b350804258 "2"^^ . _:b350804269 "2"^^ . . _:b350803664 . . _:b350804268 "2"^^ . . . _:b350804271 "2"^^ . _:b2303185 . _:b350804270 "2"^^ . _:b350803636 . . _:b350804265 "2"^^ . . _:b350803849 . _:b350804264 "2"^^ . _:b350804267 "2"^^ . _:b350804381 . . _:b350804266 "2"^^ . . _:b350804277 "2"^^ . _:b350804053 . _:b2303200 "From these data and the previously reported notion that the phosphorylation of Thr2609 does not take place in Ku-deficient cells (Falck et al., >>2005<<), we conclude that phosphorylation/autophosphorylation of DNA-PKCS is a Ku-dependent process." . . _:b350804276 "2"^^ . _:b350803676 . _:b350804279 "2"^^ . . _:b350803884 . _:b350804278 "2"^^ . _:b350803844 . _:b350803756 . _:b350803741 . _:b350804273 "2"^^ . _:b350804026 . _:b350803985 . _:b350804322 . _:b350803873 . _:b350804272 "2"^^ . _:b2303158 "to autophosphorylate itself at a cluster of 6 phosphorylation sites between the Thr2609 and Thr2647 amino acid residues (Douglas et al., 2002), as well as at an additional site outside this cluster, the Ser2056 residue (Chen et al., >>2005<<). This activity possibly leads to alteration of the protein's affinity for DNA and to inactivation of its kinase activity." . _:b350804275 "2"^^ . _:b2303232 . _:b350804325 . _:b2303225 "has demonstrated the recruitment of DSB repair proteins (NBS1, BRCA1, RAD52, and WRN) toward regions of the cell nucleus that were treated with this laser system, indicating that the system is capable of generating DSBs (Lan et al., >>2005<<). In addition, we show that the laser system generates DNA ends and that microirradiated areas colocalize with \u03B3H2AX, thereby demonstrating the formation of DSBs." . _:b350804188 . _:b350804274 "2"^^ . _:b350803850 . _:b350804221 . . . _:b350804285 "2"^^ . _:b350804316 . _:b2303190 "Using the same microirradiation system that was used by Mari et al. (>>2006<<; facility of the Erasmus Medical Center, Rotterdam, Netherlands) and following the same protocol, a small disk within the nucleus was irradiated with a pulsed 800-nm laser." . _:b350804139 . _:b350804237 . _:b350804284 "2"^^ . _:b350804032 . _:b350804287 "2"^^ . _:b350803625 "20"^^ . _:b350804286 "2"^^ . . _:b350803627 "19"^^ . _:b350804281 "2"^^ . _:b350804389 . _:b350803626 "19"^^ . _:b350804280 "2"^^ . _:b350803629 "18"^^ . _:b350804337 . . _:b350804283 "2"^^ . _:b350803628 "19"^^ . _:b350804282 "2"^^ . _:b350804052 . _:b350804293 "2"^^ . _:b350803630 "16"^^ . . . _:b350803631 "15"^^ . _:b350804292 "2"^^ . _:b350803632 "14"^^ . _:b350803935 . _:b350804261 . _:b350804295 "2"^^ . _:b350803639 "12"^^ . _:b350803633 "14"^^ . _:b350804089 . _:b350804294 "2"^^ . _:b350804431 . _:b350803638 "13"^^ . _:b350803634 "14"^^ . _:b350804289 "2"^^ . _:b350803637 "13"^^ . _:b350804288 "2"^^ . _:b350803955 . _:b350804357 . _:b350803636 "13"^^ . _:b350803919 . _:b350804291 "2"^^ . _:b350803635 "13"^^ . . . _:b350804290 "2"^^ . _:b350804011 . . _:b2303174 "These mutations are known to interfere with efficient DSB repair (Kurimasa et al., 1999; Kienker et al., >>2000<<; Chan et al., 2002; Ding et al., 2003; Soubeyrand et al., 2003)." . _:b350804004 . _:b2303222 . _:b350804059 . _:b350804301 "2"^^ . _:b350804300 "2"^^ . . _:b350803647 "10"^^ . . _:b350804303 "2"^^ . _:b350803646 "10"^^ . . _:b350804302 "2"^^ . _:b350804081 . _:b350803645 "10"^^ . _:b350804290 . _:b350804297 "2"^^ . _:b350804142 . _:b350803644 "10"^^ . . _:b350804296 "2"^^ . . _:b350803773 . _:b350803643 "11"^^ . _:b350804299 "2"^^ . . _:b350804046 . _:b350803642 "11"^^ . _:b350804298 "2"^^ . _:b350803641 "11"^^ . _:b350804309 "2"^^ . . _:b350803640 "11"^^ . _:b350804308 "2"^^ . _:b350804311 "2"^^ . _:b350803654 "9"^^ . _:b350804310 "2"^^ . . _:b350803655 "9"^^ . . _:b350804305 "2"^^ . _:b350803652 "10"^^ . _:b350804304 "2"^^ . _:b350803653 "9"^^ . _:b350803651 "10"^^ . . _:b350804307 "2"^^ . _:b350803650 "10"^^ . . _:b350804306 "2"^^ . _:b350803649 "10"^^ . _:b350804317 "2"^^ . . _:b350803648 "10"^^ . _:b350804316 "2"^^ . _:b350804319 "2"^^ . _:b350803662 "8"^^ . _:b350804318 "2"^^ . _:b2303207 . _:b350803663 "8"^^ . . _:b350804313 "2"^^ . _:b350803660 "8"^^ . _:b350804313 . _:b350804312 "2"^^ . _:b2303201 . . . _:b350803661 "8"^^ . _:b350804315 "2"^^ . . _:b350804179 . _:b350803658 "9"^^ . . _:b350804134 . _:b350804314 "2"^^ . _:b350803659 "8"^^ . _:b2303223 "We chose to use a 365-nm laser system, which is known for its ability to generate DNA damage in a controlled fashion (Lan et al., 2004, >>2005<<). The type and amount of DNA damage that is introduced by this laser can be controlled by adjusting the laser output intensity and the pulse frequency (Lan et al., 2005). A previous study has demonstrated the recruitment of DSB repair" . _:b350804030 . _:b350803720 . _:b350804325 "2"^^ . _:b350803656 "9"^^ . _:b350804216 . _:b350804324 "2"^^ . _:b2303227 . _:b350803990 . _:b350803657 "9"^^ . _:b350803670 "7"^^ . _:b350804380 . _:b350804327 "2"^^ . _:b350804094 . _:b350803722 . . _:b350804168 . _:b350803671 "7"^^ . _:b350804326 "2"^^ . _:b350804126 . _:b350804321 "2"^^ . _:b350803668 "8"^^ . . _:b350803836 . _:b350804320 "2"^^ . _:b350803669 "7"^^ . _:b350804323 "2"^^ . _:b350803666 "8"^^ . _:b350804322 "2"^^ . _:b2303211 . _:b350803667 "8"^^ . _:b350804333 "2"^^ . _:b350803664 "8"^^ . _:b350804293 . . . _:b350804332 "2"^^ . _:b350803665 "8"^^ . _:b350804298 . _:b350803988 . _:b350804003 . _:b350803678 "7"^^ . _:b350804335 "2"^^ . _:b350804307 . _:b350803679 "7"^^ . _:b350804334 "2"^^ . . _:b350803676 "7"^^ . _:b350804329 "2"^^ . _:b350804255 . _:b350804315 . _:b350803677 "7"^^ . _:b350804328 "2"^^ . _:b350803674 "7"^^ . _:b350804331 "2"^^ . _:b350804164 . _:b350803675 "7"^^ . _:b350804330 "2"^^ . . . _:b350804341 "2"^^ . . _:b350803672 "7"^^ . _:b350803673 "7"^^ . _:b350804340 "2"^^ . _:b350803686 "6"^^ . _:b350804084 . _:b350804343 "2"^^ . _:b2303204 . _:b350803687 "6"^^ . _:b350804342 "2"^^ . . _:b350803684 "7"^^ . _:b350804337 "2"^^ . . _:b350803685 "6"^^ . _:b350803653 . _:b350804336 "2"^^ . . _:b350803682 "7"^^ . _:b350804339 "2"^^ . _:b350804146 . _:b350803683 "7"^^ . _:b350804338 "2"^^ . _:b350803680 "7"^^ . _:b350804349 "2"^^ . _:b2303210 . _:b350803681 "7"^^ . _:b2303173 . _:b350804348 "2"^^ . _:b350803694 "6"^^ . _:b350804392 . . . _:b350804351 "2"^^ . . _:b350803695 "6"^^ . _:b350804350 "2"^^ . _:b350803864 . . _:b350803692 "6"^^ . _:b350803952 . _:b350804345 "2"^^ . _:b2303214 . _:b350803693 "6"^^ . . _:b350804344 "2"^^ . _:b350803690 "6"^^ . . _:b350804347 "2"^^ . . . _:b350803691 "6"^^ . _:b350804346 "2"^^ . . _:b350803688 "6"^^ . _:b350804357 "2"^^ . . _:b2303218 . _:b350803689 "6"^^ . _:b350804356 "2"^^ . . _:b350803702 "6"^^ . _:b350804359 "2"^^ . _:b350803703 "6"^^ . _:b350804358 "2"^^ . _:b350803700 "6"^^ . . _:b350804353 "2"^^ . _:b350804350 . _:b350803701 "6"^^ . . _:b350804352 "2"^^ . _:b350803698 "6"^^ . _:b350804355 "2"^^ . _:b350803699 "6"^^ . _:b350804354 "2"^^ . . _:b350803845 . _:b350803696 "6"^^ . _:b350804365 "2"^^ . _:b350803697 "6"^^ . . _:b350804364 "2"^^ . _:b350803710 "6"^^ . . _:b350804367 "2"^^ . . _:b350803711 "6"^^ . _:b350804366 "2"^^ . _:b350804415 . _:b350803708 "6"^^ . _:b350804361 "2"^^ . _:b350803709 "6"^^ . _:b350804360 "2"^^ . _:b350804229 . _:b350804245 . _:b350803706 "6"^^ . . _:b350804363 "2"^^ . _:b350804228 . _:b350803707 "6"^^ . _:b350804362 "2"^^ . _:b350804396 . _:b350804231 . _:b350803704 "6"^^ . _:b350804108 . _:b350804411 . _:b350804373 "2"^^ . _:b350804230 . . _:b350803705 "6"^^ . . _:b350804372 "2"^^ . . _:b350804225 . _:b350803718 "6"^^ . _:b350804375 "2"^^ . _:b350804224 . _:b350804346 . _:b350803719 "6"^^ . _:b350804200 . _:b350804374 "2"^^ . _:b350804227 . _:b350803716 "6"^^ . _:b350804369 "2"^^ . _:b350804226 . _:b350803717 "6"^^ . _:b350804368 "2"^^ . _:b350804237 . _:b350803714 "6"^^ . _:b350804371 "2"^^ . _:b350804181 . _:b350804236 . _:b2303176 . _:b350803715 "6"^^ . _:b350804370 "2"^^ . _:b350804239 . _:b350803628 . _:b350803712 "6"^^ . _:b350804381 "2"^^ . _:b350804238 . _:b350803713 "6"^^ . . _:b350804380 "2"^^ . _:b350804233 . _:b350804219 . _:b350804383 "2"^^ . _:b350804232 . _:b350803725 "5"^^ . _:b350804382 "2"^^ . . _:b350804235 . _:b350803724 "5"^^ . . _:b350804377 "2"^^ . . _:b350804234 . _:b350803727 "5"^^ . _:b350803702 . _:b350804376 "2"^^ . _:b350804245 . _:b350803726 "5"^^ . _:b350803818 . . _:b350804379 "2"^^ . _:b350804244 . _:b350803721 "5"^^ . _:b350804401 . _:b350804378 "2"^^ . _:b350804247 . _:b350803720 "6"^^ . . _:b350804389 "2"^^ . _:b350803629 . _:b350804246 . _:b350803723 "5"^^ . _:b350804388 "2"^^ . . _:b350804241 . _:b350803822 . _:b350803722 "5"^^ . _:b350804354 . _:b350804275 . _:b350804328 . _:b350804214 . _:b350804391 "2"^^ . _:b350804240 . _:b350803733 "5"^^ . _:b350804390 "2"^^ . _:b350804243 . _:b350803732 "5"^^ . _:b350803690 . _:b350804385 "2"^^ . _:b350804242 . _:b350803735 "5"^^ . . . _:b350803841 . _:b350804384 "2"^^ . _:b350804253 . _:b350803734 "5"^^ . _:b350804387 "2"^^ . . _:b350804252 . _:b350803637 . _:b350803729 "5"^^ . _:b350804039 . . _:b350804386 "2"^^ . . _:b350804255 . _:b350803728 "5"^^ . . _:b350804395 . . _:b350804397 "2"^^ . . _:b350804254 . _:b350803731 "5"^^ . . _:b350804396 "2"^^ . . _:b350804249 . _:b350803730 "5"^^ . _:b350804174 . _:b350804399 "2"^^ . _:b350804248 . _:b350804015 . _:b350804136 . _:b350803741 "5"^^ . _:b350804398 "2"^^ . _:b350804251 . _:b350803740 "5"^^ . _:b350804393 "2"^^ . _:b350804250 . _:b350803743 "5"^^ . _:b350804267 . . _:b350804392 "2"^^ . _:b350804261 . _:b350803742 "5"^^ . _:b350804395 "2"^^ . _:b350804260 . _:b350803737 "5"^^ . . _:b350804394 "2"^^ . _:b350804263 . _:b350803736 "5"^^ . _:b350804405 "2"^^ . _:b350804262 . _:b350803739 "5"^^ . . _:b350804404 "2"^^ . _:b350804257 . _:b350803738 "5"^^ . _:b350804187 . . _:b350804332 . _:b350804407 "2"^^ . _:b350803749 "4"^^ . _:b350804256 . _:b350804077 . . _:b350803706 . _:b350804406 "2"^^ . _:b350803748 "4"^^ . _:b350804259 . _:b350804401 "2"^^ . _:b350803751 "4"^^ . _:b350804338 . _:b350804258 . _:b350804400 "2"^^ . _:b350803750 "4"^^ . _:b350804269 . . . _:b350804403 "2"^^ . _:b350804268 . _:b350803745 "5"^^ . _:b350804402 "2"^^ . _:b350804271 . _:b350803744 "5"^^ . . _:b350804413 "2"^^ . _:b350804270 . _:b350803747 "5"^^ . _:b350804405 . . _:b350804412 "2"^^ . _:b350804265 . _:b350803746 "5"^^ . _:b350804415 "2"^^ . _:b350803757 "4"^^ . _:b350804264 . . _:b350804414 "2"^^ . _:b350803756 "4"^^ . _:b350804267 . _:b350804409 "2"^^ . _:b350803759 "4"^^ . _:b350804266 . _:b350803984 . _:b350804408 "2"^^ . _:b350803758 "4"^^ . _:b350804277 . . _:b2303167 "This inhibition of ligation can be relieved by DNA-PKCS autophosphorylation, indicating that autophosphorylation induces a conformational change in the DNA-PKCS molecule that liberates DNA ends (Weterings et al., 2003; Block et al., >>2004<<; Reddy et al., 2004; Cui et al., 2005)." . _:b350803683 . _:b350804411 "2"^^ . _:b350803753 "4"^^ . _:b350804276 . . _:b350804410 "2"^^ . _:b350803752 "4"^^ . _:b350804279 . _:b350803901 . _:b350804421 "2"^^ . _:b350803755 "4"^^ . _:b350804278 . . _:b350804420 "2"^^ . _:b350803754 "4"^^ . _:b350804273 . _:b350804423 "2"^^ . _:b350803765 "4"^^ . _:b350804272 . _:b350804422 "2"^^ . _:b350803764 "4"^^ . _:b350804275 . . . _:b350804417 "2"^^ . _:b350803767 "4"^^ . _:b350804274 . . _:b350804416 "2"^^ . _:b350803766 "4"^^ . _:b350804285 . _:b350804419 "2"^^ . _:b350803761 "4"^^ . _:b350804284 . _:b2303160 . _:b350804418 "2"^^ . _:b350803760 "4"^^ . _:b350804287 . . _:b350804429 "2"^^ . _:b350803763 "4"^^ . _:b350804286 . _:b350804428 "2"^^ . _:b350803762 "4"^^ . _:b350804382 . _:b350804281 . _:b350804006 . _:b350804431 "2"^^ . _:b350803773 "4"^^ . _:b350804280 . . _:b350804430 "2"^^ . _:b350803772 "4"^^ . _:b350804283 . _:b350803649 . . _:b350804425 "2"^^ . _:b350803775 "4"^^ . _:b350804282 . _:b350804424 "2"^^ . _:b350803774 "4"^^ . _:b350804293 . . _:b350804427 "2"^^ . _:b350803769 "4"^^ . _:b350804292 . . _:b350804426 "2"^^ . _:b350803768 "4"^^ . _:b350804295 . _:b350803771 "4"^^ . _:b350804294 . _:b350803770 "4"^^ . _:b350804289 . _:b350804410 . _:b350803781 "4"^^ . _:b350804288 . _:b350803783 . _:b350803780 "4"^^ . _:b350804291 . _:b350804375 . _:b350803783 "4"^^ . _:b350804290 . _:b350803832 . _:b350803782 "4"^^ . _:b350804301 . _:b350803777 "4"^^ . _:b350804300 . _:b2303169 "by DNA-PKCS autophosphorylation, indicating that autophosphorylation induces a conformational change in the DNA-PKCS molecule that liberates DNA ends (Weterings et al., 2003; Block et al., 2004; Reddy et al., 2004; Cui et al., >>2005<<). These findings gave rise to the current notion that DNA-PKCS functions as a \u201Cgatekeeper,\u201D which protects DNA ends from premature processing and ligation, until the two DNA ends are properly juxtaposed and DNA-PKCS autophosphorylation" . _:b350803729 . _:b350803776 "4"^^ . _:b350804303 . . _:b2303175 "These mutations are known to interfere with efficient DSB repair (Kurimasa et al., 1999; Kienker et al., 2000; Chan et al., >>2002<<; Ding et al., 2003; Soubeyrand et al., 2003)." . _:b350803779 "4"^^ . _:b350804302 . _:b350803778 "4"^^ . _:b350804297 . _:b350803789 "4"^^ . _:b350804296 . . _:b350804379 . . _:b350803788 "4"^^ . _:b2303154 "Two protein complexes make up the catalytic core of the NHEJ process: the DNA-dependent protein kinase holoenzyme (DNA-PK) and the DNA ligase IV\u2013XRCC4 complex (Lees-Miller and Meek, 2003; Weterings and van Gent, >>2004<<). Ligase IV\u2013XRCC4 mediates ligation of the juxtaposed DNA ends in the final NHEJ step." . _:b350804335 . _:b350804299 . _:b350803887 . _:b350803791 "4"^^ . _:b350804298 . _:b350803790 "4"^^ . _:b350804309 . . . _:b350803785 "4"^^ . _:b350804308 . . _:b350804311 . _:b350803784 "4"^^ . _:b350803910 . . . _:b350803787 "4"^^ . _:b350804310 . _:b350803694 . _:b350803786 "4"^^ . _:b350804305 . . _:b350803758 . _:b350803797 "4"^^ . _:b350804304 . _:b350803796 "4"^^ . _:b350804307 . _:b350804166 . _:b350803799 "4"^^ . _:b350804306 . _:b350803798 "4"^^ . _:b350804317 . _:b350803793 "4"^^ . _:b350804316 . _:b350803792 "4"^^ . _:b350804319 . _:b350803795 "4"^^ . _:b350804318 . . _:b350804218 . _:b350803794 "4"^^ . _:b350804313 . _:b350803805 "4"^^ . _:b350804312 . . _:b350803928 . _:b350803804 "4"^^ . _:b350804315 . _:b350803820 . . _:b2303151 "At least two distinct pathways have evolved that mediate the repair of DSBs: homologous recombination (HR) and nonhomologous end-joining (NHEJ; Critchlow and Jackson, 1998; Kanaar et al., 1998; van Gent et al., >>2001<<; Lieber et al., 2003). NHEJ is considered to be the prevailing pathway during the G0 and G1 phases of the cell cycle in mammalian cells because this repair pathway does not require the presence of an intact DNA template." . . _:b350803807 "4"^^ . _:b350804314 . _:b350803806 "4"^^ . _:b350804325 . . _:b350804111 . _:b350803801 "4"^^ . _:b350804324 . _:b350804074 . _:b2303213 "in mutant cells most likely reflects the inability of these cells to quickly repair the introduced DSBs because it is known that 7A and KD mutants display deficient DSB repair (Kurimasa et al., 1999; Kienker et al., 2000; Chan et al., >>2002<<; Ding et al., 2003; Soubeyrand et al., 2003). To verify this, we generated an XRCC4-deficient (XR1) cell line that expressed YFP-tagged WT DNA-PKCS." . _:b350803962 . _:b350804327 . _:b350803800 "4"^^ . _:b350804050 . _:b350803803 "4"^^ . _:b350804326 . _:b350803802 "4"^^ . _:b350804321 . . . _:b350803838 . . _:b350804204 . _:b350804033 . _:b350803813 "4"^^ . _:b350804320 . _:b350803906 . _:b350803812 "4"^^ . _:b350804323 . _:b350803815 "4"^^ . _:b350804322 . _:b350803814 "4"^^ . _:b350804333 . . _:b350803809 "4"^^ . _:b350804332 . . _:b350803808 "4"^^ . _:b350804335 . _:b350803626 . _:b350803811 "4"^^ . _:b350804334 . _:b350804239 . _:b350803810 "4"^^ . _:b350804329 . . . _:b350803821 "3"^^ . _:b350804328 . _:b350804202 . _:b350804101 . _:b350803820 "3"^^ . _:b350804331 . _:b350803823 "3"^^ . . _:b350804330 . _:b350804106 . _:b350803822 "3"^^ . . _:b350804341 . . _:b350803817 "3"^^ . _:b350804340 . . _:b350803816 "3"^^ . . _:b350804343 . _:b350803819 "3"^^ . _:b350804294 . _:b350804342 . _:b350803818 "3"^^ . _:b350804337 . _:b350803829 "3"^^ . _:b350804421 . . _:b350804336 . _:b2303152 . _:b350803828 "3"^^ . _:b350804339 . _:b350804072 . _:b350803831 "3"^^ . . _:b350804338 . _:b350803798 . _:b350803830 "3"^^ . _:b350804349 . _:b350803654 . _:b350803825 "3"^^ . . _:b350804348 . _:b350803824 "3"^^ . _:b2303227 "It is generally assumed that the Ku70/80 heterodimer is the first NHEJ factor to bind to a broken DNA strand and that DNA-PKCS is subsequently attracted to the DSB sites (Chen et al., 1996; Singleton et al., >>1999<<). Recent studies have demonstrated that conserved C-terminal sequence motifs in Ku80 have the ability to interact with DNA-PKCS (Falck et al., 2005)." . _:b350804125 . _:b350804351 . _:b2303172 "A recent study examined the in vivo recruitment of NHEJ core enzymes to DSBs after the introduction of DNA damage by the use of a pulsed, near-infrared laser (Mari et al., >>2006<<), and it showed that the binding of Ku70/80 to DNA ends is a highly dynamic equilibrium of association and dissociation." . _:b350803705 . _:b350803827 "3"^^ . . _:b350804350 . _:b350803826 "3"^^ . . _:b350804345 . _:b350803802 . _:b350803837 "3"^^ . _:b350804119 . _:b350804344 . _:b350803936 . _:b350803836 "3"^^ . _:b350804258 . _:b350804347 . _:b350803839 "3"^^ . _:b350804346 . _:b350804124 . . _:b350803838 "3"^^ . _:b350804357 . _:b350803718 . . _:b350803833 "3"^^ . _:b350804356 . . _:b350803832 "3"^^ . _:b350804359 . _:b350803835 "3"^^ . _:b350804358 . _:b350803834 "3"^^ . _:b350804353 . _:b350803845 "3"^^ . _:b350804256 . _:b350804352 . _:b350804317 . _:b350803823 . _:b350803844 "3"^^ . _:b350804355 . _:b350803847 "3"^^ . . _:b350804354 . . _:b350803846 "3"^^ . _:b350804365 . . _:b350803841 "3"^^ . _:b350804364 . _:b350803840 "3"^^ . _:b350804367 . _:b350803843 "3"^^ . . _:b350804366 . _:b2303157 . _:b350803842 "3"^^ . _:b350804361 . _:b350803853 "3"^^ . . _:b350804360 . _:b2303231 "These findings are in agreement with recent observations of the in vivo behavior of Ku80 at DSB sites (Mari et al., >>2006<<). The 7A