Ionizing radiations cause DNA double strand breaks (DSBs) which are potentially the most lethal lesions causing genomic rearrangements and cancer if left unrepaired. Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ) pathways are the major repair mechanisms observed in mammalian cells. HR is usually dominant in S and G2 phases of the cell cycle as it requires a pair of sister chromatids for error-free replication; whereas NHEJ is observed in all the stages.
According to the classical model, Ku70/80 heterodimer is recognized as a sensor to first bind the DSB site and recruit DNA dependent protein kinase catalytic subunit (DNA-PKcs) which brings the broken ends in synapsis. The nucleases like Artemis and DNA polymerases like λ and μ process the DNA ends to attain adequate homology before ligation. XRCC4-DNA Ligase IV complex along with the newly identified molecule, XLF/Cernunnos (XRCC4-like factor), plays an important role in the final end-joining step.
The dynamics of this repair machinery is yet to be clarified. We have been using detergent fractionation method to capture the radiation induced chromatin bound complex. 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. This chromatin recruitment of XRCC4 could be seen immediately after irradiation and remained almost constant up to 4hr after 20Gy irradiation. Quantitative estimation revealed that a very small percentage of the XRCC4-Ligase IV complex was recruited to each DNA end; thus explaining the complications involved in the detection of the NHEJ machinery at the damaged site.
In addition, we are exploring the mechanism for recruitment of the ligation complex considering the plausible roles of protein modifications induced by radiation. We have also found some evidence for the requirement of DNA-Ligase IV for the recruitment of XRCC4 at the site of damaged chromatin.
Currently, we are also isolating a higher order chromatin bound complex associated with XRCC4. This complex suggestively congregates with some histone molecules. Such a striking association of histone molecules with XRCC4 on the damaged chromatin site leads to a speculation that they are structurally reorganized and may be involved in the recruitment dynamics.