**7. Conclusion**

166 Selected Topics in DNA Repair

manifestation of radiation-induced genomic instability. Then the question should be about the mechanism. One possible explanation of the defective induction of some delayed phenotypes is that error-free DNA repair, such as homologous recombination, reduced the incidence of transmissible damage in the absence of error-prone non-homologous endjoining repair. If so, delayed induction of DNA double strand breaks should be lower in Ku80-defective cells than the control CHO cells. Therefore, we checked whether delayed DNA damage was less frequent in xrs5 cells. The results clearly indicated that it was not the case. Thus, even without Ku80-dependent repair, genomic instability by itself could be induced in the progenies of surviving cells. The second possibility is that defective DNA repair in xrs5 cells decreased the chance of mis-rejoining of the broken ends that occurred many generations after the initial insult. In fact, delayed induction of chromosome bridges between two daughter cells was significantly reduce in xrs-5 and xrs-6 cells (Figure 6). Furthermore, delayed induction of dicentric chromosomes was completely absent in both xrs-5 and xrs-6 cells (Figure 8). Although several studies have reported that chromosome breakages are more frequent in Ku80-deficient cells (Darroudi and Natarajan, 1987, Kemp and Jeggo, 1986), the frequency of dicentric chromosome was relatively low considering the frequency of chromosome breaks. These results support our conclusion that the formation of dicentric chromosome caused by delayed DNA damage was compromised in Ku80-deficient cells. Although a back-up non-homologous end-joining may undertake mis-rejoining of broken ends in the absence of Ku80-dependent DNA repair (Iliakis et al., 2004), it is highly likely that a major pathway of illegitimate rejoining the DNA breaks is Ku80-dependent process (Liang et al., 1996). We also confirmed that radiation-induced genomic instability was manifested in cells derived from DNA-PKcs-defective *Scid* mouse. Moreover, delayed dicentric formation was normally detected in *Scid* cells. Therefore, DNA-PK-independent rejoining, which was suggested previously (Gao et al., 1998), is involved in delayed dicentric formation. Recently, it has been postulated that XRCC4/DNA Ligase IV-dependent but DNA-PKcs-independent rejoining needs Ku80/70 complex. Thus, it is highly possible that Ku80-dependent mis-rejoining is involved in delayed generation of dicentric chromosomes, by which chromosome bridges is generated. Such mis-rejoining inhibits segregation of two daughter cells, which results in delayed induction of giant cells as well as delayed

It has been well described that genomic instability, which is known as gross chromosomal rearrangement, is commonly observed in yeast. Gross chromosomal rearrangements manifest as translocations, chromosomal deletions, and inversions, indicating that they could be means to accelerate multiple genetic alterations associated with carcinogenesis (Kolodner et al., 2002). Although multiple pathways cooperate to suppress gross chromosomal rearrangement, homologous recombination plays a pivotal role in avoiding gross chromosomal rearrangement (Myung et al., 2001). Moreover, the restrained recruitment of homologous recombination proteins has been reported to promote gross chromosomal rearrangement. Thus, nonhomologous end-joining has little effect on gross chromosomal rearrangement in yeast.

It should be very interesting to know the consequence of cells harboring mis-rejoined chromosomes. Previously, it was reported that ionizing radiation induced genomic instability in the progeny of surviving CHO cells, which resulted in a heritable mutator phenotypes. For example, mutation frequency at the hypoxanthine-guanine phosphoribosyltransferase locus in surviving clones was persistently higher than the unirradiated progenies (Lim et al., 2000). It was expected that such mis-rejoining caused large deletions at the gene locus, however, multiplex polymerase chain reaction analysis revealed that point mutations are the

reproductive death.

Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, it was reported that delayed cell death was absent in Ku80-deficient cells. We have proved that this is because delayed induction of dicentric chromosomes is significantly compromised in those cells. In fact, reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and

Involvement of Non-Homologous End-Joining in Radiation-Induced Genomic Instability 169

Darroudi, F. & Natarajan, A.T. (1987). Cytogenetical characterization of Chinese hamster ovary

Downs, J.A.; Nussenzweig, M.C. & Nussenzweig, A. (2007). Chromatin dynamics and the

Gao, Y.; Chaudhuri, J., Zhu, C., Davidson, L., Weaver, D.T. & Alt, F.W. (1998). A targeted