and KD mutations cause a dramatic decrease in the exchange rate of bound and free molecules. In other words, these mutant DNA-PKCS molecules are more stably bound to DNA ends than WT molecules. Obviously, there is still exchange" . _:b350803852 "3"^^ . . _:b350804363 . . _:b350803855 "3"^^ . _:b350803780 . _:b350804362 . _:b350803854 "3"^^ . _:b350804373 . _:b350803849 "3"^^ . _:b350804372 . _:b350803717 . _:b350803848 "3"^^ . . _:b350804375 . _:b350803716 . _:b350803851 "3"^^ . . _:b350804374 . _:b350803719 . _:b350803850 "3"^^ . _:b350804369 . _:b350803718 . . _:b350803861 "3"^^ . _:b350804368 . _:b350803960 . . _:b350804250 . _:b350803713 . _:b350803860 "3"^^ . _:b350804371 . _:b350803712 . _:b350803863 "3"^^ . _:b350803731 . _:b350804370 . . _:b350803715 . _:b350804333 . _:b350803862 "3"^^ . _:b350804381 . _:b350803714 . _:b350803857 "3"^^ . _:b350804380 . . _:b350803725 . _:b350803856 "3"^^ . _:b350804284 . _:b350804383 . . _:b350803724 . _:b350803859 "3"^^ . _:b350803883 . _:b350804382 . . . _:b350804159 . _:b350803727 . _:b350803858 "3"^^ . _:b350804377 . _:b350803726 . _:b350803869 "3"^^ . _:b350804376 . _:b2303224 "The type and amount of DNA damage that is introduced by this laser can be controlled by adjusting the laser output intensity and the pulse frequency (Lan et al., >>2005<<). A previous study has demonstrated the recruitment of DSB repair proteins (NBS1, BRCA1, RAD52, and WRN) toward regions of the cell nucleus that were treated with this laser system, indicating that the system is capable of generating DSBs" . _:b350803656 . _:b350803721 . _:b350803868 "3"^^ . _:b350804265 . _:b350804379 . _:b350804361 . _:b350803720 . _:b350804318 . _:b350803871 "3"^^ . . _:b350804378 . . _:b350803870 "3"^^ . _:b350803723 . _:b350804389 . _:b350803722 . _:b350803865 "3"^^ . _:b350804388 . _:b350803828 . _:b350804034 . _:b350803733 . _:b350803864 "3"^^ . . _:b350804391 . _:b350803732 . _:b350803867 "3"^^ . _:b350804390 . _:b350803735 . _:b350803866 "3"^^ . . _:b350804385 . _:b350803792 . _:b350803764 . _:b350804163 . _:b350803734 . _:b350803877 "3"^^ . _:b350804384 . _:b350804310 . _:b350803729 . _:b350803876 "3"^^ . _:b350804387 . _:b350804025 . _:b350803879 "3"^^ . _:b350803728 . _:b350804386 . . _:b350804234 . _:b350803731 . _:b350804272 . _:b350803878 "3"^^ . _:b350804397 . _:b350803730 . _:b350803873 "3"^^ . _:b350804396 . _:b2303211 "intensity of the accumulation area in mutant cells most likely reflects the inability of these cells to quickly repair the introduced DSBs because it is known that 7A and KD mutants display deficient DSB repair (Kurimasa et al., >>1999<<; Kienker et al., 2000; Chan et al., 2002; Ding et al., 2003; Soubeyrand et al., 2003)." . _:b350804093 . _:b350803741 . _:b350803872 "3"^^ . _:b350804399 . _:b350803740 . _:b350803875 "3"^^ . _:b350804241 . _:b350804398 . _:b350804224 . _:b350803743 . _:b350803874 "3"^^ . _:b350804073 . _:b350804393 . _:b350803742 . _:b350803885 "3"^^ . _:b350804037 . _:b350804392 . _:b350804334 . _:b350803737 . _:b350803884 "3"^^ . _:b350804395 . _:b350803736 . _:b350803887 "3"^^ . _:b350804394 . _:b350804047 . _:b350803739 . _:b350803886 "3"^^ . . _:b350804405 . _:b350803738 . _:b350803881 "3"^^ . . _:b350804404 . . _:b350804369 . . _:b350803749 . _:b350803754 . _:b350803880 "3"^^ . _:b350804407 . _:b350803748 . _:b350803883 "3"^^ . _:b350804406 . . _:b2303196 "In brief, we treated YFP-DNA-PKCS\u2013expressing cells with either our 365-nm laser system or with the 800-nm multiphoton laser system used by Mari et al. (>>2006<<), which is known to introduce between 1,000 and 1,500 DSBs." . _:b350803751 . _:b350803882 "3"^^ . . _:b350804401 . _:b350803750 . _:b2303220 "Because phosphorylation of both Thr2609 and Ser2056 is thought to be important for DSB repair via the NHEJ pathway (Chan et al., 2002; Ding et al., 2003; Chen et al., 2005; Cui et al., >>2005<<), we generated V3 cell lines that stably expressed YFP-tagged mutant forms of DNA-PKCS that could not be phosphorylated at those sites (Fig." . _:b350803644 . . _:b350803893 "3"^^ . _:b350804248 . _:b350804400 . . _:b350803745 . _:b350803892 "3"^^ . . _:b350804403 . . . _:b350803744 . _:b350803895 "3"^^ . . . _:b350804402 . _:b350803747 . _:b350803894 "3"^^ . . _:b350804413 . _:b350804064 . _:b350803746 . _:b350803889 "3"^^ . _:b350804412 . _:b350804035 . _:b350804292 . _:b350803757 . _:b350803888 "3"^^ . _:b350804415 . _:b350803756 . _:b350803891 "3"^^ . _:b350803949 . _:b350804414 . . . _:b350803759 . . _:b350803890 "3"^^ . _:b350803816 . _:b350804409 . _:b350803758 . _:b350803901 "3"^^ . _:b350804408 . _:b350803753 . _:b350803900 "3"^^ . _:b350804411 . . . _:b350803752 . _:b350803903 "3"^^ . _:b350804410 . . _:b350803755 . _:b350803902 "3"^^ . . _:b350804421 . _:b350804407 . _:b350803869 . _:b350803754 . _:b350804394 . _:b350803897 "3"^^ . _:b350804420 . _:b350803765 . . _:b350803896 "3"^^ . _:b350804423 . . . _:b350803764 . _:b350803899 "3"^^ . _:b350804422 . _:b2303209 "The Thr2609 and Thr2647 residues, which were previously reported to be autophosphorylation sites (Chan et al., >>2002<<; Ding et al., 2003), apparently do not require the DNA-PKCS kinase to be phosphorylated." . . _:b350804167 . _:b350803767 . _:b350803898 "3"^^ . _:b350803767 . _:b350804417 . _:b350804017 . _:b350803766 . _:b350803909 "3"^^ . _:b350804416 . _:b350803761 . _:b350803908 "3"^^ . _:b350804419 . _:b350804424 . . _:b350803760 . _:b350804083 . . _:b350803911 "3"^^ . _:b350804418 . _:b350803663 . _:b350803763 . _:b350803910 "3"^^ . _:b350804429 . "10.1083%2Fjcb.200608077" . _:b350803762 . _:b350803905 "3"^^ . . _:b350804428 . _:b350804120 . _:b350803773 . _:b350803904 "3"^^ . _:b350804431 . _:b350803772 . _:b350803907 "3"^^ . _:b350804430 . . . . . _:b350803775 . _:b350803691 . _:b350803906 "3"^^ . _:b350804339 . _:b350804425 . _:b350803774 . _:b350803917 "3"^^ . . _:b350804424 . . _:b350803769 . _:b350804117 . _:b350803916 "3"^^ . _:b350804427 . _:b350803796 . _:b350803768 . _:b350804385 . _:b350803919 "3"^^ . . _:b350804426 . _:b350804023 . _:b350804363 . _:b350803771 . _:b350803918 "3"^^ . _:b2303214 "likely reflects the inability of these cells to quickly repair the introduced DSBs because it is known that 7A and KD mutants display deficient DSB repair (Kurimasa et al., 1999; Kienker et al., 2000; Chan et al., 2002; Ding et al., >>2003<<; Soubeyrand et al., 2003). To verify this, we generated an XRCC4-deficient (XR1) cell line that expressed YFP-tagged WT DNA-PKCS." . _:b350803770 . _:b350804373 . _:b350803913 "3"^^ . _:b350803946 . _:b350803781 . _:b350803912 "3"^^ . . _:b350803855 . . _:b350803780 . _:b350803915 "3"^^ . . _:b350804417 . _:b350803783 . _:b350804348 . _:b350804135 . _:b350803914 "3"^^ . _:b350803782 . . _:b350803925 "3"^^ . . _:b350803777 . _:b350803924 "3"^^ . _:b350803755 . _:b350803776 . _:b350803927 "3"^^ . _:b350803779 . _:b350803926 "3"^^ . _:b350803778 . . . _:b350803921 "3"^^ . . . . _:b350803789 . _:b350803920 "3"^^ . . . _:b350803788 . . _:b350803923 "3"^^ . _:b350803791 . _:b350803922 "3"^^ . _:b2303162 "Several studies have shown that the presence of DNA-PKCS at DNA ends interferes with efficient ligation, most likely caused by the large dimensions of the protein molecule (Calsou et al., >>1999<<; Weterings et al., 2003; Block et al., 2004; Cui et al., 2005)." . _:b350803830 . _:b350803790 . _:b350803933 "3"^^ . _:b2303181 "Fluorescent immunostaining was performed as previously described (Chen et al., >>2005<<). In brief, the irradiated cells were fixed with cold methanol for 20 min on ice and permeabilized for 10 min in PBS containing 0.5% Triton X-100." . _:b350803785 . _:b350803932 "3"^^ . _:b350803753 . _:b350803784 . _:b350803935 "3"^^ . . _:b350803713 . _:b350803787 . _:b350803934 "3"^^ . _:b350803786 . _:b350803929 "3"^^ . _:b350803797 . _:b350803928 "3"^^ . _:b350803796 . _:b350803931 "3"^^ . _:b350804341 . _:b2303168 . _:b350803799 . _:b350803870 . _:b350803930 "3"^^ . _:b350804054 . _:b350803798 . _:b350803941 "3"^^ . _:b350803921 . _:b350803793 . _:b350803940 "3"^^ . _:b350803792 . _:b350803943 "3"^^ . _:b350803795 . _:b350804238 . . _:b350803942 "3"^^ . . _:b350803794 . _:b350803937 "3"^^ . . _:b350803763 . . _:b350803805 . _:b350803936 "3"^^ . . . _:b350803804 . _:b350803939 "3"^^ . _:b350803821 . _:b350803807 . _:b350803938 "3"^^ . _:b350803991 . _:b350803806 . _:b350803949 "3"^^ . _:b350803801 . _:b350803948 "3"^^ . _:b350803800 . _:b350803951 "3"^^ . . _:b350803803 . _:b350803950 "3"^^ . . _:b350803916 . _:b2303232 . _:b350803802 . _:b350803945 "3"^^ . . _:b350804113 . _:b2303233 . _:b350803813 . _:b350803944 "3"^^ . . _:b2303234 . . _:b350803633 . _:b350803812 . _:b350803947 "3"^^ . . . _:b2303235 . _:b350803815 . _:b350803946 "3"^^ . _:b350803742 . . _:b350803814 . _:b350803957 "3"^^ . _:b350803809 . _:b350803956 "3"^^ . _:b350803809 . _:b350803808 . _:b350803959 "3"^^ . _:b350803811 . _:b350803958 "3"^^ . _:b350803810 . _:b350803953 "3"^^ . _:b350803821 . _:b350803952 "3"^^ . . . . . _:b350804220 . _:b350803820 . _:b350803955 "3"^^ . _:b350803920 . _:b350803823 . _:b350803954 "3"^^ . . . . _:b350803822 . _:b350803965 "3"^^ . _:b350803719 . _:b350803817 . _:b350803964 "3"^^ . . _:b350803816 . _:b350803967 "3"^^ . _:b2303191 . _:b350803819 . _:b350803966 "3"^^ . _:b350803973 . _:b350803888 . _:b350803818 . _:b350803961 "3"^^ . _:b350803972 . _:b350803829 . _:b350803960 "3"^^ . _:b350803975 . _:b350803828 . _:b350803963 "3"^^ . _:b350803635 . _:b350803974 . _:b350803831 . _:b350803962 "3"^^ . _:b350803969 . _:b350803975 . _:b350804260 . . _:b350803830 . _:b350803968 . _:b350803825 . _:b350803971 . . _:b350803824 . _:b350803970 . _:b350804320 . _:b350803827 . _:b350803981 . . _:b350803826 . _:b350803974 . _:b350803980 . . . _:b350804140 . _:b350803837 . . _:b350803983 . _:b350804203 . . _:b350803836 . _:b350803982 . _:b350803839 . _:b2303226 . _:b350803977 . . _:b350803838 . _:b350803749 . _:b350803976 . _:b350803833 . _:b350803979 . . . _:b350804340 . _:b350803832 . _:b2303231 . _:b2303221 _:b2303232 . _:b350803978 . _:b2303221 _:b2303233 . _:b2303221 _:b2303234 . _:b2303221 _:b2303235 . _:b350803835 . . _:b350803989 . . _:b350803834 . _:b350803988 . _:b350803845 . _:b350803991 . _:b350803844 . _:b350804045 . _:b350803990 . _:b350803847 . _:b350803985 . _:b350803625 . _:b2303160 "Such phosphorylation-induced alterations are important during DSB repair in vivo because mutations in the phosphorylation cluster cause severely increased radiation sensitivity and decreased DNA repair (Chan et al., 2002; Ding et al., >>2003<<; Soubeyrand et al., 2003)." . . _:b350803846 . . _:b350803984 . _:b350803891 . _:b350803841 . _:b350803987 . _:b350803725 . _:b350803840 . _:b350803986 . . . _:b350803843 . _:b350803776 . _:b350803997 . _:b350803842 . _:b350803996 . . _:b350803853 . _:b2303234 "Subsequently, when the DNA ends are correctly juxtaposed (Cui et al., >>2005<<), the DNA-PKCS molecules will autophosphorylate each other." . _:b350803999 . . _:b350803852 . _:b350803998 . _:b350803855 . . . _:b350803993 . _:b350803854 . _:b350803692 . _:b350803992 . _:b2303179 . _:b350803849 . _:b350803995 . _:b350803848 . . _:b350803994 . . _:b350803851 . _:b350804005 . _:b350803850 . _:b350804004 . _:b350803727 . _:b350803861 . _:b350804007 . _:b350803860 . . _:b350804006 . . _:b350803863 . _:b350804001 . _:b350803862 . _:b350804000 . _:b350803857 . _:b2303172 . _:b350804003 . _:b350803856 . _:b2303148 _:b2303172 . _:b350804002 . _:b2303148 _:b2303173 . _:b2303148 _:b2303174 . . _:b350804031 . _:b2303148 _:b2303175 . _:b350803730 . _:b350803859 . _:b2303148 _:b2303168 . _:b350804013 . _:b2303148 _:b2303169 . _:b350803677 . . _:b2303148 _:b2303170 . _:b2303148 _:b2303171 . _:b350803858 . _:b350804012 . _:b350804104 . _:b350803869 . _:b2303148 _:b2303176 . _:b350804015 . _:b2303148 _:b2303177 . _:b350803943 . _:b350803868 . _:b2303195 . _:b350804014 . _:b350803925 . _:b350803980 . _:b350803871 . _:b350804009 . _:b350803870 . _:b350804008 . . _:b350803865 . _:b2303196 . _:b350804011 . _:b350804085 . _:b350803864 . _:b350804010 . . _:b350803867 . _:b350804021 . . . _:b350803866 . . _:b350804020 . . _:b350803877 . _:b350803854 . _:b350804023 . _:b350803896 . _:b350803876 . _:b2303187 . _:b350804022 . _:b350804273 . _:b350803879 . . _:b350804017 . . _:b350803878 . _:b2303189 . _:b350804016 . _:b350804186 . . _:b350804308 . _:b350803873 . _:b2303188 . . _:b350804019 . . _:b350803872 . _:b350804018 . _:b350803681 . _:b350803875 . _:b2303190 . . _:b350804029 . . _:b350803965 . _:b350803874 . _:b350804028 . . _:b350803885 . _:b350804031 . _:b350803884 . _:b350803808 . _:b350804030 . _:b2303148 _:b2303156 . _:b2303148 _:b2303157 . _:b2303148 _:b2303158 . _:b2303148 _:b2303159 . _:b350803887 . _:b2303148 _:b2303152 . _:b2303195 "of DSBs that was produced during microirradiation, we compared the DNA-PKCS accumulation that was caused by our laser-system with the DNA-PKCS accumulation that was caused by an already calibrated microirradiation system (Mari et al., >>2006<<). In brief, we treated YFP-DNA-PKCS\u2013expressing cells with either our 365-nm laser system or with the 800-nm multiphoton laser system used by Mari et al. (2006), which is known to introduce between 1,000 and 1,500 DSBs." . _:b350803912 . _:b350804025 . _:b2303148 _:b2303153 . . _:b2303148 _:b2303154 . _:b2303148 _:b2303155 . _:b350803886 . _:b2303148 _:b2303164 . _:b2303235 . _:b2303210 "The Thr2609 and Thr2647 residues, which were previously reported to be autophosphorylation sites (Chan et al., 2002; Ding et al., >>2003<<), apparently do not require the DNA-PKCS kinase to be phosphorylated." . . _:b350804024 . . _:b2303148 _:b2303165 . . _:b2303148 _:b2303166 . _:b2303148 _:b2303167 . _:b350803881 . _:b2303148 _:b2303160 . _:b350804027 . _:b2303148 _:b2303161 . _:b350804425 . _:b2303148 _:b2303162 . _:b2303148 _:b2303163 . _:b350803880 . _:b350804026 . . _:b2303204 "Because mutation of DNA-PKCS phosphorylation sites is also known to interfere with DSB repair, clustered phosphorylation of DNA-PKCS is likely to play an important role during NHEJ (Chan et al., 2002; Ding et al., >>2003<<; Chen et al., 2005; Cui et al., 2005)." . _:b350803883 . _:b350804037 . _:b350804193 . _:b350803882 . . _:b350804036 . _:b2303148 _:b2303149 . . _:b2303148 _:b2303150 . _:b350804243 . _:b2303148 _:b2303151 . _:b350803893 . _:b350804039 . _:b2303188 "Mari et al. (>>2006<<) used an 800-nm multiphoton laser to induce DNA damage." . _:b350803892 . . _:b350804038 . _:b2303178 _:b2303180 . _:b2303178 _:b2303181 . _:b350803811 . _:b2303178 _:b2303182 . _:b350803951 . _:b2303178 _:b2303183 . _:b350803895 . . _:b350804033 . _:b2303178 _:b2303179 . _:b350803894 . _:b350803948 . . _:b350804032 . . . _:b350803889 . _:b350804035 . _:b2303178 "materials and methods" . _:b350804001 . _:b350803888 . . _:b350804034 . . _:b350803891 . _:b350803977 . _:b2303178 _:b2303192 . _:b350804045 . . _:b2303178 _:b2303193 . _:b350804153 . _:b350804345 . _:b350803890 . _:b2303178 _:b2303188 . _:b350804044 . _:b2303178 _:b2303189 . _:b2303178 _:b2303190 . _:b2303178 _:b2303191 . _:b350803901 . _:b2303178 _:b2303184 . _:b350804047 . _:b2303178 _:b2303185 . _:b2303178 _:b2303186 . . _:b2303178 _:b2303187 . _:b350803734 . _:b350803900 . _:b350804046 . _:b350803903 . . _:b350804041 . _:b350803902 . _:b350804040 . _:b350803737 . _:b2303235 "For example, we did not mutate the Ser3205 residue that has previously been identified as an autophosphorylation site (Douglas et al., >>2002<<). This site can still be phosphorylated in the 7A mutant, but not in the KD mutant." . _:b350803897 . _:b350804043 . _:b350803896 . _:b350804042 . . _:b350803899 . _:b350804053 . . _:b350803898 . _:b350804052 . _:b350803909 . _:b350804055 . _:b350803908 . _:b350804054 . _:b350803911 . . _:b350804165 . _:b350804049 . _:b350803723 . _:b350803910 . _:b350804048 . _:b350803856 . . _:b350803905 . _:b350804051 . _:b350803904 . _:b2303194 _:b2303196 . _:b350804050 . _:b2303194 _:b2303197 . _:b350803815 . _:b2303194 _:b2303198 . _:b2303194 _:b2303199 . _:b350803907 . _:b350804061 . . _:b2303194 _:b2303195 . _:b350803906 . _:b350804060 . _:b350803917 . _:b350804063 . _:b350803916 . _:b2303194 _:b2303212 . _:b350804062 . _:b2303194 _:b2303213 . _:b2303194 _:b2303214 . _:b2303194 _:b2303215 . _:b350803919 . _:b2303194 _:b2303208 . _:b350804057 . _:b2303194 _:b2303209 . _:b2303194 _:b2303210 . _:b2303194 _:b2303211 . _:b350803918 . _:b2303194 _:b2303204 . _:b350804056 . _:b2303194 _:b2303205 . _:b2303194 _:b2303206 . . _:b2303194 _:b2303207 . _:b350803913 . _:b2303194 _:b2303200 . _:b350804059 . . _:b2303194 _:b2303201 . _:b2303194 _:b2303202 . _:b2303194 _:b2303203 . _:b350803912 . . _:b350804058 . . _:b350803959 . _:b350803929 . _:b350804171 . _:b350803915 . _:b350804143 . . _:b350804069 . _:b350803914 . _:b350804364 . _:b350803698 . _:b2303194 _:b2303220 . _:b350804068 . . _:b350803925 . . _:b350803880 . _:b350804071 . _:b2303194 _:b2303216 . . _:b2303194 _:b2303217 . _:b2303194 _:b2303218 . _:b2303194 _:b2303219 . _:b350803924 . . _:b350804070 . . . . . _:b350803927 . _:b350804065 . _:b350803926 . _:b350804064 . _:b350803921 . _:b350804067 . _:b350803716 . . _:b350803920 . . _:b350804066 . . _:b350803686 . _:b350803923 . _:b350804077 . _:b350804192 . _:b350803922 . _:b350804076 . _:b350803740 . . _:b350803933 . _:b350804079 . . _:b350803932 . _:b2303170 "notion that DNA-PKCS functions as a \u201Cgatekeeper,\u201D which protects DNA ends from premature processing and ligation, until the two DNA ends are properly juxtaposed and DNA-PKCS autophosphorylation can take place (Weterings and van Gent, >>2004<<)." . _:b350804078 . . _:b350803935 . _:b350804107 . _:b350804073 . . . _:b350803934 . . _:b350804072 . . _:b350804061 . _:b350803929 . _:b350803812 . _:b350804075 . _:b350803928 . . _:b350804074 . _:b350803860 . _:b350803931 . _:b350804085 . _:b350804100 . _:b350803930 . _:b350804084 . _:b350803738 . _:b350803941 . _:b350804229 . . _:b350804087 . . _:b350803940 . _:b350804086 . _:b350803639 . _:b2303216 "Because XR1 cells are unable to efficiently repair DSBs (Li et al., >>1995<<), we expected the presence of DNA-PKCS to be