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8
Involvement of Non-Homologous
End-Joining in Radiation-Induced
Genomic Instability
Keiji Suzuki, Motohiro Yamauchi, Masatoshi Suzuki,
Yasuyoshi Oka and Shunichi Yamashita
Nagasaki University Graduate School of Biomedical Sciences,
Atomic Bomb Disease Institute, Department of Radiation Medical Sciences,
Japan
1. Introduction
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
Selected Topics in DNA Repair
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manifestation of radiation effects and the integrity of the genome in the cells surviving
radiation exposure will be discussed.
2. Radiation-induced genomic instability
It has been well established that ionizing radiation induces delayed effects in the progeny of
surviving cells (Little, 2003, Lorimore et al., 2003, Morgan et al., 1996, Suzuki et al., 2003).
This phenomenon is now called radiation-induced genomic instability, which is manifested
as the delayed expression of various radiation effects, such as delayed reproductive death,
delayed chromosomal instability, and delayed mutagenesis (Figure 1). Radiation-induced
genomic instability has been commonly observed in many cell culture systems as well as in
various animals (Lorimore et al., 2003, Morgan, 2003). In addition, there are a series of
studies showing that radiation-induced genomic instability is attributed to transgenerational
effects in mice using hypervariable minisatellite sequences, which have been renamed as
expanded simple tandem repeats (Niwa, 2006). Radiation-induced genomic instability
results in accumulating gene mutations and chromosomal rearrangements in addition to the
direct genome damage caused by the primary radiation exposure. Therefore, it has been
thought to play a pivotal role in accelerating the process of radiation-induced carcinogenesis
(Huang et al., 2003, Kadhim et al., 1992, Niwa, 2003, Suzuki, 1997).
Fig. 1. Radiation-induced genomic instability.
Because radiation-induced genomic instability is induced in a certain fraction of the
progenies originated from a single survived cell, not a single gene mutation but some
epigenetic changes could be involved in the initiation of radiation-induced genomic
instability (Wright, 2010). Persistently elevated levels of oxidative stress was found is
association with radiation-induced genomic instability (Azzam et al., 2003, Kim et al., 2006a,
Limoli et al., 2003). Further association was confirmed in studies where chronic hydrogen
peroxide treatment initiated instability. Administration of free-radical scavengers,
antioxidant treatment, and reducing oxygen tension each reduced delayed chromosomal
instability, suggesting a role of oxidative stress in perpetuating genomic instability (Roy et
al., 2000). Moreover, unstable clones showed mitochondrial dysfunction, which might
explain elevated levels of oxidative stress (Kim et al., 2006b). An inflammatory response has
also been proposed to be involved in radiation-induced genomic instability particularly in
bone marrow (Wright, 2010). Furthermore, possible involvement of bystander effects has
also been discussed. Bystander effects are responses observed in unirradiated cells as a
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
mitochondrial complex IV (Kim et al., 2006b).
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 X-
rays. 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
amending delayed DNA double-strand breaks in surviving cells.
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 NHEJ-
defective 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
defective in the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs).
3. Non-homologous end-joining and its mutants
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,
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including Ku80, Ku70, XRCC4, Artemis, DNA ligase IV and DNA-PKcs itself (Mahaney et
al., 2009, Weterings and Chen, 2007). Autophosphorylation alters the conformation of DNA-
PKcs, allowing the recruitment of DNA end-processing enzymes, such as MRE11 and
Artemis (Huertas, 2010). MRE11 exhibits a 3' to 5' exonuclease activity and plays a critical
role in homologous recombination, but its function in non-homologous end-joining is still to
be determined. On the other hand, Artemis shows a 5' to 3' exonuclease activity, and it is
suggested to be involved in a subset of DNA double-strand breaks. In fact, cells defective in
both nucleases do not show significant radiation sensitivity. Processing of broken ends
might create DNA single-strand breaks, which are amended by the DNA polymerase X
family, such as polymerase mu and polymerase lambda, and terminal
deoxyribonucleotidyltransferase. DNA double-strand breaks are finally rejoined by a
complex composed of DNA ligase IV, XRCC4, and XLF.
Until now, several mutants defective in DNA double-strand break repair have been cloned
(Jeggo, 1998). They are highly sensitive to radiation and show chromosome instability in
response to DNA damaging agents. As shown in Table 1, at least four independent groups
were identified. XR-1 mutant belong to X-ray repair complementing defective repair in
Chinese hamster cells 4 (XRCC4) group holds mutations in the XRCC4 gene. Both xrs-5 and
xrs-6 cells are the members of the XRCC5 group, and they show profound defect in the Ku80
function (Singleton et al., 1997). In xrs-5 cells, the expression of the Ku80 gene is significantly
compromised. In xrs-6 cells, a 13-base pair insertion causes a truncation of KU80 protein,
which accelerates degradation of KU80 protein. The responsible gene for the XRCC7 group
is the DNA-PKcs (Zdzienicka, 1999). Both scid and V-3 mutants harbor premature
termination of the protein in the C-terminus. In irs-20 a mutation that causes substitution of
the amino acid located in the C-terminal region.
Table 1. Rodent cell mutants with defective non-homologous end-joining repair.
Radiation sensitivity of these mutants was determined by colony formation assay. A single
cell is able to form a cluster of progenitor cells, namely, a colony, when it is incubated for 10
days or more. After X-irradiation, numbers of colonies are decreased in a dose dependent
manner. Consequently, cell survival can be calculated by dividing the number of colonies
after X-irradiation by the number of colonies formed by the control cells. As shown in
Figure 2, both xrs-5 (closed diamond), xrs-6 (closed triangle), and scid (closed square) cells
show significant reduction of cell survival as compared to CHO cells (open circle). These
cells lose the shoulder of the survival curve observed in CHO cells, indicating that they lose
DNA repair capacity. To compliment the defect in xrs5 cells, the human Ku86 gene was
introduced by electroporation, and as a result, radiation sensitivity was significantly
recovered (open diamond), confirming that a single gene mutation causes profound DNA
repair defect.