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  • Mutation in BRCT II domain W R either in the


    Mutation in BRCT-II domain (W893R) either in the full-length context or the C-terminal context greatly reduced the expression of LIG4 (Fig. 2B, Fig. 3B), suggesting the importance of BRCT-II domain in the maintenance of LIG4. In this regard, it might be noted that LIG4 protein was undetectable in Ligase IV syndrome patients harboring ccr2 inhibitor heterozygote mutation R580X and R814X [33]. The former mutation would result in the production of LIG4 protein completely lacking both of BRCT-I and BRCT-II domains and the latter mutation would result in the production of LIG4 protein lacking most part of BRCT-II domain. Moreover, it was recently reported that BRCT-II domain, in addition to XIR domain, was required for stable interaction between LIG4 and XRCC4 [34].
    Acknowledgments We thank Mr. Isao Yoda for cell irradiation and the laboratory members, especially Ms. Masami Kitamura, for assistance in cell culture and ccr2 inhibitor experiments. This work was supported by Grant-in-Aid for Young Scientists (A) (21689033). Initial part of this study is the result of “Study on Initiating Events in the Recognition and Repair of DNA Double-Strand Breaks” carried out under the Strategic Promotion Program for Basic Nuclear Research by the Ministry of Education, Culture, Sports, Science and Technology of Japan.
    Introduction DNA damage repair is essential life processes for maintaining the integrity and stability of genomic DNA, in which many enzymes, bio-molecules and ribozymes participate (Friedberg, 2003, Sancar et al., 2004, McCabe et al., 2006). In particular, the ligation reaction of 5′-phosphate and 3′-hydroxyl terminus by DNA ligase is the key step during DNA damage repair (Karimi-Busheri et al., 1998). However, many DNA breaks which are generated by ionizing radiation, toxic agents or nucleases often results in DNA strands with 3′-phosphate and 5′-hydroxyl termini (Allinson, 2010, Caldecott, 2008, Hu et al., 1998). 5′-hydroxylated termini and 3′-phosphate can not be ligated by DNA ligase and failed to ligation repair, resulting in some serious consequences (Whitehouse et al., 2001). Therefore, the pre-phosphorylated of 5′-hydroxyl termini by polynucleotide kinase (PNK), and dephosphorylated of 3′-phosphate termini by phosphatase are indispensable before ligase-mediated DNA repair. Nevertheless, alterations of ligase activity or PNK activity, as well as phosphatase activity could influence the DNA ligation repair and involved in the pathogeny of various diseases (such as Bloom's syndrome, Werner syndrome et al.) (Nishizuka, 1984, Yu et al., 2009; Blundred et al., 2011; Orimo, 2010). Therefore, in order to further understand the ligase-mediated DNA repair progress and the biological functions of DNA phosphorylation, and screen DNA repair enzymes-targeted drugs, it is crucial to develop some simple, convenient, and sensitive detection methods for monitoring these repair enzymes activity. The traditional methods for these enzymes assays are mainly based on radio isotope 32P-labeling, polyacrylamide gel electrophoresis, and autoradiography (Bernstein et al., 2005, Karimi-Busheri et al., 1999, Meijer et al., 2002, Odell and Shuman, 1999). Although these methods work well both in vitro and in vivo detecting systems, complicated procedures and the potential radioactive health hazards issues, vastly limit their applications. Recently, to solve these problems, great efforts have been made and various sensing strategies have been explored, such as fluorescence, colorimetry, chemiluminescence, bioluminescence and electrochemistry (Hou et al., 2013, Jiang et al., 2014, He et al., 2014, Du et al., 2014, Zhang et al., 2016). Notably, the fluorescent method is the most popular and attractive due to their operation easy and intuitive, design flexibility, and ease of signal readout. Up to now, the fluorescence-based strategies mainly depend on organic dyes directly or indirectly. For instance, Song and co-workers described a novel fluorescence approach for real-time monitoring PNK activity based on singly FAM labeled DNA-hairpin smart probe coupled with λ exonuclease cleavage (Song and Zhao, 2009). Guo et al. reported a novel fluorescence approach for detecting DNA ligase and polynucleotide kinase activity based on toehold-mediated strand displacement and split G-quadruplex probes (Guo et al., 2015). Our groups have investigated the progresses of nucleic acid phosphorylation, nucleic acid dephosphorylation, and nucleic acid ligation using TAMRA labeled molecular beacon with high selectivity (Tang et al., 2003, Tang et al., 2005, Ma et al., 2007). Although these methods have their own advantage, they usually rely on toxic and costly dye-labeled DNA probes or complicated probe design. Therefore, more avenues still need to be explored for monitoring DNA repair enzymes activity.