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  • In our recent study we established a procedure to examine


    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
    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 Agarose GPG/ME sale 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. LIG4-CT contains two BRCT domains and XIR in between. BRCT domain is shown to be a phosphopeptide binding module [30], [31]. ATM and DNA-PKcs are thought to phosphorylate a number of proteins, including histone H2AX, in response to DNA damage, phosphorylated H2AX or other protein might be a bait of BRCT domains in LIG4 (Fig. 4D). If this is the case, it is expected that depletion of ATM and/or DNA-PKcs would reduce chromatin binding of XRCC4/LIG4 complex. However, we and others demonstrated that the chromatin binding of XRCC4 was not affected by wortmannin, a potent inhibitor of both of ATM and DNA-PK [21], [22]. As another possibility, it was reported that LIG4 interacts with Ku via BRCT-I [19] (Fig. 4D). In the present study, however, W725R mutation in BRCT-I did not affect the chromatin binding of LIG4-CT but the chromatin binding of XRCC4 was diminished (Fig. 3B). It might be expected that the interaction between LIG4-CT and XRCC4 is weakened by W725R mutation. However, the interaction between XRCC4 with W725R mutant and non-mutated LIG4-CT was similar in co-immunoprecipitation experiments (data not shown). Considering this, BRCT-I might have a role in stabilizing XRCC4 on chromatin. XRCC4 can then interact with XLF (Fig. 4D). Nevertheless, as we introduced mutation in putative phosphopeptide binding pocket, it remains possible that another interface of BRCT-I mediates the interaction between LIG4 and Ku. It may appear contradictory that the chromatin binding of LIG4 was considerably reduced by W725R mutation in the full-length context, but not in the C-terminal context. In this regard, we would note that an electron microscopy study suggested the juxtaposition of LIG4-NT to XRCC4 [10]. Although it is currently unclear whether there is a direct, physical interaction between LIG4-NT and XRCC4, possible tertiary interaction among LIG4-CT, XRCC4 and LIG4-NT, which would be lost in W725R mutant, might firm the complex.