Another emerging approach is the

Another emerging approach is the live cell imaging, tracking the behavior of the fluorescently labeled proteins after laser micro irradiation [2]. Mari et al. demonstrated that the accumulation of XRCC4 in irradiated area was dependent on Ku but not on DNA-PKcs [23]. Yano et al. showed that the accumulation of XRCC4 in irradiated area could be observed, but was significantly reduced, in Erastin lacking DNA-PKcs, indicating the role of DNA-PKcs in stabilizing XRCC4 on chromatin [24]. Moreover, XRCC4 kinetics in kinase-dead DNA-PKcs-expressing cells were similar to normal DNA-PKcs expressing cells, suggesting that DNA-PKcs might play a scaffolding, rather than a catalytic, role [24]. Recent studies by Rulten et al. and Grundy et al. indicated that APLF is recruited to damage site via interaction with Ku and/or PARP-3 and, in turn, promotes the recruitment or retention of XRCC4 [25], [26]. In our recent study, we established a procedure to examine the chromatin binding of XRCC4 using a biochemical fractionation analysis using a detergent Nonidet P-40 [22]. In this study, we investigated the role of LIG4 and its subdomains in the recruitment of XRCC4/LIG4 complex to chromatin.
Materials and methods
Results
Discussion In the present study, we first demonstrated that the chromatin binding was diminished in LIG4−/− and could be restored by introduction of LIG4 cDNA (Fig. 1). This is in agreement with a study by Drouet et al. showing that the chromatin binding of XRCC4 was reduced in cells from Ligase IV syndrome patient [21]. Their study and the present study indicated that the chromatin binding of XRCC4 is dependent on LIG4. Thus, the mechanisms driving XRCC4/LIG4 complex to the chromatin would lie in LIG4 rather than XRCC4. LIG4-CT could bind to chromatin and also restore chromatin binding of XRCC4 in LIG4−/− (Fig. 3). The ability of C-terminal region, in addition to DNA binding domain in N-terminus, to interact with chromatin might have an implication in the mechanisms of DSB repair through NHEJ. Presence of dual DNA binding domain would enable “hooking” another DNA end (Fig. 4B). It might be noted that a recent electron microscopic study showed the end-bridging by human or yeast XRCC4/LIG4 complex, engaging two Ku-bound DNA ends [32]. Alternatively, LIG4 may move along DNA with DNA-binding domain and then anchor at DSB via C-terminal region (Fig. 4C). In both of these models, we infer that LIG4-CT might bind selectively or preferentially to the DSB. This is based on the observation that the chromatin binding of LIG4-CT was enhanced by irradiation (Fig. 3A and C) and also on the lines of evidence suggesting the potential of LIG4-CT to interact with proteins, which are assumed to exist at DSB (see the next paragraph and Fig. 4D). However, the chromatin binding of LIG4-CT could be observed even in unirradiated cells. There are two possible explanations for this observation. First, LIG4-CT might bind to spontaneously damaged chromatin. Second, LIG4-CT can bind to and slide along undamaged chromatin but binds more strongly to damaged chromatin. We cannot presently distinguish these possibilities.