Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV.
XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4(K271R) mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4(K210R) mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3'-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4(K271R) was defective in the nuclear localization of itself and LIG4, whereas XRCC4(K210R) was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4(K271R), but not M10-XRCC4(K210R), showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4(WT). The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4(K271R) than in M10-XRCC4(WT), whereas it was only marginally increased in M10-XRCC4(K210R) as compared to M10-XRCC4(WT). The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4.
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Mikoto
M
Fukuchi
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Rujira
R
Wanotayan
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Sicheng
S
Liu
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Shoji
S
Imamichi
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Mukesh Kumar
MK
Sharma
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan. Electronic address: yoshim@nr.titech.ac.jp.
Yoshihisa
Y
Matsumoto
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C101467:XRCC4 protein, human; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP; D008239:Lysine
DNA double-strand break repair; DNA ligase IV; Non-homologous end-joining; Nuclear localization; XRCC4