maintained at the DSB site." . _:b350803943 . _:b350804331 . _:b350804091 . . _:b350804081 . _:b2303221 _:b2303222 . _:b2303221 _:b2303223 . _:b350803942 . _:b2303221 _:b2303224 . _:b350804080 . _:b2303221 _:b2303225 . _:b2303221 _:b2303226 . _:b2303221 _:b2303227 . _:b350803937 . _:b2303221 _:b2303228 . _:b350804083 . _:b2303221 _:b2303229 . _:b2303221 _:b2303230 . _:b2303221 _:b2303231 . _:b350803936 . _:b350804082 . _:b350803939 . _:b350804093 . _:b350804367 . _:b350803885 . _:b350803938 . _:b350804092 . . _:b350803949 . . _:b350804095 . _:b350803761 . . _:b350803948 . . _:b350804094 . _:b350803951 . _:b350804089 . . _:b350803950 . _:b350804088 . _:b350803945 . _:b350804091 . _:b350803944 . . _:b350804090 . _:b350803825 . . _:b350803947 . _:b350804101 . . _:b350803946 . _:b350804100 . _:b350803957 . . _:b350804103 . _:b2303168 "ligation can be relieved by DNA-PKCS autophosphorylation, indicating that autophosphorylation induces a conformational change in the DNA-PKCS molecule that liberates DNA ends (Weterings et al., 2003; Block et al., 2004; Reddy et al., >>2004<<; Cui et al., 2005)." . _:b350804195 . _:b350803956 . . _:b350804102 . _:b350803959 . _:b350804097 . _:b350803958 . _:b350804096 . _:b350803953 . _:b350804099 . _:b350803952 . _:b350804098 . _:b350804342 . _:b350803955 . _:b350803833 . _:b350804109 . . . . . _:b350803954 . _:b350804108 . "PMC0" . _:b350803965 . _:b350804111 . _:b350804209 . _:b350803964 . . _:b350804110 . _:b350803967 . _:b350804105 . _:b350803684 . . . . _:b350803966 . _:b350804104 . _:b350804366 . _:b350803961 . _:b350804107 . . _:b350803960 . _:b350804106 . _:b350803631 . _:b350803963 . _:b350804309 . . _:b350804117 . _:b350803962 . _:b350804116 . _:b350804119 . . _:b350804118 . . _:b350804113 . . _:b350804112 . _:b350804299 . _:b350804372 . . _:b350804115 . . _:b350803927 . _:b2303221 . . _:b350804114 . . _:b350803744 . _:b350804125 . _:b350804211 . _:b350804115 . _:b350804124 . _:b350804127 . . _:b350804126 . _:b350803630 . _:b350804121 . _:b350804120 . _:b350804123 . _:b350803926 . . _:b350804122 . . _:b2303194 . _:b350804095 . _:b350804133 . . _:b350804282 . . _:b350804132 . _:b350803790 . _:b350804135 . _:b350804134 . _:b2303178 . _:b350804129 . . _:b350804129 . _:b350804128 . _:b350803800 . _:b350804131 . . . _:b350803827 . _:b350804130 . _:b350803717 . _:b350803708 . _:b350804141 . . _:b350804406 . _:b350803724 . _:b350804140 . _:b350803971 . . _:b350804178 . . . _:b350804143 . _:b350803879 . _:b350804109 . _:b350804142 . _:b2303148 . _:b350804137 . _:b350804283 . . _:b350804136 . _:b350803904 . _:b350804139 . . _:b2303151 . _:b350804138 . _:b350803837 . . _:b350804149 . _:b350804230 . . _:b350804148 . . . _:b350804151 . . . _:b350804038 . _:b350804150 . _:b350804145 . . _:b350804413 . _:b350804144 . _:b350804253 . _:b350804147 . _:b350804146 . . _:b350804368 . . _:b350804213 . _:b350804157 . _:b350804156 . . . . _:b350804159 . . _:b350804158 . _:b350804153 . _:b350804222 . _:b350804152 . _:b350803641 . _:b350804155 . _:b350804154 . . _:b350804165 . _:b350803709 . _:b350804105 . _:b350803924 . _:b350804423 . _:b350804164 . _:b350803969 . . _:b350804167 . _:b350804352 . _:b350804066 . _:b350804166 . _:b350804161 . _:b350803770 . . _:b350804160 . _:b350803831 . _:b350804205 . _:b350804163 . _:b350803789 . _:b350804162 . _:b350804096 . _:b350804173 . . _:b350804172 . _:b350804251 . _:b350804013 . _:b350804175 . _:b350803979 . _:b350804174 . _:b350804227 . _:b350804169 . _:b350804168 . . _:b350804171 . _:b2303155 "The formation of a kinase-competent DNA-PK complex by Ku70/80 and DNA-PKCS requires simultaneous binding of these enzymes to a DNA terminus (Lees-Miller and Meek, >>2003<<; Weterings and van Gent, 2004). Because Ku70/80 has much higher affinity for DNA ends than DNA-PKCS, this heterodimer most likely binds to DNA termini first and subsequently attracts DNA-PKCS toward the DSB." . . . _:b350804170 . _:b350803772 . . _:b350804181 . _:b350804180 . . _:b350804183 . _:b350803987 . _:b350804182 . _:b350804177 . _:b350803857 . _:b350804176 . _:b350804180 . _:b350803668 . _:b350804179 . . . _:b350804178 . _:b350804138 . . _:b350804189 . _:b350803907 . _:b350804188 . _:b350803903 . . . _:b350804191 . . _:b350804190 . . _:b350804185 . _:b350804279 . _:b350804252 . . . _:b350804184 . . _:b350804187 . . _:b350804186 . _:b2303186 . _:b350804197 . _:b350804196 . . . _:b350804199 . _:b350804198 . _:b350803994 . _:b350804193 . . _:b350804192 . . _:b350803726 . _:b350804195 . . _:b350803699 . _:b350804194 . . _:b350804297 . _:b350804360 . _:b350804205 . _:b350804204 . _:b350804207 . _:b350804206 . _:b350804201 . _:b350804200 . _:b350804203 . _:b350803911 . _:b350804409 . _:b350804148 . . _:b350804055 . . _:b350804202 . _:b350804347 . _:b350804213 . . _:b350804212 . _:b350804197 . _:b350804215 . _:b350804214 . _:b2303219 "Because phosphorylation of both Thr2609 and Ser2056 is thought to be important for DSB repair via the NHEJ pathway (Chan et al., 2002; Ding et al., 2003; Chen et al., >>2005<<; Cui et al., 2005), we generated V3 cell lines that stably expressed YFP-tagged mutant forms of DNA-PKCS that could not be phosphorylated at those sites (Fig." . _:b350804209 . _:b350804324 . _:b350804223 . _:b350804208 . . _:b350804211 . _:b350804182 . _:b350803819 . _:b350804210 . _:b350804221 . . _:b350804220 . _:b350804383 . _:b350804288 . _:b350804212 . _:b350804223 . _:b350804222 . _:b350804226 . _:b350804303 . . _:b350804217 . _:b2303215 "of these cells to quickly repair the introduced DSBs because it is known that 7A and KD mutants display deficient DSB repair (Kurimasa et al., 1999; Kienker et al., 2000; Chan et al., 2002; Ding et al., 2003; Soubeyrand et al., >>2003<<). To verify this, we generated an XRCC4-deficient (XR1) cell line that expressed YFP-tagged WT DNA-PKCS." . _:b350803647 . _:b350804216 . _:b350804219 . _:b350804218 . _:b2303223 . _:b350803842 . . . . . _:b350803701 . _:b2303225 . _:b2303224 . . . . _:b350804110 . _:b350804208 . . . . . . _:b350804157 . . . _:b350804060 . . . . . . _:b350803863 . _:b350803886 . _:b350803824 . _:b350803703 . _:b350803865 . _:b350803851 . . . . _:b350804419 . . . _:b350803671 . _:b350804078 . _:b350803956 . _:b350804080 . _:b2303233 . _:b350803750 . . . . _:b350803868 . . . _:b350804198 . . _:b350804277 . _:b350804199 . _:b350803897 . _:b350803739 . . _:b350804268 . . _:b350804304 . . _:b350803876 . . . . . . _:b2303185 "DSBs were introduced in the nuclei of cultured cells by microirradiation with a pulsed nitrogen laser (Spectra-Physics; 365 nm, 10 Hz pulse) as previously described (Lan et al., >>2004<<, 2005). The laser system was directly coupled (Micropoint Ablation Laser System; Photonic Instruments, Inc." . . _:b350803752 . _:b2303232 "Biochemical experiments have demonstrated that unphosphorylated DNA-PKCS blocks DNA ends, and thereby inhibits efficient ligation (Weterings et al., >>2003<<; Block et al., 2004). This ligation block can be relieved by DNA-PKCS autophosphorylation, which induces a conformational change in the DNA\u2013protein complex that liberates the DNA ends." . _:b350804149 . _:b350803782 . . _:b350803839 . _:b350804191 . . . _:b350803642 . . . _:b350803787 . _:b2303228 . _:b350804266 . _:b350803679 . _:b350803950 . _:b350804161 . . . . _:b2303200 . . _:b2303182 "Anti-pS2056 and -pT2647 polyclonal antibodies and anti-pT2609 and -\u03B3H2AX monoclonal antibodies were generated as described previously (Chan et al., >>2002<<; Chen et al., 2005; Yajima et al., 2006)." . _:b350803931 . . . _:b350803786 . . . _:b2303163 "Several studies have shown that the presence of DNA-PKCS at DNA ends interferes with efficient ligation, most likely caused by the large dimensions of the protein molecule (Calsou et al., 1999; Weterings et al., >>2003<<; Block et al., 2004; Cui et al., 2005)." . . _:b350804257 . _:b350804233 . . . _:b350803629 . . . . . _:b350804044 . _:b350803628 . _:b350803840 . _:b350803631 . _:b2303208 "This is consistent with our previous finding that Ser2056 is an autophosphorylation site and that phosphorylation of this residue therefore requires the DNA-PKCS kinase activity (Chen et al., >>2005<<). The Thr2609 and Thr2647 residues, which were previously reported to be autophosphorylation sites (Chan et al., 2002; Ding et al., 2003), apparently do not require the DNA-PKCS kinase to be phosphorylated. It has recently been shown that" . _:b350803670 . _:b350803630 . . _:b350803748 . _:b350803779 . . _:b350803625 . . _:b350803627 . _:b350804398 . _:b350803626 . _:b350804075 . _:b2303161 "alterations are important during DSB repair in vivo because mutations in the phosphorylation cluster cause severely increased radiation sensitivity and decreased DNA repair (Chan et al., 2002; Ding et al., 2003; Soubeyrand et al., >>2003<<)." . _:b350803637 . . _:b350803636 . _:b350803639 . _:b350803638 . . _:b2303171 . _:b350803633 . _:b350803632 . _:b350803995 . _:b350803843 . _:b350803635 . _:b350803675 . _:b350803900 . _:b350803634 . . _:b350803645 . _:b2303226 "A recent study, in which cells were exposed to a comparable