**5. Role of non-homologous end-joining on radiation-induced genomic instability**

Radiation-induced genomic instability has been reported commonly in various cell systems including human and rodent cells (Little, 2003, Lorimore et al., 2003, Morgan et al., 1996, Suzuki et al., 2003). However, Chang and Little demonstrated that delayed reproductive death, one characteristic manifestation of radiation-induced genomic instability, was not observed in Ku80-deficient xrs5 cells (Chang and Little, 1992a). The authors suggested that the cellular processing of DNA double strand breaks during repair must play a role in delayed reproductive death. In fact, our current study confirmed their results and found that not only delayed cell death but also delayed induction of giant cells and chromosome bridge were absent in xrs-5 cells (Figures 5 and 6). Furthermore, other Ku80-deficient cell line, xrs6, also revealed deficiency in the induction of those delayed phenotypes. Thus, it becomes clear that Ku80-dependent non-homologous end-joining is involved in the

Involvement of Non-Homologous End-Joining in Radiation-Induced Genomic Instability 167

predominant type of genetic alterations in the mutants (Chang and Little, 1992b). Because cells with micronuclei were frequently observed among the surviving cells, persistent break induction, which may not be involved in delayed cell death under the p53-dysfunctional condition (Tzang et al., 1999), may be resulted from mis-rejoined chromosomes. In fact, previous study demonstrated that delayed chromosomal instability was mediated by bridgebreakage-fusion cycle, which could also be involved in the perpetuation of radiation-induced genomic instability. It has been proved that ionizing radiation induces delayed genomic instability, which accumulates genetic alterations including gene mutations, loss of heterozygosity, and chromosome rearrangements, concurrently with delayed reproductive death. As Ku80-deficiency compromised delayed cell death through the formation of dicentric chromosomes, it is likely that such cells with defective DNA repair capacity are more

**6. Molecular nature of radiation signature involved in radiation-induced** 

Radiation-induced genomic instability is transmitted through many generations after the initial X-irradiation, indicating that there should be some mechanism(s), by which the initial radiation insults are memorized (Suzuki, 1997). As delayed DNA double-strand breaks are identified in surviving cells, persistent DNA damage is one candidate for radiation signature. In fact, a recent study has proved that DNA double-strand breaks induced by very low dose of X-rays remain unrepaired for many days in confluent cells (Rothkamm and Lobrich, 2003). Furthermore, if persistent DNA is radiation signature, radiation-induced genomic instability is more prevailing in xrs-5 and xrs-6 cells compared with CHO cells. But, we observed radiationinduced genomic instability at similar level between these cells (Suzuki et al., 2009). Thus, these results exclude the possibility that radiation signature is DNA double-strand breaks *per se*. Previously, we compared delayed aberrations of X chromosome with or without large deletion at the hypoxanthine-quanine-phosphorybosyltransferase gene, which is located at Xq26.1 (Toyokuni et al., 2009, Suzuki et al., 2003). Because cells defective in the gene were able to grow in the presence of 6-thioguanine, we cloned 6-thioguanine resistant clones after 3 Gy of X-rays, and examine gene deletion. We found some of the resistant clones had large deletions expanding over several megabases. Interestingly, these clones with large deletions showed higher probability to induce delayed chromosomal instability. Thus, altered higherorder chromatin structure could be a candidate for radiation signature. It is possible that such altered higher-order chromatin structure results in replication stress, which causes DNA

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

susceptible to carcinogenesis induced by DNA damaging agents.

**genomic instability** 

double-strand breaks.

**7. Conclusion** 

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 reproductive death.

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 predominant type of genetic alterations in the mutants (Chang and Little, 1992b). Because cells with micronuclei were frequently observed among the surviving cells, persistent break induction, which may not be involved in delayed cell death under the p53-dysfunctional condition (Tzang et al., 1999), may be resulted from mis-rejoined chromosomes. In fact, previous study demonstrated that delayed chromosomal instability was mediated by bridgebreakage-fusion cycle, which could also be involved in the perpetuation of radiation-induced genomic instability. It has been proved that ionizing radiation induces delayed genomic instability, which accumulates genetic alterations including gene mutations, loss of heterozygosity, and chromosome rearrangements, concurrently with delayed reproductive death. As Ku80-deficiency compromised delayed cell death through the formation of dicentric chromosomes, it is likely that such cells with defective DNA repair capacity are more susceptible to carcinogenesis induced by DNA damaging agents.
