Carcinogenesis vol.23 no.5 pp.687–696, 2002
Carcinogenesis Young Investigator Award
Sensing and repairing DNA double-strand breaks
Wellcome Trust and Cancer Research UK Institute of Cancer and
Developmental Biology, Tennis Court Road, Cambridge CB2 1QR, UK and
Department of Zoology, University of Cambridge, Downing Street,
Cambridge CB2 3EJ, UK.
The DNA double-strand break (DSB) is the principle
cytotoxic lesion for ionizing radiation and radio-mimetic
chemicals but can also be caused by mechanical stress
on chromosomes or when a replicative DNA polymerase
encounters a DNA single-strand break or other type of
DNA lesion. DSBs also occur as intermediates in various
biological events, such as V(D)J recombination in develop-
ing lymphoid cells. Inaccurate repair or lack of repair of
a DSB can lead to mutations or to larger-scale genomic
instability through the generation of dicentric or acentric
chromosomal fragments. Such genome changes may have
tumourigenic potential. In other instances, DSBs can be
sufficient to induce apoptosis. Because of the threats posed
by DSBs, eukaryotic cells have evolved complex and highly
conserved systems to rapidly and efficiently detect these
lesions, signal their presence and bring about their repair.
Here, I provide an overview of these systems, with
particular emphasis on the two major pathways of DSB
repair: non-homologous end-joining and homologous
recombination. Inherited or acquired defects in these path-
ways may lead to cancer or to other human diseases, and
may affect the sensitivity of patients or tumour cells to
radiotherapy and certain chemotherapies. An increased
knowledge of DSB repair and of other DNA DSB responses
may therefore provide opportunities for developing more
effective treatments for cancer.
The DNA within our cells is continually being exposed to
DNA-damaging agents. These include ultraviolet light, natural
and man-made mutagenic chemicals and reactive oxygen
species generated by ionizing radiation (IR) or by processes
such as redox cycling by heavy metal ions and radio-mimetic
drugs (1,2). Of the various forms of damage that are inflicted
by these mutagens, probably the most dangerous is the DNA
double-strand break (DSB). DNA DSBs are generated when
the two complementary stands of the DNA double helix are
broken simultaneously at sites that are sufficiently close to
one another that base-pairing and chromatin structure are
insufficient to keep the two DNA ends juxtaposed. As a
consequence, the two DNA ends generated by a DSB are
liable to become physically dissociated from one another,
Abbreviations: A–T, ataxia-telangioectaria; DSB, double-strand break; HR,
homologous recombination; IR, ionizing radiation; NBS, Nijmegen breakage
syndrome; NHEJ, non-homologous end-joining; SCID, severe combined
© Oxford University Press
making ensuing repair difficult to perform and providing the
opportunity for inappropriate recombination with other sites
in the genome. Another barrier to rapid and error-free DSB
repair is the fact that the DNA termini have often also sustained
base damage, meaning that DSB ligation cannot occur until
processing by DNA polymerases and/or nucleases has taken
Despite posing major threats to genomic integrity (see
below), DSBs are nevertheless sometimes generated deliber-
ately and for a defined biological purpose. Probably the best
characterized example of this in higher eukaryotes is the
pathway of V(D)J recombination, which occurs in developing
B- and T-lymphocytes to provide the basis for the antigen-
binding diversity of the immunoglobulin and T-cell receptor
proteins. In this pathway, DNA DSBs are generated at specific
loci by a site-specific nuclease composed of the RAG1 and
RAG2 proteins, and the DSBs are subsequently repaired by
proteins that also function in repair of DSBs that have been
generated by mutagenic agents (for reviews see refs 3,4).
Although tight controls are imposed on events such as V(D)J
recombination, they can sometimes go awry, with potentially
devastating consequences for the cell or for the organism.
DSBs are potent inducers of mutations and of cell death. In
metazoa, just one DSB can kill a cell if it leads to the
inactivation of an essential gene or, more commonly, triggers
apoptosis (5). Furthermore, there is experimental evidence for
a causal link between the generation of DSBs and the induction
of mutations and chromosomal translocations with tumouri-
genic potential (6–10). Indeed, it is generally accepted that
such chromosomal translocations must have arisen through the
generation of one or more chromosomal DNA DSBs that were
subsequently ligated together by a cellular DNA repair system.
Many cancers of lymphoid origin bear oncogenic chromosomal
rearrangements that have arisen as a consequence of the
defective DSB repair of V(D)J recombination intermediates
(11–13). A classical example of this is provided by the B-cell
malignancy, Burkitt’s lymphoma, where the c-MYC gene
is often juxtaposed by genome rearrangement to the
immunoglobulin heavy-chain genes. Furthermore, the loss
and/or amplification of chromosomal material that is charac-
teristic of many cancer cells—and is associated with the
inactivation of tumour suppressor loci and activation of proto-
oncogenes, respectively—is most easily explained as having
arisen through inappropriate DSB repair events. In addition,
and as discussed further below, mutations in many of the
factors involved in DSB signalling and repair lead to increased
predisposition to cancer in people and in animal models.
Indeed, defects in cellular responses to DSBs may be a frequent
initiating event of carcinogenesis (4,7).
The DNA-damage response
As shown in Figure 1, cells respond to DNA DSBs through
the actions of systems that detect the DNA lesion and then
trigger various downstream events. At least in some cases,
Fig. 1. Schematic representation of cellular response to DNA DSBs.
Multiple sensor proteins are shown that physically recognize DNA damage.