number of DSBs, showed conclusively that these cells were still able to complete cell division (Mari et al., >>2006<<). In addition, we would like to stipulate that the recruitment of DNA-PKCS in our system fully depends on the presence of Ku, ruling out the possibility of artificial, aspecific aggregation." . _:b350803846 . _:b350803644 . _:b350803647 . . _:b350803646 . _:b350804151 . . . _:b350803641 . _:b350804247 . . _:b2303229 "Some experiments have even suggested that DNA-PKCS is able to bind to DNA ends in the absence of Ku70/80 (Hammarsten and Chu, >>1998<<; Merkle et al., 2002)." . _:b350803640 . _:b350803810 . _:b350803643 . . _:b350803642 . . _:b350803653 . . _:b350803652 . . _:b350803655 . _:b350804087 . _:b350803654 . _:b350804400 . _:b350804285 . _:b350803649 . _:b350804370 . _:b350803648 . _:b350803784 . . _:b350803696 . _:b350803651 . _:b2303194 "results" . _:b350803650 . _:b350803661 . _:b350803774 . _:b350803660 . . _:b350803663 . _:b350803662 . _:b350803983 . _:b350803657 . _:b350803656 . _:b350803970 . _:b2303152 "At least two distinct pathways have evolved that mediate the repair of DSBs: homologous recombination (HR) and nonhomologous end-joining (NHEJ; Critchlow and Jackson, 1998; Kanaar et al., 1998; van Gent et al., 2001; Lieber et al., >>2003<<). NHEJ is considered to be the prevailing pathway during the G0 and G1 phases of the cell cycle in mammalian cells because this repair pathway does not require the presence of an intact DNA template." . . _:b350803659 . _:b350803658 . _:b350803669 . . _:b350803668 . _:b2303191 "expressing YFP-tagged DNA-PKCS, were irradiated with uranium particles with an initial energy of 11.4 MeV/nucleon (energy on the target cells, 3.8 MeV/nucleon) at the UNILAC facility at GSI, as previously described (Jakob et al., >>2000<<). Cells were cultured in a Petri dish on 40-\u03BCm-thick polycarbonate sheets. Duration of irradiation was <5 ms per sample." . . _:b350803671 . . _:b350803670 . _:b350804058 . _:b350803665 . _:b350804326 . _:b350804040 . _:b350803664 . _:b350804276 . _:b350803667 . _:b2303230 "Some experiments have even suggested that DNA-PKCS is able to bind to DNA ends in the absence of Ku70/80 (Hammarsten and Chu, 1998; Merkle et al., >>2002<<). We directly demonstrate that DNA-PKCS does not visibly accumulate at induced DSB sites in Ku80-deficient cells. Complementing these cells with Ku80 restores the ability of DNA-PKCS to accumulate. Collectively, our findings indicate that" . _:b350803666 . _:b350803944 . . . _:b350803677 . . . _:b350804069 . _:b350803676 . . . _:b350803679 . _:b350803961 . _:b350803678 . _:b350803905 . _:b2303193 . _:b350804428 . _:b350803673 . _:b2303192 . _:b350803672 . _:b350804225 . _:b350804183 . _:b350803675 . _:b350803674 . . _:b350804086 . . . _:b350803685 . _:b350803999 . _:b350803684 . _:b350803687 . _:b350803686 . _:b350803681 . _:b2303162 . _:b350803680 . _:b350803683 . . _:b350804160 . _:b350803682 . _:b350803693 . _:b2303153 "Two protein complexes make up the catalytic core of the NHEJ process: the DNA-dependent protein kinase holoenzyme (DNA-PK) and the DNA ligase IV\u2013XRCC4 complex (Lees-Miller and Meek, >>2003<<; Weterings and van Gent, 2004). Ligase IV\u2013XRCC4 mediates ligation of the juxtaposed DNA ends in the final NHEJ step." . . . _:b350803692 . . _:b2303203 "Because mutation of DNA-PKCS phosphorylation sites is also known to interfere with DSB repair, clustered phosphorylation of DNA-PKCS is likely to play an important role during NHEJ (Chan et al., >>2002<<; Ding et al., 2003; Chen et al., 2005; Cui et al., 2005)." . _:b350803695 . . _:b350804156 . . _:b350803694 . _:b350803814 . _:b350803689 . _:b350804016 . _:b350804319 . _:b350803688 . _:b350803691 . _:b350803690 . _:b350803701 . _:b350803700 . . _:b350803703 . . _:b350803702 . _:b350803895 . _:b350803697 . _:b350803982 . . . _:b350803696 . _:b350803769 . _:b350803699 . _:b2303173 "These mutations are known to interfere with efficient DSB repair (Kurimasa et al., >>1999<<; Kienker et al., 2000; Chan et al., 2002; Ding et al., 2003; Soubeyrand et al., 2003)." . _:b350803698 . . _:b350803872 . . _:b350803709 . . . _:b350803708 . _:b350803711 . _:b350803710 . . . . _:b350803705 . _:b350803704 . . _:b350803707 . _:b350803706 . _:b350804010 . _:b2303197 "We have previously shown that IR induces phosphorylation/autophosphorylation of the Ser2056 and Thr2609 amino acid residues of DNA-PKCS (Chan et al., >>2002<<; Chen et al., 2005)." . . _:b350804270 . . _:b2303148 . _:b350803804 . . _:b2303149 . . _:b2303150 . . . . _:b350804071 . _:b2303177 "These mutations are known to interfere with efficient DSB repair (Kurimasa et al., 1999; Kienker et al., 2000; Chan et al., 2002; Ding et al., 2003; Soubeyrand et al., >>2003<<)." . _:b2303151 . . . _:b350803710 . _:b350803871 . _:b350804384 . _:b350803805 . _:b350804385 . _:b350804386 . . _:b350804387 . _:b350804388 . _:b350804070 . _:b350804389 . _:b350804390 . _:b2303170 . _:b350804391 . _:b2303156 . _:b350803813 . _:b350804392 . _:b350804393 . _:b350804394 . _:b350804141 . . _:b2303157 . _:b350804395 . _:b350804396 . _:b350804397 . _:b350804398 . _:b350804399 . _:b2303158 . _:b350804400 . . _:b350804401 . _:b350804402 . _:b350804403 . _:b2303159 . _:b350804404 . _:b350804405 . . _:b350804406 . . _:b2303152 . _:b350804407 . _:b350803899 . _:b350804408 . _:b350804409 . _:b350804410 . . _:b2303153 . _:b350804411 . _:b350804412 . _:b350804067 . _:b350804413 . _:b350804414 . _:b350804415 . _:b2303154 . _:b350804352 . _:b350804353 . _:b350804354 . _:b350804399 . _:b350804355 . _:b2303155 . _:b350804356 . _:b350804121 . _:b350804102 . _:b350804357 . _:b350804358 . _:b350804359 . _:b2303164 . . _:b350804360 . _:b350804024 . _:b350804361 . . _:b350804362 . _:b350804363 . _:b2303165 . _:b350804364 . _:b350804365 . _:b350804366 . _:b350804367 . _:b2303166 . _:b350804368 . _:b350804369 . _:b350804370 . _:b350804371 . _:b2303167 . _:b350804372 . _:b350804373 . _:b350804374 . _:b350804375 . _:b2303160 . _:b350804376 . _:b350804377 . _:b350804378 . _:b350804379 . _:b2303161 . _:b350804380 . _:b350804381 . _:b350804382 . _:b350804383 . _:b2303162 . . . _:b2303163 . _:b350804027 . _:b2303172 . _:b350803914 . _:b350803759 . _:b2303173 . _:b350804300 . _:b350804323 . _:b2303174 . _:b2303175 . _:b350804391 . _:b2303168 . _:b2303169 . . _:b350804355 . . _:b2303170 . _:b350804416 . . _:b350804417 . . _:b350804418 . . _:b350804419 . _:b2303171 . _:b350804420 . _:b350804421 . _:b350804422 . _:b350804423 . _:b2303180 . . _:b350804424 . . _:b350803853 . _:b350804425 . _:b350804426 . . _:b350804427 . _:b2303181 . _:b350804428 . _:b350803733 . _:b350804429 . _:b350803996 . _:b350804430 . _:b350804431 . _:b2303182 . . _:b350803712 . _:b350804358 . _:b2303183 . _:b2303192 "Kinetic measurements of the recruitment of YFP-DNA-PKCS at sites of ion traversals were performed using a beamline microscope (Jakob et al., >>2005<<) equipped with a 7190\u201353 EBCCD-camera (Hamamatsu)." . _:b2303176 . . _:b350804232 . _:b2303177 . . _:b350804301 . _:b350804236 . _:b350804256 . _:b2303178 . _:b350804257 . _:b350804258 . . _:b350804259 . _:b2303179 . _:b350804260 . _:b350804261 . _:b350804262 . _:b350804263 . _:b2303188 . _:b350803667 . _:b350804264 . _:b350804265 . . _:b350804266 . . _:b350803972 . _:b350803967 . _:b350804267 . _:b2303189 . _:b350804268 . _:b350804269 . _:b2303166 "This inhibition of ligation can be relieved by DNA-PKCS autophosphorylation, indicating that autophosphorylation induces a conformational change in the DNA-PKCS molecule that liberates DNA ends (Weterings et al., >>2003<<; Block et al., 2004; Reddy et al., 2004; Cui et al., 2005)." . _:b350804270 . _:b350804271 . _:b2303190 . _:b350804272 . _:b2303189 "We microirradiated several YFP-DNA-PKCS\u2013expressing V3 cells with either our 365-nm laser system or with the microirradiation system used by Mari et al. (>>2006<<; both systems are based on LSM 510 confocal microscopes, set to provide comparable background and fluorescence intensity levels)." . _:b350804273 . _:b350803672 . _:b350804274 . _:b350803973 . _:b350804275 . _:b2303191 . _:b350804276 . _:b350804277 . . _:b350804278 . _:b350804049 . . _:b350804279 . _:b2303184 . _:b2303150 . . _:b350804280 . _:b350804281 . _:b350804418 . _:b350804282 . _:b350804283 . _:b2303185 . _:b350804284 . _:b350804285 . _:b350804286 . _:b350804287 . _:b2303186 . _:b350804224 . _:b350804206 . _:b350804225 . _:b350804226 . _:b350804227 . _:b2303187 . _:b350803859 . _:b350804228 . _:b350803938 . _:b350804229 . _:b2303184 . _:b350804230 . _:b350804231 . _:b2303196 . . _:b350804232 . _:b350804162 . _:b350804233 . _:b350804234 . _:b350804235 . _:b2303197 . _:b350804236 . _:b350804237 . _:b350804238 . _:b350804239 . _:b2303198 . _:b350804240 . _:b350804241 . _:b350804242 . . _:b350804243 . _:b2303199 . _:b350804244 . _:b350804245 . _:b350804246 . _:b350804247 . _:b2303192 . _:b350804248 . _:b350804249 . _:b350804250 . _:b350804251 . _:b2303193 . _:b350804252 . . _:b350804253 . _:b350804254 . _:b350804255 . _:b350804320 . _:b2303194 . _:b350804321 . _:b350804322 . . _:b350804323 . _:b2303195 . . _:b350803918 . _:b350804324 . _:b350804325 . _:b350804326 . _:b350804327 . _:b2303204 . _:b350804365 . _:b350804185 . _:b350804328 . _:b350804329 . _:b350804330 . _:b350804331 . _:b2303205 . _:b350804332 . _:b350804333 . _:b350804334 . _:b350804335 . _:b2303206 . . _:b350804336 . _:b350804337 . _:b350804338 . _:b350804339 . _:b2303207 . _:b350804327 . _:b350803947 . _:b350804020 . _:b350804340 . _:b350804341 . _:b350804342 . _:b350804343 . _:b2303200 . _:b2303179 "CHO cell lines AA8 (WT), V3 cells (defective in DNA-PKcs expression; Whitmore et al., >>1989<<), and Xrs6 cells (defective in Ku80 expression; Denekamp et al., 1989) were maintained at 37\u00B0C in \u03B1-minimum Eagle's medium with 10% FCS, 100 units/ml penicillin, and 100 \u03BCg/ml streptomycin (HyClone)." . _:b350804344 . _:b350803909 . _:b350804345 . _:b350804346 . _:b350804278 . _:b350804347 . _:b2303201 . _:b350804127 . _:b350804348 . _:b350804349 . _:b350804350 . _:b350804351 . _:b2303202 . _:b350804288 . _:b350804289 . _:b350804290 . _:b350804291 . _:b2303203 . _:b350804292 . _:b350804293 . _:b350804294 . _:b350803715 . _:b350804295 . _:b2303212 . _:b350804296 . _:b350804297 . _:b350803655 . _:b350804298 . _:b350804299 . _:b2303213 . _:b350804300 . _:b350804301 . . _:b350804302 . _:b350804303 . _:b2303214 . . _:b350804304 . _:b350804305 . . _:b350804306 . . _:b350804307 . _:b2303215 . _:b350803682 . _:b350804308 . _:b2303164 "Several studies have shown that the presence of DNA-PKCS at DNA ends interferes with efficient ligation, most likely caused by the large dimensions of the protein molecule (Calsou et al., 1999; Weterings et al., 2003; Block et al., >>2004<<; Cui et al., 2005)." . _:b350804309 . _:b350804310 . . _:b350803908 . _:b350804311 . _:b2303208 . _:b350804312 . _:b350804313 . . . _:b350804314 . . _:b350804315 . _:b2303209 . . _:b350804316 . _:b350804254 . _:b350804150 . . _:b350804317 . _:b350804318 . _:b350804319 . _:b2303210 . _:b350804128 . _:b350803954 . _:b350804129 . _:b350804130 . _:b350804131 . _:b2303211 . _:b350804132 . _:b350804133 . _:b350804134 . _:b350803768 . _:b350804135 . _:b2303220 . _:b350804136 . _:b350804137 . _:b350804138 . _:b350804384 . _:b350804139 . _:b350804140 . _:b2303221 . _:b350804141 . . . _:b350804142 . _:b350804143 . _:b2303222 . _:b350804144 . . _:b350804145 . _:b350804146 . _:b350804147 . _:b2303223 . _:b350803775 . _:b350803834 . _:b350804148 . _:b350804246 . _:b350804149 . _:b350804150 . . _:b350804151 . _:b2303216 . _:b350804210 . _:b350804152 . . _:b350804153 . . _:b350804154 . _:b350804155 . _:b2303217 . _:b350804156 . _:b350804157 . _:b350804158 . _:b350804281 . _:b350804159 . _:b2303218 . . _:b350804096 . _:b350804097 . _:b350804098 . _:b350803669 . _:b350803932 . _:b2303219 . _:b350804099 . _:b350804100 . . . _:b350804101 . _:b350803922 . . _:b350804102 . _:b2303150 "At least two distinct pathways have evolved that mediate the repair of DSBs: homologous recombination (HR) and nonhomologous end-joining (NHEJ; Critchlow and Jackson, 1998; Kanaar et al., >>1998<<; van Gent et al., 2001; Lieber et al., 2003)." . _:b350804103 . _:b2303228 . _:b350804104 . _:b350804105 . _:b350803803 . _:b350804106 . _:b350804107 . _:b2303229 . _:b350804108 . _:b350804109 . _:b350804110 . _:b350804111 . _:b2303230 . _:b350804112 . . _:b350804113 . _:b350804114 . _:b350804175 . _:b350804115 . _:b2303231 . _:b350804116 . _:b350803981 . _:b350804117 . _:b350804118 . _:b350803852 . _:b350804119 . _:b2303224 . _:b350804120 . _:b350804121 . _:b350804122 . _:b2303215 . _:b350804123 . _:b2303225 . _:b350804124 . _:b350804125 . _:b350804126 . . _:b350804127 . _:b2303226 . _:b350804192 . _:b350804193 . _:b350804194 . _:b350804195 . _:b2303227 . _:b350804196 . _:b350804197 . . _:b350804198 . _:b350804199 . _:b350804200 . _:b350804201 . _:b350804202 . _:b350804203 . . _:b350804204 . _:b350804205 . . _:b350804206 . . _:b350804207 . _:b350804208 . _:b350804209 . _:b350804210 . _:b350804211 . _:b350804212 . _:b350804213 . _:b350804214 . _:b350804215 . . _:b350804216 . _:b350804217 . _:b350804218 . _:b350804219 . _:b350804220 . _:b350804221 . _:b350804222 . . . _:b350804223 . _:b350804160 . . _:b350803923 . _:b350804161 . _:b350804162 . _:b350804163 . _:b350804164 . . _:b350804165 . _:b350804166 . _:b350804167 . _:b350804168 . . _:b350804169 . _:b350804170 . _:b350804171 . _:b350804172 . _:b350804173 . _:b350804174 . _:b350804144 . _:b350804175 . _:b350804176 . _:b350804043 . _:b350804177 . _:b350804178 . . _:b350804179 . _:b350804180 . _:b350804181 . . . _:b350804182 . _:b2303199 "Many in vitro studies have shown that the Ku70/80 heterodimer directs DNA-PKCS to DNA ends and stabilizes the DNA\u2013DNA-PKCS interaction (Smith and Jackson, >>1999<<). To investigate whether the recruitment of DNA-PKCS to DSB sites is dependent on the Ku protein in vivo, we generated a Ku80-deficient Xrs6 cell line that stably expressed YFP-DNA-PKCS." . _:b350804183 . _:b350804408 . _:b350804184 . _:b350804185 . _:b350804186 . _:b350804187 . _:b350804188 . _:b350804189 . _:b350804190 . _:b350804191 . _:b350804000 . _:b350804001 . _:b350804002 . _:b350804003 . _:b350804004 . . _:b350804005 . _:b350804306 . _:b350804302 . . _:b350804006 . _:b350804007 . . _:b350804008 . _:b350803704 . . _:b350804009 . _:b350804010 . _:b350804011 . . _:b350804012 . _:b350804013 . _:b350804014 . _:b350804015 . . _:b350804016 . . . _:b350804017 . . _:b350804018 . _:b350804019 . _:b350804020 . _:b350804021 . _:b350804022 . _:b350804023 . _:b350804024 . _:b350804025 . _:b350804026 . _:b350804027 . _:b350804028 . . _:b350804029 . _:b350804030 . _:b350804031 . _:b350803968 . _:b350803969 . _:b350803970 . _:b350803971 . _:b350803972 . _:b350803973 . _:b350803974 . _:b350803975 . _:b350803976 . _:b350803977 . . _:b350803978 . _:b350803979 . . . _:b350803980 . _:b350803981 . _:b350803982 . _:b350803983 . _:b350803984 . _:b350803894 . _:b2303221 "discussion" . _:b350803985 . _:b350803986 . . _:b350803987 . _:b350804291 . _:b350803988 . _:b350803989 . . _:b350803990 . . . _:b350803991 . _:b350803992 . _:b350803993 . _:b350804062 . _:b350803994 . _:b350804173 . _:b350803995 . _:b350803996 . _:b350803997 . . _:b350803998 . _:b2303156 . _:b350803826 . _:b350803662 . _:b350803999 . _:b350804064 . _:b350804065 . _:b350804066 . _:b2303177 . _:b350804067 . _:b350804068 . _:b350804069 . . _:b2303154 . _:b350804070 . _:b350804071 . _:b350804072 . _:b350804073 . _:b350804074 . _:b350804075 . _:b350804076 . _:b350804077 . _:b350804078 . . _:b350804079 . _:b350804080 . _:b350804081 . _:b350804082 . _:b350803964 . _:b350804083 . _:b350804084 . _:b350804085 . _:b350804086 . _:b350804087 . _:b350804088 . _:b350804089 . _:b2303176 "These mutations are known to interfere with efficient DSB repair (Kurimasa et al., 1999; Kienker et al., 2000; Chan et al., 2002; Ding et al., >>2003<<; Soubeyrand et al., 2003)." . _:b350804090 . _:b350804091 . _:b350804092 . _:b350804093 . _:b350804094 . . _:b350804095 . _:b350804032 . _:b350804033 . _:b350804034 . _:b350803673 . _:b350804035 . . _:b350804036 . . _:b350804037 . _:b350804038 . . _:b350804039 . _:b350804040 . _:b350804041 . _:b350804042 . _:b350804043 . _:b350804044 . _:b350804045 . _:b350804046 . _:b350804047 . _:b350804048 . _:b350804049 . . _:b350804050 . _:b350804051 . _:b350804052 . _:b350804053 . _:b350804036 . _:b350804054 . _:b350804055 . _:b350804056 . _:b350804057 . _:b350804058 . _:b350804059 . _:b350804060 . _:b350803835 . _:b350804061 . _:b350803693 . _:b350804062 . . _:b350804063 . . _:b2303161 . _:b350803963 . _:b350803646 . _:b350804295 . . . . . _:b350804123 . . _:b350804414 . . _:b2303180 "CHO cell lines AA8 (WT), V3 cells (defective in DNA-PKcs expression; Whitmore et al., 1989), and Xrs6 cells (defective in Ku80 expression; Denekamp et al., >>1989<<) were maintained at 37\u00B0C in \u03B1-minimum Eagle's medium with 10% FCS, 100 units/ml penicillin, and 100 \u03BCg/ml streptomycin (HyClone)." . . . . . _:b350803721 . . _:b350804170 . _:b2303166 . . _:b350804274 . _:b350804217 . _:b350803689 . . _:b350804371 . _:b2303163 . . . _:b2303198 "We have previously shown that IR induces phosphorylation/autophosphorylation of the Ser2056 and Thr2609 amino acid residues of DNA-PKCS (Chan et al., 2002; Chen et al., >>2005<<). Immunostaining with phosphospecific antibodies revealed that both the Ser-2056 and Thr-2609 residues were phosphorylated in the microirradiated region (Fig. 1 E), indicating that the laser-induced DNA lesions are capable of inducing a" . _:b350804116 . _:b350803801 . . _:b350804330 . _:b350803747 . _:b350803989 . . . . . _:b2303217 "Because phosphorylation of both Thr2609 and Ser2056 is thought to be important for DSB repair via the NHEJ pathway (Chan et al., >>2002<<; Ding et al., 2003; Chen et al., 2005; Cui et al., 2005), we generated V3 cell lines that stably expressed YFP-tagged mutant forms of DNA-PKCS that could not be phosphorylated at those sites (Fig." . _:b350803992 . _:b350803957 . _:b2303205 "Because mutation of DNA-PKCS phosphorylation