抄録
Ionizing radiations cause DNA double strand breaks (DSBs), potentially the most lethal lesions if left unrepaired. Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ) pathways are the major repair mechanisms observed in mammalian cells.
The core players of NHEJ are recognized as the Ku70/80 heterodimer as one of the initial sensors to bind the DSB site and recruit DNA dependent protein kinase catalytic subunit (DNA-PKcs) which brings the broken DNA ends in synapsis. The nucleases like Artemis and DNA polymerases like λ and μ process the DNA ends to attain adequate homology before the final end-joining step by XRCC4-DNA Ligase IV-XLF complex.
The exact hierarchy and the complex dynamics of this repair machinery are yet to be clarified. We have been using a detergent fractionation method to capture the radiation induced chromatin bound complex. This revealed that a subpopulation of XRCC4 changed into an extraction resistant form that was liberated by micrococcal nuclease treatment, indicating that it had been tethered to chromatin DNA.
We further isolated the higher order chromatin bound complex associated with XRCC4 by immunoprecipitation. Western blotting and mass spectrometric analyses of the immunoprecipitate demonstrated the presence of DNA ligase IV, histone and its interactor. Several ribonucleoproteins were also observed. Such striking associations of XRCC4 on the damaged chromatin site leads to a speculation that the NHEJ repair pathway is much more sophisticated and complex than hitherto comprehended.