Multiple transducer proteins then amplify and diversify the DNA-damage
signal, and a range of downstream effectors regulate various aspects of
these systems can be viewed as classical signal-transduction
cascades in which a ‘signal’ (DNA damage) is detected by a
‘sensor’ (DNA-damage binding protein) that then triggers the
activation of a ‘transducer’ system (protein kinase cascade),
which amplifies and diversifies the signal by targeting a series
of downstream ‘effectors’ of the DNA-damage response.
Clearly, such systems need to be exquisitely sensitive and
selective, as they must be triggered rapidly and efficiently by
low numbers of, and maybe just one, chromosomal DNA
DSB, yet must remain inactive under other conditions.
One cellular response to DSBs is to activate and/or induce
the levels of DNA repair proteins, which are then physically
recruited to the site of the DNA lesion to bring about its repair
(Figure 1). In addition, dividing cells respond to DNA DSBs
by slowing down progression through the cell cycle. When
damage arises in the G1or S cell-cycle phases, for example,
entry into S-phase is prevented or progress through S-phase
is slowed, respectively. This presumably provides time to
allow DNA repair to occur before the lesions are encountered
by a replicative DNA polymerase. Similarly, DNA DSBs
present in G2-phase prevent entry into mitosis, thereby pre-
venting the mis-segregation of chromosomal fragments during
cytokinesis (for recent reviews see refs 7,14,15). Such pauses
in cell-cycle progression are often termed ‘cell-cycle check-
points’. Recently, however, it has become clear that DNA
damage-induced cell-cycle checkpoint pathways actually regu-
late several other events and, moreover, that various aspects
of these responses may even take place in cells that are not
actively dividing. Therefore, in some circumstances, a more
appropriate term for these events is simply ‘the DNA-damage
A crucial component of the DNA DSB signalling cascade
in mammalian cells is the protein kinase, ATM (for reviews
see refs 16,17). ATM deficiency leads to the human cancer
predisposition and neurodegenerative syndrome ataxia-telang-
iectasia (A-T). At the cellular level, ATM deficiency is mani-
fested by increased sensitivity to ionizing radiation and other
agents that yield DNA DSBs but little or no hypersensitivity
to other forms of DNA damage. In addition, A-T cells are
markedly impaired in ionizing radiation-induced G1–S, intra-
S and G2–M cell-cycle checkpoints (16,17). Recent data
suggest that ATM is recruited to and activated at sites of DNA
DSBs (18). Once activated, ATM then phosphorylates various
downstream substrates, including p53, the checkpoint kinase
CHK2, BRCA1 and NBS1, leading to a variety of effects on
DNA repair, cell-cycle progression and apoptosis (for detailed
reviews see refs 7,14–17; also see below). ATM homologues
also exist in Saccharomyces cerevisiae (Tel1p) and Schizosac-
charomyces pombe (Tel1), where they are also involved in
genome surveillance and controlling telomere length.
Another DNA-damage surveillance protein that is related to
ATM is ATR (for review see ref. 17). Disruption of the gene
for ATR leads to early embryonic lethality in the mouse (19,20)
and to cellular inviability in mouse or chicken DT40 B-
lymphocyte cells. The reason for this lethality is not yet clear
but is likely to reflect a role for ATR in the recognition and
repair of DNA replication complexes that have stalled at sites
of DNA damage. Overexpression of catalytically inactive
dominant-negative mutants of ATR leads to hypersensitivity
to several DNA-damaging agents and to the DNA replication
inhibitor hydroxyurea (21,22). Homologues of ATR also exist
in S.cerevisiae and S.pombe (Mec1p and Rad3, respectively)
and play key roles in the DNA-damage response (for reviews
see refs 14,15,17). The available evidence indicates that ATR
phosphorylates an overlapping set of targets to ATM and
responds to a distinct spectrum of lesions from those that
trigger ATM activation. It is also clear that ATR plays a
particularly important role in signalling DNA damage during
S-phase (for example see ref. 23). As discussed further below,
both ATM and ATR share homology in their kinase domains
with the DNA DSB repair protein DNA-PKcs.
An aspect of the DNA-damage response that may be
particularly important for non-dividing cells is the elevation
of the levels of deoxyribonucleotides, which are necessary
for the DNA synthesis-dependent steps of DSB repair. In
mammalian cells, this is achieved at least in part by the
p53-dependent transcriptional induction of the ribonucleotide
reductase subunit p53R2 (24), whereas in S.cerevisiae it
is mediated by the post-translational modification of the
ribonucleotide reductase inhibitor, Sml1p (25). Other non-cell-
cycle aspects of the DNA-damage response include changes
in factors bound to yeast telomeric DNA (26–28) and the
reorganization of chromatin structure. In yeast, this latter
response is brought about by the phosphorylation of histone
H2A (29), whereas in mammals it is triggered by phosphoryla-
tion of the histone H2A isoform, H2AX (30,31). This could
lead to alteration of chromatin structure at the site of DNA
damage so that recruitment of DSB repair factors can take
place efficiently (29,32). Finally, under conditions where the
extent of DNA damage is too great, cells can instead enter an
apoptotic programme. Although the details of how this decision
is reached are not yet clear, it appears that this pathway
involves the actions of proteins that also function in other
aspects of the DNA-damage response (5,33,34).
DNA DSB repair pathways
There are two main pathways for DNA DSB repair—homolog-
ous recombination (HR) and non-homologous end-joining
(NHEJ). These pathways are largely distinct from one another
and function in complementary ways to effect DSB repair
(35–38). During HR, the damaged chromosome enters into
synapsis with, and retrieves genetic information from, an
undamaged DNA molecule with which it shares extensive
sequence homology. In contrast, NHEJ, which brings about
the ligation of two DNA DSBs without the requirement for
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Received January 22, 2002; accepted January 23, 2002