sites is also known to interfere with DSB repair, clustered phosphorylation of DNA-PKCS is likely to play an important role during NHEJ (Chan et al., 2002; Ding et al., 2003; Chen et al., >>2005<<; Cui et al., 2005). To test whether kinase activity and clustered phosphorylation of DNA-PKCS are important for its recruitment to DSB sites, we generated two V3 cell lines that stably expressed YFP-tagged mutant forms of DNA-PKCS." . . _:b350804362 . _:b350803785 . _:b350803736 . _:b350804172 . _:b350803666 . . . _:b350803958 . _:b350804280 . _:b350803674 . . . _:b350803648 . . _:b350804351 . . . _:b350804430 . . _:b350804190 . . . _:b350804259 . _:b350804137 . . _:b350804007 . _:b350804201 . _:b350804427 . _:b350804215 . . _:b350803791 . _:b350804353 . _:b350804079 . . _:b350804112 . . _:b350803973 "2"^^ . _:b350803972 "2"^^ . _:b350803975 "2"^^ . _:b350803974 "2"^^ . . _:b350803969 "3"^^ . _:b350804388 . _:b350804019 . . _:b350803968 "3"^^ . . _:b2303218 "Because phosphorylation of both Thr2609 and Ser2056 is thought to be important for DSB repair via the NHEJ pathway (Chan et al., 2002; Ding et al., >>2003<<; Chen et al., 2005; Cui et al., 2005), we generated V3 cell lines that stably expressed YFP-tagged mutant forms of DNA-PKCS that could not be phosphorylated at those sites (Fig." . _:b350803971 "3"^^ . _:b2303202 "It has previously been shown that the kinase activity of DNA-PKCS is essential during NHEJ-mediated DSB repair in mammalian cells (Kurimasa et al., 1999; Kienker et al., >>2000<<). Because mutation of DNA-PKCS phosphorylation sites is also known to interfere with DSB repair, clustered phosphorylation of DNA-PKCS is likely to play an important role during NHEJ (Chan et al., 2002; Ding et al., 2003; Chen et al.," . _:b350803970 "3"^^ . _:b350804390 . _:b350803981 "2"^^ . _:b2303157 "It is, however, well established that DNA-PKCS has the ability to autophosphorylate itself at a cluster of 6 phosphorylation sites between the Thr2609 and Thr2647 amino acid residues (Douglas et al., >>2002<<), as well as at an additional site outside this cluster, the Ser2056 residue (Chen et al., 2005)." . _:b350803858 . _:b350803878 . _:b350804329 . _:b350803980 "2"^^ . . _:b350803983 "2"^^ . . _:b350803982 "2"^^ . . . . . _:b350803977 "2"^^ . . _:b350803976 "2"^^ . _:b350804155 . _:b350803979 "2"^^ . . _:b350803978 "2"^^ . _:b350803913 . . _:b350804403 . _:b350803989 "2"^^ . _:b350803988 "2"^^ . _:b350803991 "2"^^ . _:b350803990 "2"^^ . _:b350803985 "2"^^ . _:b350803797 . _:b350803984 "2"^^ . . _:b350803976 . _:b350804296 . _:b350803898 . _:b350804130 . _:b350803987 "2"^^ . . _:b2303199 . _:b350803986 "2"^^ . _:b350803997 "2"^^ . _:b350803915 . _:b350803996 "2"^^ . . _:b350803999 "2"^^ . . _:b350803998 "2"^^ . . . _:b350803993 "2"^^ . _:b350803992 "2"^^ . _:b350803995 "2"^^ . _:b2303212 "accumulation area in mutant cells most likely reflects the inability of these cells to quickly repair the introduced DSBs because it is known that 7A and KD mutants display deficient DSB repair (Kurimasa et al., 1999; Kienker et al., >>2000<<; Chan et al., 2002; Ding et al., 2003; Soubeyrand et al., 2003). To verify this, we generated an XRCC4-deficient (XR1) cell line that expressed YFP-tagged WT DNA-PKCS." . _:b350804118 . _:b350804145 . _:b350803994 "2"^^ . . . _:b350804005 "2"^^ . _:b350804004 "2"^^ . _:b2303186 "DSBs were introduced in the nuclei of cultured cells by microirradiation with a pulsed nitrogen laser (Spectra-Physics; 365 nm, 10 Hz pulse) as previously described (Lan et al., 2004, >>2005<<). The laser system was directly coupled (Micropoint Ablation Laser System; Photonic Instruments, Inc." . _:b350804007 "2"^^ . _:b350804006 "2"^^ . _:b350804028 . . . _:b350803829 . _:b350804001 "2"^^ . _:b350804000 "2"^^ . _:b2303180 . . _:b350804003 "2"^^ . _:b350804002 "2"^^ . _:b350804426 . . _:b350803939 . _:b350804013 "2"^^ . . _:b350804012 "2"^^ . . . _:b350803658 . _:b350804015 "2"^^ . _:b350803760 . _:b350804402 . . _:b350804014 "2"^^ . _:b350804009 "2"^^ . _:b350803966 . . _:b350804008 "2"^^ . . _:b350804011 "2"^^ . _:b350804147 . . _:b350804010 "2"^^ . _:b350804051 . . _:b350804021 "2"^^ . _:b350804020 "2"^^ . _:b350804023 "2"^^ . _:b350804264 . _:b350804022 "2"^^ . _:b350804017 "2"^^ . _:b350804016 "2"^^ . _:b350804132 . _:b350804019 "2"^^ . . . _:b350804018 "2"^^ . _:b350804029 "2"^^ . _:b350803862 . _:b350803788 . _:b350804028 "2"^^ . _:b350804031 "2"^^ . _:b350804030 "2"^^ . _:b350803861 . _:b350803882 . _:b350803937 . _:b350804025 "2"^^ . . _:b350804024 "2"^^ . . _:b350804027 "2"^^ . _:b350804026 "2"^^ . . _:b350803657 . . _:b350804311 . _:b350804037 "2"^^ . _:b350803953 . . _:b350804036 "2"^^ . _:b350804039 "2"^^ . _:b350804242 . _:b350804038 "2"^^ . _:b350804033 "2"^^ . . _:b350804032 "2"^^ . . . _:b350804014 . _:b350804035 "2"^^ . _:b350803660 . . . _:b350804034 "2"^^ . _:b350804045 "2"^^ . _:b350803817 . _:b2303156 "The formation of a kinase-competent DNA-PK complex by Ku70/80 and DNA-PKCS requires simultaneous binding of these enzymes to a DNA terminus (Lees-Miller and Meek, 2003; Weterings and van Gent, >>2004<<). Because Ku70/80 has much higher affinity for DNA ends than DNA-PKCS, this heterodimer most likely binds to DNA termini first and subsequently attracts DNA-PKCS toward the DSB." . . _:b350804044 "2"^^ . _:b350804289 . _:b350803807 . _:b350804047 "2"^^ . . _:b350804046 "2"^^ . _:b350804393 . . _:b350804041 "2"^^ . . _:b350804021 . _:b350804040 "2"^^ . . . . _:b350803875 . _:b350804043 "2"^^ . . . _:b350804042 "2"^^ . _:b350804053 "2"^^ . _:b350803942 . _:b350804052 "2"^^ . _:b350804055 "2"^^ . . . . _:b350804054 "2"^^ . . _:b350804049 "2"^^ . _:b350804082 . _:b350804128 . _:b350804048 "2"^^ . _:b2303187 "the number of DSBs that were induced by microirradiation, we compared the DNA-PKCS accumulation that was caused by our 365-nm laser system with the DNA-PKCS accumulation that was caused by a previously calibrated system (Mari et al., >>2006<<). Mari et al. (2006) used an 800-nm multiphoton laser to induce DNA damage." . _:b350804051 "2"^^ . _:b350804386 . _:b350804050 "2"^^ . _:b350803661 . _:b350804061 "2"^^ . _:b350803650 . _:b350804060 "2"^^ . _:b350804344 . . _:b350804063 "2"^^ . _:b2303174 . _:b350803751 . . _:b350804062 "2"^^ . . _:b350804057 "2"^^ . _:b350804056 "2"^^ . _:b350803930 . _:b350804059 "2"^^ . _:b350803794 . _:b2303153 . . . _:b350804058 "2"^^ . _:b2303158 . _:b2303175 . _:b350804069 "2"^^ . _:b350803893 . _:b2303155 . _:b350804068 "2"^^ . _:b350804189 . . _:b350804071 "2"^^ . _:b350803685 . _:b350803757 . _:b350804070 "2"^^ . _:b350804005 . _:b350804065 "2"^^ . _:b350803743 . _:b350803745 . . _:b350804064 "2"^^ . . . _:b350804067 "2"^^ . _:b350804269 . _:b2303182 . . _:b350804066 "2"^^ . _:b350804077 "2"^^ . _:b350804356 . _:b350804076 "2"^^ . . _:b350804079 "2"^^ . _:b350804078 "2"^^ . . _:b350804073 "2"^^ . _:b350804072 "2"^^ . . _:b350804075 "2"^^ . . _:b350804387 . _:b350804056 . _:b2303233 "Biochemical experiments have demonstrated that unphosphorylated DNA-PKCS blocks DNA ends, and thereby inhibits efficient ligation (Weterings et al., 2003; Block et al., >>2004<<). This ligation block can be relieved by DNA-PKCS autophosphorylation, which induces a conformational change in the DNA\u2013protein complex that liberates the DNA ends." . _:b350804074 "2"^^ . . _:b350803697 . _:b350804085 "2"^^ . _:b2303184 "Anti-pS2056 and -pT2647 polyclonal antibodies and anti-pT2609 and -\u03B3H2AX monoclonal antibodies were generated as described previously (Chan et al., 2002; Chen et al., 2005; Yajima et al., >>2006<<). Anti-\u03B3H2AX polyclonal antibody was purchased from Cell Signaling Technology. Secondary antibodies (anti\u2013mouse or\u2013rabbit conjugated with Alexa Fluor 488/350 or rhodamine) were purchased from Invitrogen. DNA ends in irradiated cells were" . _:b350804084 "2"^^ . _:b350803632 . . _:b350804087 "2"^^ . . _:b350803890 . _:b350804086 "2"^^ . . . . . _:b2303165 . _:b350804081 "2"^^ . . . _:b2303212 . . . _:b350804080 "2"^^ . _:b2303193 "Uranium ion tracks were visualized by etching polycarbonate, as described previously (Jakob et al., >>2005<<)." . _:b350803732 . _:b350804083 "2"^^ . _:b350803652 . . _:b350804082 "2"^^ . _:b350804093 "2"^^ . _:b350803799 . . _:b2303207 "In one mutant form (KD), we impaired the kinase ability (Kurimasa et al., >>1999<<). In the other mutant (7A), we substituted all of the 7 major phosphorylation sites (Thr2609-Thr2647 and Ser2056) with alanine residues, impairing phosphorylation of these sites (Fig. 3 A). Cellular expression of both mutant proteins was" . _:b350804092 "2"^^ . _:b2303197 . _:b350804095 "2"^^ . _:b350804412 . _:b350804314 . _:b350804094 "2"^^ . _:b350803968 . _:b350804089 "2"^^ . _:b350804088 "2"^^ . . _:b350804091 "2"^^ . . _:b2303202 . _:b350803643 . _:b2303201 "It has previously been shown that the kinase activity of DNA-PKCS is essential during NHEJ-mediated DSB repair in mammalian cells (Kurimasa et al., >>1999<<; Kienker et al., 2000)." . _:b350804090 "2"^^ . _:b350804133 . _:b350804101 "2"^^ . . . _:b350804100 "2"^^ .