Hartlerode, A.J. & Scully, R. (2009). Mechanisms of double-strand break repair in somatic

Huang, L.; Snyder, A.R. & Morgan, W.F. (2003). Radiation-induced genomic instability and

Huang, L.; Kim, P.M., Nickoloff, J.A. & Morgan, W.F. (2007). Targeted and nontargeted

Iliakis, G.; Wang, H., Perrault, A.R., Boecker, W., Rosidi, B., Windhofer, F., Wu, W., Guan, J.,

Jackson S.P. & Bartek, J. (2009). The DNA-damage response in human biology and diseases. *Nature*, Vo.461, No.7267, (October 2009), pp. 1071-1078, ISSN 0028-0836 Jeggo, P.A. (1998). Identification of genes involved in repair of DNA double-strand breaks in

Kadhim, M.A.; MacDonald, D.A., Goodhead, D.T., Lorimore, S.A., Marsden, S.J. & Wright,

Kastan, M.B. & Bartek, J. (2004). Cell-cycle checkpoints and cancer. *Nature*, Vol.432, No.7015,

Kemp, L.M. & Jeggo, P.A. (1986). Radiation-induced chromosome damage in X-ray-sensitive

Kim, G.J.; Chandrasekaran, K. & Morgan, W.F. (2006a). Mitochondrial dysfunction,

No.1-4, (November 2004), pp. 14-20, ISSN 1424-8581

(November 2004), pp. 316-323, ISSN 0028-0836

No.3, (November 1986), pp. 255-263, ISSN 0027-5107

*Research*, Vol.177, No.1, (March 1987), pp. 133-148, ISSN 0027-5107

958, ISSN 0028-0836

ISSN 0264-6021

ISSN 0033-7587

368, ISSN 0267-8357

9993

0072

2003), pp. 5848-5854, ISSN 0950-9232

X-ray-sensitive mutant cells xrs 5 and xrs 6. I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine. *Mutation* 

preservation of genetic information. *Nature*, Vol.447, No.7147, (June 2007), pp. 951-

DNA-PKcs-null mutation reveals DNA-PK-independent functions for KU in V(D)J recombination. *Immunity*, Vol.9, No.3, (September 1998), pp. 367-376, ISSN 1074-7613

mammalian cells. *The Biochemical Journal*, Vol.423, (September 2009), pp. 157-168,

its implications for radiation carcinogenesis. *Oncogene*, Vol.22, No.45, (October

effects of low-dose ionizing radiation on delayed genomic instability in human cells. *Cancer Research*, Vol.67, No.3, (February 2007), pp. 1099-1104, ISSN 0008-5472 Huertas, P. (2010). DNA resection in eukaryotes: deciding how to fix the break. *Nature* 

*Structual and Molecular Biology*, Vol.17, No. 1, (January 2010), pp. 11-16, ISSN 1545-

Terzoudi, G. & Pantelias, G. (2004). Mechanisms of DNA double strand break repair and chromosome aberration formation. *Cytogenetic Genome Research*, Vol.104,

mammalian cells. *Radiation Research*, Vol.150, No.5, (November 1998), pp. 80-91,

E.G. (1992). Transmission of chromosomal instability after plutonium alpha-particle irradiation. *Nature*, Vol.355, No.6362, (February 1992), pp. 738-740, ISSN 0028-0836 Kastan, M.B. & Lim, D.S. (2000). The many substrates and functions of ATM. *Nature Reviews.* 

*Molecular and Cellular Biology*, Vol.1, No.3, (December 2000), pp. 179-186, ISSN 1471-

mutants (xrs) of the Chinese hamster ovary cell line. *Mutation Research*, Vol.166,

persistently elevated levels of reactive oxygen species and radiation-induced genomic instability: a review. *Mutagenesis*, Vol.21, No.6, (October 2006), pp. 361-

delayed cell death. Thus, our current study demonstrated that DNA repair pathway is an important determinant of cellular response to ionizing radiation not only in the immediate response but also in cells surviving radiation exposure. Survived cells induced DNA double strand breaks many generations after the initial insult. Although the mechanism of delayed DNA damage induction has to be determined, delayed dicentric formation indicated that delayed DNA damage was induced in G1 phase. Such delayed DNA damage could be repaired by NHEJ repair, but it also provided a chance to engender mis-rejoining. These results should bring a new insight into how DNA repair protects the integrity of the genome from the insults of DNA damaging agents.
