Ku recruits XLF to DNA double-strand breaks.
XRCC4-like factor (XLF)--also known as Cernunnos--has recently been shown to be involved in non-homologous end-joining (NHEJ), which is the main pathway for the repair of DNA double-strand breaks (DSBs) in mammalian cells. XLF is likely to enhance NHEJ by stimulating XRCC4-ligase IV-mediated joining of DSBs. Here, we report mechanistic details of XLF recruitment to DSBs. Live cell imaging combined with laser micro-irradiation showed that XLF is an early responder to DSBs and that Ku is essential for XLF recruitment to DSBs. Biochemical analysis showed that Ku-XLF interaction occurs on DNA and that Ku stimulates XLF binding to DNA. Unexpectedly, XRCC4 is dispensable for XLF recruitment to DSBs, although photobleaching analysis showed that XRCC4 stabilizes the binding of XLF to DSBs. Our observations showed the direct involvement of XLF in the dynamic assembly of the NHEJ machinery and provide mechanistic insights into DSB recognition.
Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, 5801 Forest Park Road, Dallas, TX 75390-9187, USA.
Ken-ichi
K
Yano
Keiko
K
Morotomi-Yano
Shih-Ya
SY
Wang
Naoya
N
Uematsu
Kyung-Jong
KJ
Lee
Aroumougame
A
Asaithamby
Eric
E
Weterings
David J
DJ
Chen
D034961:Antigens, Nuclear / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D007834:Lasers; D011485:Protein Binding; D013816:Thermodynamics
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D004247:DNA; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D045643:DNA Repair Enzymes