**1. Introduction**

156 Selected Topics in DNA Repair

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DNA double-strand breaks are the most detrimental form of DNA damage induced by either endogenous and exogenous sources. DNA double-strand breaks are generated in response to ionizing radiation, radiomimic drugs, and topoisomerase inhibitors. They also created during V(D)J and class switch recombination in lymphocytes, meiotic recombination in germ cells, and by retroviral integrations (Downs et al., 2007, Polo and Jackson, 2011). Since DNA double-strand breaks discontinue chromosome structure, they may result in cell death that are associated with radiosensitivity, immunodeficiency, neurodegeneration, and developmental defects (Jackson and Bartek, 2009, Mahaney et al., 2009, O'Driscoll and Jeggo, 2006, Weterings and Chen, 2007, Wyman and Kanaar, 2006). Thus, cells have evolved sophisticated mechanisms, by which DNA double-strand breaks are repaired. Two major pathways to repair DNA double-strand breaks are non-homologous end-joining and homologous and homologous recombination (Hartlerode and Scully, 2009, Mahaney et al., 2009, Weterings and Chen, 2007, Wyman and Kanaar, 2006). While rejoining of DNA breaks are indispensable for the survival of cells, DNA repair, by itself, may threaten the stability of the genome. (Burma et al., 2006, Pastink et al., 2001, Sonoda et al., 2006, van Gent et al., 2001). In particular, non-homologous end-joining, which is the primary DNA repair pathway functions in G1 phase, is error-prone (Hartlerode and Scully, 2009, Lieber, 2010). It causes loss or rearrangement of the genetic information through mis-rejoining of DNA double strand breaks. Processing of DNA broken ends by exonucleases and endonucleases also provide another chance to alter DNA sequences. Consequently, surviving cells can avoid lethal effects of DNA double-strand breaks but it results in a loss of heterozygosity as well as gross genome rearrangements that are associated with cancer predisposition. Although most genome rearrangements have been thought to be generated directly by the initial radiation exposure (Leonhardt et al., 1999), recent findings have demonstrated that the integrity of the genome is also endangered eventually, if the cells were survived exposure to DNA damaging agents. In this chapter, the results showing that delayed DNA double-strand breaks are induced several generations after the initial insult in the progenies of surviving cells are presented, and a role of non-homologous end-joining on delayed

Involvement of Non-Homologous End-Joining in Radiation-Induced Genomic Instability 159

result of receiving signals from irradiated cells (Mothersill and Seymour, 2004, Prise and O'Sullivan, 2009). A variety of responses have been described including DNA damage induction, chromosomal instability, and cell death. As bystander effects have been observed in coculture of irradiated and unirradiated cells, and after the transfer of medium from irradiated cells to unirradiated cells, secreted factor(s) may be involved in transducing the bystander signals (Sowa and Morgan, 2004). It has been hypothesized that increased secretion of transforming growth factor beta results in stimulation of production of reactive oxygen species through a membrane NADPH oxidase. In fact, previous study demonstrated that transforming growth factor beta increased oxidative stress through decreased activity of

Although oxidative stress is surely involved in perpetuation of radiation-induced genomic instability (Azzam et al., 2003, Coates et al., 2008, Miller et al., 2008, Kim et al., 2006a, Limoli et al., 2003, Wright, 2007), alternative mechanisms could be associated with manifestation of radiation-induced genomic instability in non-hematopoietic cells. We have shown that delayed unscheduled induction of DNA double strand breaks is involved in the manifestation of delayed phenotypes (Suzuki et al., 2003). In fact, our study indicated that increased phosphorylated histone H2AX foci, which correspond to DNA double-strand breaks, were frequently detected in the progeny of normal human diploid cells surviving Xrays. Moreover, delayed reactivation of p53 in response to DNA damage was manifested in the surviving clones. Delayed induction of DNA double strand breaks was also confirmed by delayed induction of chromosomal aberrations (Toyokuni et al., 2009). Thus, it is evidenced that induction of DNA double strand breaks is induced indirectly in surviving cells from exposure to radiation, indicating that DNA repair pathways could play roles in

Previously, Chang and Little reported that radiation-induced genomic instability was absent in xrs5 cells, which are NHEJ-deficient Chinese hamster cells defective in Ku80 protein (Chang and Little, 1992a). Interestingly, delayed reproductive death was not observed in these cells, even though they harbor sufficient amount of DNA double-strand breaks. Since the mechanism of delayed reproductive death has not been fully described yet, we have hypothesized that defective NHEJ in xrs5 cells decreases the chance of mis-rejoining of the broken ends, which result in the formation of dicentric chromosomes involved in division halt. To test this possibility we examined delayed chromosomal instability in two NHEJdefective cells, xrs-5 and xrs-6 cells, and compared the frequency with the wild-type CHO cells. Furthermore, delayed induction of dicentric chromosomes was examined in cells

Non-homologous end-joining is one of the two major pathways involved in amending DNA double-strand breaks in multicellular eukaryotes. It primarily plays a critical role during G0-, G1- and early S-phase of cell cycle (Lieber 2010, Polo and Jackson, 2011). Non-homologous end-joining is initiated by binding a heterodimeric protein complex consists of Ku80 and Ku70 to both ends of DNA double-strand breaks (Jackson and Bartek, 2009, Mahaney et al., 2009, O'Driscoll and Jeggo, 2006, Weterings and Chen, 2007, Wyman and Kanaar, 2006). Then, DNA-PKcs, a catalytic subunit of DNA-dependent protein kinase, is recruited to Ku-DNA complex, which results in activation of the protein kinase activity of DNA-PKcs and tethering two broken DNA ends. DNA-PKcs phosphorylates a number of proteins,

defective in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).

mitochondrial complex IV (Kim et al., 2006b).

amending delayed DNA double-strand breaks in surviving cells.

**3. Non-homologous end-joining and its mutants** 

manifestation of radiation effects and the integrity of the genome in the cells surviving radiation exposure will be discussed.
