title: XRCC4 controls nuclear import and distribution of Ligase IV and exchanges faster at damaged DNA in complex with Ligase IV.
abstract: Non-homologous end-joining (NHEJ) is one major pathway for the repair of double-stranded DNA breaks in mammals. Following break recognition, alignment and processing, broken DNA ends are finally rejoined by the essential DNA Ligase IV. In the cell, Ligase IV is unable to function without its constitutive interaction partner XRCC4 and becomes unstable when it is missing, and it has been assumed that XRCC4 may also be required for recruitment of Ligase IV to repair sites. To investigate the function of complex formation between both proteins directly in the living cell, we stably expressed them as bio-fluorescent fusion proteins in human HT-1080 cell clones. Ligase IV or XRCC4 were expressed either alone or both were co-expressed at a roughly equimolar ratio. Labelled proteins were overexpressed manifold in comparison to endogenously expressed proteins. We show that over-expressed Ligase IV was only partially imported into the nucleus and showed a diffuse distribution there, whereas XRCC4 expressed alone was entirely nuclear with a distinct exclusion from nucleoli. When Ligase IV was co-expressed with XRCC4, both proteins formed the natural complex, and Ligase IV was not only efficiently imported but also resembled the sub-nuclear distribution of XRCC4. In addition, Ligase IV, when in complex with XRCC4, acquired a delayed nuclear reimport after mitotic cell division of XRCC4. We further determined by photobleaching the kinetics with which the proteins exchange at UVA laser-irradiated nuclear sites between damage-bound and diffusing states. We found that the dynamic exchange rate of the Ligase IV/XRCC4 complex at micro-irradiated sites was faster than that of XRCC4 expressed alone. In summary, our findings demonstrate a novel function of XRCC4 in controlling nuclear import and sub-nuclear distribution of Ligase IV, and they suggest that XRCC4 modulates the dynamic interaction of the Ligase IV/XRCC4 complex with the NHEJ machinery at double-stranded DNA breaks.
authors: Institute of Clinical Chemistry and Laboratory Diagnostics, Univ Düsseldorf, Medical Faculty, D-40225 Düsseldorf, Germany.
authors: Elke
authors: E
authors: Berg
authors: Morten O
authors: MO
authors: Christensen
authors: Ilaria
authors: I
authors: Dalla Rosa
authors: Ellen
authors: E
authors: Wannagat
authors: Reiner U
authors: RU
authors: Jänicke
authors: Lennart M
authors: LM
authors: Rösner
authors: Wilhelm G
authors: WG
authors: Dirks
authors: Fritz
authors: F
authors: Boege
authors: Christian
authors: C
authors: Mielke
mesh_terms: D021581:Active Transport, Cell Nucleus; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D059766:DNA End-Joining Repair; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D055785:Gene Knockdown Techniques; D006801:Humans; D007700:Kinetics
chemical_list: D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C101467:XRCC4 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP