Nature Reviews Cancer | AOP, published online 19 February 2015; doi:10.1038/nrc3892
REVIEWS
Topoisomerase-mediated
chromosomal break repair:
an emerging player in many games
Mohamed E. Ashour1,2, Reham Atteya2 and Sherif F. El-Khamisy1,2
Abstract | The mammalian genome is constantly challenged by exogenous and endogenous
threats. Although much is known about the mechanisms that maintain DNA and RNA
integrity, we know surprisingly little about the mechanisms that underpin the pathology and
tissue specificity of many disorders caused by defective responses to DNA or RNA damage.
Of the different types of endogenous damage, protein-linked DNA breaks (PDBs) are
emerging as an important player in cancer development and therapy. PDBs can arise during
the abortive activity of DNA topoisomerases, a class of enzymes that modulate DNA
topology during several chromosomal transactions, such as gene transcription and
DNA replication, recombination and repair. In this Review, we discuss the mechanisms
underpinning topoisomerase-induced PDB formation and repair with a focus on their role
during gene transcription and the development of tissue-specific cancers.
Topological entanglements
The coiling and winding of the
DNA double helix.
Transesterification reaction
The process of exchanging the
organic group of an ester with
the organic group of an
alcohol.
Krebs Institute,
Department of Molecular
Biology and Biotechnology,
University of Sheffield,
Sheffield, S10 2TN, UK.
2
Center for Genomics,
Helmy Institute, Zewail City of
Science and Technology,
Giza 12588, Egypt.
Correspondence to S.F.E.‑K. e-mail: s.el‑khamisy@
sheffield.ac.uk
doi:10.1038/nrc3892
Published online
19 February 2015
1
The intertwined nature of two complementary DNA
strands is a unique feature to ensure the transmission,
storage and expression of valuable genetic information.
However, almost all types of DNA transactions, such
as chromatin compaction, the formation of higherorder structures, gene transcription, replication and
recombination, lead to topological entanglements that
must be resolved to maintain cell function1. DNA
topoisomerases are elegant biological tools that have
evolved to resolve such DNA entanglements2,3. The
human genome encodes six topoisomerases organized
into three types4: type IA (DNA topoisomerase IIIα
(TOP3α) and TOP3β), type IB (nuclear TOP1 and mitochondrial TOP1) and type IIA (TOP2α and TOP2β).
Several genetic, biochemical and biological studies
have demonstrated the importance of topo­isomerases
in numerous cellular processes, such as DNA replication, transcription and chromatin remodelling 5–7. More
recently, a broader role for topoisomerase activity on
RNA during translation has emerged8,9.
Topoisomerases cleave and rapidly reseal one
(type I) or two (type II) nucleic acid strands through a
transesterification reaction that uses a Tyr residue in the
active site of the topoisomerase as a nucleophile, generating a transient break through which topological changes
can occur 7,10. This smart mode of catalysis is rather ‘dangerous’ because it generates an intermediate in which
the topoisomerase becomes covalently linked to the 3′
(type IB) or 5′ (type IA and IIA) terminus of nucleic
acids through a phosphotyrosine linkage called topo­
isomerase cleavage complex (TOPcc)2. Failure to complete the topoisomerase catalytic cycle results in trapping
of topoisomerases on DNA termini, generating proteinlinked DNA breaks (PDBs)11,12. The generation of PDBs
is probably a frequent event, as suggested by the presence of most PDB repair activities in all forms of life5,13–16.
Thus, although topoisomerase activity is crucial for cell
function, it must be closely monitored by a number of
repair mechanisms to avoid the consequences of inappropriate topoisomerase activity. This Review provides
a brief overview of targeting topoisomerases in cancer
therapy, which has been covered in depth in several
other reviews17–20, and we then discuss multiple modes
of PDB repair with a focus on the role of topoisomerases during gene transcription and the development
of tissue-specific cancers.
Targeting topoisomerases in cancer therapy
Manipulating topoisomerase activity by catalytic inhibition or poisoning has been widely exploited to kill
cancer cells19–22. When considering the cellular consequences of targeting topoisomerases, one should distinguish between inhibiting the activity versus ‘poisoning’
or ‘trapping’ the enzyme in an inactive state on DNA.
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Supercoiling
The over- or underwinding of
the DNA double helix.
R‑loops
The association of a stretch of
single-stranded RNA with a
complementary region of DNA,
whereby one DNA strand is
displaced as a loop.
Topoisomerase catalytic inhibitors interfere with any step
of the enzyme catalytic cycle, such as binding to DNA
or the binding and hydrolysis of ATP21,23. Conversely,
topoisomerase poisons stabilize the topoisomerase–DNA
reversible intermediate (TOPcc) by misaligning the
DNA ends, precluding re-ligation and thereby trapping
the enzyme on DNA, creating a PDB.
The presence of topoisomerase is a prerequisite for
the cytotoxic effect of topoisomerase poisons4,24. Cancer
cells can adapt to treatment with topoisomerase poisons through the downregulation of the topoisomerase involved4. Conversely, co‑amplification of TOP2A
(which encodes TOP2α) and ERBB2 (also known as
HER2) in breast cancer has been suggested to contribute to the efficacy of doxorubicin, a drug that targets
TOP2α25. PDBs can also be triggered by anticancer protocols that do not involve topoisomerase poisons. For
example, intermediates of DNA base excision repair,
such as 5′- or 3′-nicked abasic sites or DNA flaps containing deoxyribose-phosphate, have been reported to
trap TOP2 on DNA, creating PDBs26,27. Similarly, in vitro
studies demonstrated trapping of TOP1 by nicks or DNA
secondary structures28. More recent in vivo work has
shown that ionizing radiation and chemotherapies such
as alkylating agents can also generate PDBs, at least a
subset of which are repaired by activities that liberate
TOP1 from DNA termini29–31.
Cancer cytotoxicity induced by topoisomerase poisons is primarily caused by the generation of cytotoxic
PDBs (primarily double-strand breaks (DSBs)) during
DNA replication19,32,33. The generation of DSBs can be
direct, as a result of the collision of TOPcc with replication forks, or indirect, as a result of the accumulation of positive supercoiling ahead of replication forks
as a consequence of inhibiting topoisomerase activity 34.
Topoisomerase poisons also elicit profound effects on
transcription. The TOP1 poison camptothecin (CPT)
downregulates ~20% of expressed genes in cancer
cell lines, with preferential effects on long genes35,36.
TOP1 poisons may also promote the formation of
DNA–RNA hybrids (R‑loops), causing DNA breakage
and cytotoxicity 37. The mechanism of topoisomerasemediated cytotoxicity depends on the cell type (cycling
versus non-cycling), duration of treatment and drug
concentration. In cancer cells, replication-dependent
DSBs are responsible for cytotoxicity with low doses
of TOP1 poison, whereas higher doses trigger cytotoxicity in an S‑phase-independent manner 38, which
is probably due to differential effects on replication
and transcription. TOP1 poisons do not induce cytotoxicity by TOP1‑independent mechanisms, whereas
some, but not all, TOP2 poisons can induce cytotoxicity through mechanisms that are independent of TOP2.
This is a reflection of the individual drugs rather than
an intrinsic property of TOP2 poisons39,40. For example, anthracyclines induce cytotoxicity by trapping
TOP2cc on DNA and/or by the generation of free radicals41. Anthracyclines have also been shown to promote
nucleo­some eviction at the promoter of active genes,
causing promoter fragility and potentially attenuating
the DNA repair response42,43.
Chromosomal PDB repair
The repair of PDBs involves an intricate coordination
of signalling and repair factors that detect the PDB and
precisely disjoin the covalently linked topoisomerase
from PDBs or endonucleolytically cut the DNA, releasing the topoisomerase and a fragment of DNA. This is
then followed by the filling in of missing nucleotides and
the ligation of the remaining nick to restore the intact
nucleic acid backbone. We briefly summarize current
views on signalling and discuss the roles of key PDB
repair enzymes.
PDB signalling. TOP1 poisons activate the two main
checkpoint cascades mediated by ataxia telangiectasia and Rad3‑related (ATR)–checkpoint kinase 1
(CHK1) and ataxia telangiectasia mutated (ATM)–CHK2;
ATR–CHK1 is the main pathway for checkpoint activation. Consequently, depletion of ATR or CHK1 sensitizes mammalian cells to CPT12,44. Although ATM is not
considered to be the main signalling pathway induced
by TOP1 poisons in cancer cells, ATM participates in
many other aspects of DNA damage responses, such
as the coordination of homologous recombination
(HR)45–47 and the phosphorylation of downstream PDB
repair factors, including tyrosyl-DNA phosphodiesterase 1 (TDP1) and polynucleotide kinase phosphatase
(PNKP)48–51. In contrast to cancer cells, in post-mitotic
cells ATM has a key role in the response to TOP1 poisons37,52. ATM deficiency in neuronal cells leads to the
accumulation of TOP1cc, most likely due to the role
of ATM in controlling TOP1 degradation during transcription, a prerequisite for subsequent PDB repair conducted by TDP1 (REFS 53,54). Whether a similar role for
ATM exists in the response to TOP1 poisons in cancer
cells remains to be tested.
Similar to TOP1 poisons, TOP2 poisons induce
ATR–CHK1 and ATM–CHK2 activation55. The role of
ATM seems to be particularly important in cells lacking TDP2, which is the enzyme that disjoins TOP2 from
PDBs56. Because some TOP2 poisons are not restricted
to targeting TOP2, it is difficult to pinpoint the signalling pathway involved. For example, anthracyclines
induce ATM activation57, but depletion of ATM leads
to a minimal increase in sensitivity to this class of TOP2
poisons58,59. This is probably because anthracyclines can
crosslink DNA independently of their ability to generate TOP2‑mediated PDBs58. Although the role of PDB
signalling in response to topoisomerase poisons is well
established, its role in PDB-mediated carcinogenesis is
not clear.
Precision scissors: TDPs. TDP1 was the first enzyme
reported to be capable of liberating a stalled topo­
isomerase from DNA termini without cleaving DNA60
(FIG. 1). Subsequent studies showed that TDP1 is a broadspectrum DNA end-processing enzyme (FIG. 2a,b)
that can resolve various 3′-blocking termini, such as
3′-phosphoglycolate produced during radiotherapy 29,61,
apurinic or apyrimidinic (AP) sites generated by chemo­
therapeutic alkylating agents (for example, temozolomide)30,62 and chemotherapeutic chain-terminating
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Landmarks of TDP1 research
Discovery of TOP1 as
the molecular target
for the anticancer
agent camptothecin171
1985
1996
Cloning of Tdp1 in
Saccharomyces cerevisiae,
which encodes the enzyme
that cleaves 3′-tyrosyl-DNA
phosphodiester linkages15
1999
Nash and co-workers
report the first activity
that specifically cleaves
Top1 from the 3′ end of
DNA in Saccharomyces
cerevisiae60
2002
TDP1 depletion and PARP
inhibitors are reported to
be epistatic with TOP1
poisons but synergistic
with alkylating agents30
2005
Mutant TDP1
identified as the
causative agent of
SCAN1 in humans72
and the first crystal
structure for TDP1
is solved73
2007
2014
Generation of the first
Tdp1-knockout mouse173
Mutant TDP1 is linked to deregulated
gene transcription in SCAN1 owing
to persistence of TOP1-induced
chromosomal PDBs172
Landmarks of TDP2 research
Discovery of
TOP2 as the
molecular target
of the antitumour
agent m-AMSA174
1984
• The crystal structure of zebrafish, Caenorhabditis elegans and mouse TDP2 are solved84,87
• TDP2 is implicated in the pathogenesis of
picornavirus infections, in which it cleaves a
protein–RNA linkage during viral replication79
2009
Human TTRAP is shown
to be the 5′-tyrosyl-DNA
phosphodiesterase that
repairs TOP2 PDBs and
was named TDP2 (REF. 77)
2012
TDP2 is identified
as a transcriptional
target of mutant
p53 and is found to
be increased in
lung cancer82
2013
The first
association
between defects
in TDP2 and
human disease132
2014
The generation of the first
Tdp2-knockout mouse
and the demonstration
that TDP2 functions in
NHEJ to repair
TOP2-induced PDBs66
Reviews
| Cancer
Figure 1 | Precision scissors for PDB repair: TDP1 and TDP2.Nature
m‑AMSA,
amsacrine;
NHEJ, non-homologous end-joining; PARP, poly(ADP-ribose) polymerase; PDB,
protein-linked DNA break; SCAN1, spinocerebellar ataxia with axonal neuropathy;
TDP, tyrosyl-DNA phosphodiesterase; TOP, topoisomerase; TTRAP, TRAF and TNF
receptor-associated protein.
nucleoside analogues63. TDP1 has also been reported
to liberate TOP2 from PDBs in yeast (FIG. 2b), but it is
debatable whether TDP1 has a similar role in vertebrate cells64–66. TDP1 function is tightly regulated by
post-translational modifications. Phosphorylation of
TDP1 at Ser81 by ATM or DNA-dependent protein
kinase (DNA‑PK) promotes TDP1 stability, interaction with X-ray repair cross-complementing protein 1
(XRCC1)–DNA ligase 3α (LIG3α) and PDB repair in
cycling cells48,49. SUMOylation at Lys111 promotes
TDP1 recruitment to transcription stalling sites, which
is important for non-cycling cells67,68. Poly(ADP)ribosylation (PARylation) of TDP1 enhances its stability and
recruitment to DNA damage sites68. Although a role for
PARylation is conceivable in sensing conventional singlestrand breaks (SSBs), such as those with damaged
sugar moieties, it is less obvious why a ‘sensor’ role for
PARylation might be required for dealing with PDBs.
Such breaks are the products of topoisomerase abortive activity and are thus more likely to be ‘sensed’ by
the topoisomerase stalling event. In addition to being a
canonical damage sensor, poly(ADP-ribose) polymerase
(PARP) might act as a molecular switch that channels
PDBs to the TDP1 pathway, and as such might protect DNA from non-specific endonucleolytic cleavage
(FIG. 3a–c). This role of PARP might explain the synergistic effect of PARP inhibitors and TOP1 poisons, and the
absence of additional sensitization by TDP1 depletion30,68.
Targeting TDP1 for cancer therapy is clinically attractive for many reasons. First, TDP1 is a broad-spectrum
3′ DNA end-processing enzyme: therefore, suppressing
its activity is predicted to potentiate a number of cancer therapy protocols that induce PDBs either directly
or indirectly, such as TOP1 poisons, alkylating agents,
radiotherapy and chain terminators 22,29,53,61,63,69–71.
Second, TDP1 deficiency is well tolerated in vertebrates
and thus targeting TDP1 is likely to result in minimal
toxicity. Tdp1‑knockout mice, Tdp1−/− mouse embryonic
fibroblasts and Tdp1−/− DT40 cells are viable31,65, and
TDP1 deficiency in humans is also compatible with viability 72. Third, the crystal structure of TDP1 was solved
many years ago73. Despite multiple attempts to develop
TDP1 inhibitors in a number of laboratories, including
ours, none of the available compounds has progressed
to clinical trials as a result of their poor physicochemical
properties71,74–76. This demonstrates the need for alternative libraries and screening methods coupled with new
biophysical and virtual screening tools.
Another recently discovered ‘precision scissor’ is
TDP2 (FIG. 1). Although TDP2 does not share structural similarity with TDP1, it functions in a remarkably similar way by disjoining stalled TOP2 and other
5′‑linked phospho­tyrosine adducts from PDBs65,77–79
(FIG. 2a,c). TOP2 proteo­lysis has also been reported to
be a prerequisite for TDP2 action80. In contrast to TDP1
catalytic activity, which is independent of divalent metals
and produces unligatable DNA termini, TDP2 activity
requires the availability of divalent metals and produces
DNA with readily ligatable termini77,81 (FIG. 2a; TABLE 1).
The products of TDP2 activity are substrates for nonhomologous end-joining (NHEJ) (FIG. 3d). Consequently,
TDP2 is epistatic with core components of the NHEJ
pathway, such as KU70 (also known as XRCC6), in
response to TOP2 poisons66. How TDP2 is recruited to
sites of PDBs and whether the recruitment mechanisms
promote PDB repair during replication, transcription or
both is not known. TDP2 is also an attractive target for
cancer therapy. Its depletion in avian, mouse or human
cells increases sensitivity to different classes of TOP2
poisons65,66,77,81. Expression of TDP2 is increased in nonsmall-cell lung cancer cells and is transcriptionally upregulated in mutant p53 cells82,83. The crystal structure of
TDP2 has also been solved, and efforts to develop TDP2
inhibitors are ongoing 84–87.
PDB repair nucleases. Instead of precisely liberating
the topoisomerase without cleaving the DNA to which
it is linked, DNA can be non-specifically cut to resolve
PDBs. It is not clear how the pathway choice is determined, but recent evidence suggests the involvement
of PARP in channelling TOP1‑mediated PDBs to the
non-nucleolytic pathway 30,68. It is also not fully clear
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a
DNA
DNA
O
O
O
DNA
O
O
Base
DNA
P
N+
O
H2O
O–
O
O
Covalent
intermediate
TOP1
Base
O
O
3′
O–
P
O–
Unligatable end
TDP1
Base
O
3′
HN
TDP1
O–
P
O
His263
O
O
O–
P
O
N:
HN
O
O
TOP2
5′
O
O
Mg2+ – O
TOP2
DNA
O– H
Asp272
O
P
5′
O
O
O
O
O:
:
O
TOP1
Base
O
O
3′
5′
Base
H
O–
P
O–
O
Base
O
O
O
TDP2
Base
DNA
Ligatable end
DNA
b TDP1 substrates
O
5′ DNA
O
Base
O
c TDP2 substrates
3′-phosphotyrosyl
linkages
TOP2α
TOP2β
TOP3α ?
P protein
O
O
P
O–
TOP1
3′ R
O
O
O
OH
O
O
Base
O
OH
OH
O
O
O
O
OH
O
DNA
O
O
3′-phospho- 3′-abasic
glycolate
sites
O
TOP3β ?
VPG
3′-phosphoα,β-unsaturated
aldehyde
Base
Non-phosphorylated
RNA or DNA
OH (OH ) nucleotide
O
P
O–
O
O
Base
O P
O
O–
O
O
Base
O
P
O–
O
O–
O
O
O
O
O
DNA
DNA
RNA
5′-phosphotyrosyl
linkages
P
Base
OH
TOP1
5′-phosphotyrosine 5′-phosphotyrosine 3′-phosphotyrosine
lesions on DNA
lesions on RNA
linkages
Figure 2 | The catalytic cycle and physiological substrates of TDPs. a | The abortive activity of topoisomerase I
Nature
(TOP1) results in the trapping of TOP1 on the 3′ terminus of a DNA single-strand break. His263 in the
activeReviews
site of | Cancer
tyrosyl-DNA phosphodiesterase 1 (TDP1) undertakes a nucleophilic attack on the phosphodiester bond, resulting in
the liberation of trapped TOP1 and the formation of a TDP1–DNA covalent intermediate. The TDP1–DNA covalent
intermediate is resolved by a hydrolytic reaction driven by His493 in the active pocket of TDP1 (not shown), liberating
TDP1 and leaving behind a 3′‑phosphate terminus. The abortive activity of TOP2 results in the trapping of TOP2 on the
5′ terminus of a DNA double-strand break. The Asp272 residue in TDP2 activates a water molecule to drive the hydrolytic
cleavage of the phosphodiester bond. Three additional conserved residues (not shown) and Mg2+ bind the 5′-phosphate to
stabilize a transition state in this reaction without the formation of TDP2–DNA covalent complex. The trapped TOP2 is
then released, leaving behind a ligatable 5′-phosphate terminus. The proposed model for catalytic cycles of TDPs is
based on data from REFS 73,87. b | TDP1 is a broad-spectrum 3′ DNA end-processing enzyme, which acts on a myriad of
DNA adducts. TDP1 physiological substrates include 3′‑phosphotyrosyl linkages such as TOP1 protein-linked DNA
breaks (PDBs) arising from nuclear or mitochondrial TOP1 activity, 3′‑phosphoglycolate, 3′‑abasic sites and
3′‑phospho‑α,β‑unsaturated aldehyde. These breaks are abundant endogenous lesions that can also arise following
radiation- or alkylation-induced DNA damage during cancer therapy. TDP1 can remove the terminal non-phosphorylated
RNA or DNA nucleotide from the 3′ end through its nucleosidase activity and can also hydrolyse 3′‑phosphoamide
linkages arising from the abortive activity of TDP1 itself. Yeast, but not human, Tdp1 has been shown to process
5′‑phosphotyrosyl linkages arising from abortive TOP2 activity. c | In contrast to TDP1, TDP2 only acts on phosphotyrosinelinked DNA adducts. The principal physiological activity of TDP2 is resolving 5′‑phosphotyrosine lesions arising at TOP2
PDBs. TDP2 has been shown to remove hepatitis B viral polymerase (P protein) from relaxed circular DNA, but TOP3α
remains a putative substrate. TDP2 can remove picornavirus protein (VPG) from the 5′ end of its RNA, and TOP3β is thus a
plausible substrate. TDP2 exhibits limited activity on 3′‑phosphotyrosyl linkages of TOP1 PDBs, but this activity seems to
be important in the absence of TDP1 (REF. 65).
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whether proteolytic degradation of topoisomerases is
required prior to the action of PDB nucleases, as is the
case for TDPs. Elegant work on the meiotic recombination protein SPO11, which is covalently bound to DNA
via a 5′-phosphotyrosyl linkage that is similar to TOP2
covalent binding to DNA, suggests that proteolytic degradation might be dispensible88,89. However, recent studies
in yeast and mammalian cells suggest a broader role for
DNA-dependent proteolytic activity upstream of canonical PDB repair90,91. For example, the yeast metalloprotease
Wss1 has been recently shown to promote cell survival
on exposure to TOP1‑mediated DNA damage91. Yeast
mutants lacking Wss1 accumulate PDBs and exhibit signs
of genomic instability, suggesting a broad requirement for
proteolytic degradation during PDB repair 91. Whether
this requirement exists for all modes of PDB repair
in eukaryotes has yet to be determined. Early studies in
yeast revealed a role for Mus81–Mms4 (MUS81–essential
meiotic structure-specific endonuclease 1 (EME1) in
humans), Rad1–Rad10 (xeroderma pigmentosum group
F-complementing protein (XPF)–ERCC1 in humans),
Mre11–Xrs2 (MRE11–Nijmegen breakage syndrome
protein 1 (NBS1) in humans) and Slx4–Slx1 (SLX4–SLX1
in humans) during TOP1‑mediated PDB repair, and
recent studies have confirmed this role in vertebrate cells
(reviewed in REF. 12). In human cells, the endonuclease
MUS81–EME1 cleaves DNA at stalled replication forks
adjacent to the branched point, whereas XPF–ERCC1
endonuclease can cleave flapped structures arising outside S-phase92,93. XPF and MUS81 also carry out overlapping functions during HR after removal of TOP1 from
PDBs94. SLX4 has also been implicated in PDB repair,
during which it acts as a scaffold for three structurespecific nucleases: MUS81–EME1, XPF–ERCC1 and
SLX1 (FIG. 3b). The SLX4–MUS81 interaction is crucial
for TOP1‑mediated PDB repair, whereas SLX4–XPF
interaction is dispensable95. Another player is CtBPinteracting protein (CtIP; also known as RBBP8), which
functions at the 5′ DNA resection during the initial step of
HR. CtIP has been implicated in PDB repair in response
to both TOP1 and TOP2 poisons96,97. CtIP interacts in a
phospho-dependent manner with the tumour suppressor
protein BRCA1, which seems to be crucial for its role as a
PDB repair factor 59,98,99. Consistently, the nuclease activity of CtIP is dispensable for the resection of restriction
enzyme-generated clean breaks but is essential for the
processing of PDBs induced by TOP1 or TOP2 poisons100
(FIG. 3b,d,e). Pioneering work on SPO11 led to several valuable insights into the diverse roles of PDB nucleases88.
The MRX complex (Mre11–Rad50–Xrs2; orthologous
to the human MRE11–RAD50–NBS1 (MRN) complex)
and its functional partner Ctp1 (the orthologue in fission yeast, known as Sae2 in budding yeast) have been
shown to drive nucleolytic removal of Spo11 in budding
and fission yeast 89,97,101. In fission yeast, the MRX complex and Ctp1 are also required for the removal of Top2
from PDBs; however, the same role was not confirmed
in budding yeast89,97. In avian and mammalian cells, the
MRN complex and CtIP seem to possess specific roles in
TOP2‑mediated PDB repair that are distinct from their
resection function during HR98,102.
DNA helicases. Recent evidence suggests that TOP1
poisons induce the accumulation of positive supercoils, which hamper replication fork progression. In
this model, PDBs are not converted to DNA breaks but
instead cause replication fork slowing and reversal in
a process that is promoted by PARP activity, primarily
by PARP1 (REFS 34,103). PARP is probably suppressing
unscheduled fork restart by inhibiting RECQ1 (also
known as RECQL) helicase104. This model demonstrates fork reversal as a potential route by which cells
can tolerate TOP1 poisons and implicates RECQ1
as a putative PDB repair factor that possibly mediates resistance to TOP1 poisons (FIG. 3c). A similar
mechanism of fork reversal and restart could be envisaged for TOP2‑mediated PDB repair because depletion of RECQ1 also increases cellular sensitivity to
TOP2 poisons104.
PDBs, carcinogenesis and transcription
Although targeting topoisomerases (for example,
topoisomerase poisons) and PDB repair machines
(for example, TDP1 and TDP2 inhibitors) is useful for killing cancer cells, the inappropriate repair of
PDBs can promote cancer in a tissue-specific manner
— a process that is closely linked to gene transcription
(FIGS 4,5). Topoisomerases have crucial roles in driving
gene expression, particularly expression of long genes
(FIG. 4a,b) and in suppressing deleterious transcription
by-products, such as R-loops35,36,105. A moving RNA
polymerase generates positive supercoiling ahead of the
transcription bubble and negative supercoiling behind105.
The accumulation of positive supercoils in front of the
bubble reduces the travelling rate of RNA polymerases105, and underwound DNA behind the bubble may
pair with the nascent transcript, generating R‑loops
(FIG. 4c). R‑loops interfere not only with transcription
but also with replication fork progression, promoting
DNA breakage and genome instability (reviewed in
REFS 106,107). TOP1 prevents R‑loop formation through
the relaxation of negative supercoiling 106. In addition,
TOP3β has recently been reported to prevent R‑loop formation and suppress the rate of chromosomal translocation (for example, MYC–immuno­globulin heavy chain
locus (IGH) translocation) in conjunction with Tudor
domain–containing protein 3 (TDRD3, which increases
the stability of TOP3β and promotes its recruitment to
chromatin)108. TOP3β also possesses an RNA topo­
isomerase activity, and its depletion increases the risk
of schizophrenia and intellectual disability 8,9 (FIG. 3d).
TOP2β seems to function in the regulation of gene promoters109. Rosenfeld and colleagues have shown that
TOP2β‑mediated chromosomal breakage regulates transcription initiation at multiple regulated transcription
units110. It has been suggested that TOP2β cleavage at
promoters triggers structural or topological modulations
to facilitate dynamic changes in chromatin organization
that are required to initiate transcription110. It is also possible that the binding of the transcription factors to promoters might cause localized torsional stress, for which
TOP2β cleavage is required to initiate transcription110.
However, despite the crucial roles of topoisomerases in
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a
TOP1
5′
P
b
HO
TOP1
Proteasomal
degradation
HO
SLX4
Y
P
SLX1
MRN
P
P
HO
P
HO
PARP1
CtIP
BRCA
OH
TOP1
RECQ1
HO
PNKP
P
RAD51
HR
RECQ1
LIG3
d
e
TOP2
5′
Proteasomal
degradation
OH
P
P
P
P
OH
P
HO
TOP2
CtIP
MRN
OH
HO
P
P
HO
Y
P
P
P
HO
TOP2
Proteasomal
degradation?
CtIP
MRN
TDP2
P
TOP2
5′
Y
OH
5′
Proteasomal
degradation?
XPF
ERCC1
HO
PARP1
TDP1
OH
TOP1
MUS81 EME1
ATM
XRCC1
P
5′
c
NHEJ
HO
OH
BRCA
RAD51
HO
HR
KU70 KU80
DNA-PK
XRCC4
LIG4
Figure 3 | Overview of chromosomal protein-linked DNA break repair. a | Topoisomerase I (TOP1) at protein-linked
DNA breaks (PDBs) is subjected to proteasomal degradation, most likely controlled by ataxia telangiectasia mutated (ATM),
leaving behind a small peptide linked to the DNA through a phosphodiester bond. Tyrosyl-DNA phosphodiesterase 1 (TDP1)
hydrolyses the phosphodiester bond between TOP1 and DNA, creating 3′‑phosphate and 5′‑hydroxyl termini, which
require the action of polynucleotide kinase phosphatase (PNKP) to restore the conventional 3′‑hydroxyl and 5′‑phosphate
termini, which are suitable for extension by a polymerase, followed by nick sealing by DNA ligase III (LIG3). The role of
various post-translational modifications and core components of single-strand break repair has been omitted for
simplicity. b | PDBs can be processed by non-specific nucleolytic cleavage of DNA, releasing TOP1 and a fragment of DNA.
NatureF-complementing
Reviews | Cancer
This is conducted by structure-specific nucleases, such as MUS81, XPF (xeroderma pigmentosum group
protein) or SLX1, held by the scaffold protein SLX4. The interaction of SLX4 with MUS81, and to a lesser extent with SLX1,
is important during PDB repair, whereas its interaction with XPF is dispensable (double-headed arrow). CtBP-interacting
protein (CtIP) can also remove TOP1 from PDBs using its nuclease activity, independent from its resection role during
homologous recombination (HR). c | Poly(ADP-ribose) polymerase 1 (PARP1) and RECQ1 promote the resolution of PDBs
by inhibiting unscheduled replication fork restart through the helicase activity of RECQ1, giving more time for the repair
of PDBs, most likely by the pathway depicted in part a. d | TOP2‑mediated PDBs can be processed by two distinct
non-homologous end-joining (NHEJ) pathways. An error-free pathway mediated by TDP2 disjoins TOP2 peptide from
PDBs following partial TOP2 degradation; this pathway does not result in DNA deletions. Conversely, an error-prone
pathway mediated by nucleases (CtIP and the MRE11–RAD50–NBS1 (MRN) complex) removes TOP2 and a fragment of
DNA, resulting in loss of genetic information. e | CtIP and the MRN complex can also resolve TOP2‑mediated PDBs during
HR in proliferating cells, using sister chromatids as templates, without loss of genetic information. DNA-PK,
DNA-dependent protein kinase; EME1, essential meiotic structure-specific endonuclease 1; XRCC, X-ray repair
cross-complementing protein. Red dashed arrows indicate DNA replication.
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Table 1 | Similarities and differences between TDP1 and TDP2
TDP1
TDP2
Discovery
Identified through Saccharomyces cerevisiae
Identified through Saccharomyces cerevisiae77
Protein structure
Crystal structure solved73
Crystal structure solved84,87
68 kDa protein (608 aa)
41 kDa protein (362 aa)
Amino‑terminal regulatory domain and carboxy‑terminal
catalytic domain
Amino‑terminal regulatory domain and carboxy‑terminal
catalytic domain
Catalytic motif
Two HKN motifs
Four motifs: TWN, LQE, GDXN and SDH
Ancestor
Belongs to phospholipase D family73
Belongs to EEP-domain nuclease87
Substrate requirements
Prefers single-stranded DNA substrates
Prefers single-stranded DNA substrates
Recognizes and processes DNA substrates without base
sequence restrictions
Recognizes and processes DNA substrates without base
sequence restrictions
Processes several 3ʹ‑blocking DNA lesions (primarily TOP1
PDBs)
Limited to 5′‑tyrosyl DNA lesions (TOP2 PDBs)
Functions as a monomer
Functions as a monomer
Forms a transient covalent intermediate with the
substrate73
Does not form any covalent interactions with the
substrate
Polarity
3′ to 5′ (REF. 31)
5′ to 3′ (REF. 77)
End product
3′-phosphate ends that require further processing by
PNKP
5′-phosphate and 3′‑hydroxyl ends that can be directly
ligated
Catalysis mechanism
15,60
Dependency on metal ions No
Yes
Nucleosidase activity
Yes
No
Post-translational
modifications
Phosphorylation at Ser81 by ATM or DNA‑PK (in
proliferating cells)48, SUMOylation at Lys111 (REF. 67) and
PARylation68
None identified
Associated disease
TDP1 defects cause neurodegeneration and
spinocerebellar ataxia72
•TDP2 defects cause intellectual disability, seizures and
ataxia132
•TDP2 participates in the pathogenesis of picornaviruses
and hepatitis B infection78,79
Relevance to cancer
In response to TOP1 poisons, TDP1 depletion is epistatic
with PARP1 inhibitors and predicted to be synthetically
lethal in nuclease-deficient cancers92,93 , while in response
to alkylating agents such as temozolomide,TDP1
depletion is synergistic with PARP1 inhibitors30
In response to TOP2 poisons, TDP2 depletion is epistatic
with NHEJ (for example, KU70) and predicted to be
synthetically lethal in nuclease- or HR-deficient cancers66
aa, amino acid; ATM, ataxia telangiectasia mutated; DNA‑PK, DNA-dependent protein kinase; EEP, exonuclease-endonuclease-phosphatase; HR, homologous
recombination; NHEJ, non-homologous end-joining; PARP1, poly(ADP-ribose) polymerase 1; PARylation, poly(ADP)ribosylation; PDB, protein-linked DNA break;
PNKP, polynucleotide kinase phosphatase; TDP, tyrosyl-DNA phosphodiesterase; TOP, topoisomerase.
driving gene transcription and preventing R‑loop formation, and their putative roles during translation109,111,112,
topoisomerase activity can, ironically, also trigger
transcription-associated genome instability and cancer.
Transcription-associated PDBs and gene deletion.
TOP1 activity has recently been implicated in 5‑base
pair (bp) deletions in highly transcribed genes113,114.
The exact mechanism (or mechanisms) by which TOP1
mediates mutagenesis is not fully understood; however,
emerging evidence implicates ribose contamination of
genomic DNA in this process115. Despite the remarkable preference of DNA polymerases to incorporate
deoxyribonucleotides, the greater cellular concentration of ribonucleotides favours their erroneous incorporation in genomic DNA at high rates, with estimates
exceeding 1,000,000 ribonucleotides incorporated
in the DNA per cell division in vertebrate cells 115.
One possible mechanism of ribose removal from
DNA involves TOP1‑mediated nicking of the DNA
backbone on the 3′ side of the ribonucleotide, creating an SSB with a 3′‑terminated ribonucleotide (FIG. 4e).
Although this seminal discovery was reported by
Sekiguchi and Shuman in 1997, it took nearly 15 years
to realize the importance and consequences of ribonucleotide removal from genomic DNA116. Following
TOP1‑mediated nicking, the ribonucleotide undergoes
an internal 2′–3′ cyclization event to release TOP1,
leaving behind a nick or gap in DNA. Evidence for the
release of TOP1 by internal cyclization without the
requirement for PDB repair came from experiments
in budding yeast, in which deletion of TDP1 did not
affect the rate of TOP1‑dependent gene deletions113,114.
There are many possible scenarios for subsequent
end processing. If another TOP1 molecule creates a second nick a few bases away from the initial ribonucleotide, it releases the short stretch of DNA between the
two TOP1 nicks. This event traps the second TOP1 on
DNA because it cannot conduct the re‑ligation reaction
owing to several nucleotide gaps between the 3′ end to
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a
c
b
Hormone-driven
gene transcription
AAAA
?
Cancer development or neurological disorder
Long gene
transcription
d
R-loop
formation
FMRP
Removal of
topological stress
TDRD3
TOP2
TOP1
TOP3
Positive
supercoils
Negative
supercoils
Pol II
RNA
Pol II
R
First TOP1 cleavage
R-loop
AAAA
Pol II
Ribosome
e
5′
g
cleavage
5′
h
3′ and 5′ end
processing?
5′
5′
TOP1
Realignment
Realignment
Exo1
f Second TOP1
5′ end
processing
Srs2
3′ end
processing?
TOP1
Negative ligation (TOP1)
Negative ligation (ligase?)
• Gap filling (Pol?)
• Ligation (ligase?)
Gene
deletion
Figure 4 | Topoisomerases and transcription-associated genome instability. a | Topoisomerase II (TOP2) facilitates the
transcription of many hormone-driven genes through the induction of protein-linked DNA breaks (PDBs)
regulatory
sites
NatureatReviews
| Cancer
of target genes. Hormone-driven TOP2‑mediated PDBs could trigger chromosomal translocation in a tissue-specific
manner, leading to the development of cancer (FIG. 5). b | TOP1 and TOP2 are required during global transcription,
particularly the transcription of long genes. Accumulation of topoisomerase-mediated PDBs by depletion of core PDB
repair components, such as tyrosyl-DNA phosphodiesterase 2 (TDP2), leads to downregulation of the transcription of long
genes and the development of neurological disease132. It is not clear whether accumulation of PDBs as a result of TDP2
depletion can also trigger carcinogenesis directly through chromosomal translocation or indirectly by reducing expression
of long genes. c | A moving RNA polymerase (Pol II) generates positive supercoiling ahead of the transcription bubble, which
might reduce the travelling rate of the polymerase, and negative supercoiling behind the bubble, which induces the
annealing of the nascent RNA with the underwound DNA, producing DNA–RNA hybrids (R‑loops). R‑loops can interfere
with transcription and replication, promoting DNA breakage and genome instability. TOP1 prevents R‑loop formation
through the relaxation of negative supercoiling. TOP3β can also suppress R‑loop formation, in conjunction with Tudor
domain–containing protein 3 (TDRD3), which promotes the stability and recruitment of TOP3β to chromatin. d | TOP3β
forms a complex with TDRD3 and fragile X mental retardation protein (FMRP) that possess an additional role as an RNA
topoisomerase, with putative functions during translation. e | DNA polymerases can mistakenly incorporate ribonucleotides
(R) into genomic DNA owing to the greater cellular concentration of ribonucleotides in comparison to deoxyribonucleotides. RNaseH2 removes ribonucleotides from DNA but, in the absence of RNaseH2 (rnh2Δ), attempts by TOP1 to remove
ribonucleotides can induce mutagenic deletions. TOP1 cleaves the DNA backbone on the 3′ side of the ribonucleotide,
creating a single-strand break with a 3′‑terminated ribonucleotide, which undergoes an internal 2′‑3′ cyclization event,
creating a 3′‑cyclic sugar phosphate residue (pink hexagon). Three possible scenarios may occur. f | If another TOP1
molecule creates a second nick a few bases away from the initial ribonucleotide, it would release the short stretch of DNA
between the two TOP1 nicks, trapping TOP1 on DNA and generating a PDB. If the TOP1 cleavage site is adjacent to a short
tandem repeat sequence, strand slippage of the complementary strand mediated by the tandem repeat might occur,
bringing the 5′‑hydroxyl and the 3′‑trapped TOP1 next to each other. Ligation of these products, promoted by TOP1, results
in deletion. g | A second scenario may involve 3′ and 5′ end processing by unknown nucleases, followed by re-ligation of the
processed ends, also resulting in gene deletion. h | In contrast to the first and second scenarios, a third possibility envisages
the faithful repair of ‘dirty ends’, resulting in preservation of genetic information. Work in yeast suggests that this process is
initiated by the unwinding of DNA from the 5′‑hydroxyl side by Srs2 helicase, followed by 5′ end resection by exonuclease 1
(Exo1), trimming of the 3′‑cyclic sugar phosphate residue by a yet-to-be-discovered activity, filling in of the missing
nucleotides using the complementary DNA strand as a template and, finally, DNA ligation.
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which the enzyme is attached and the 5′ end; this generates a PDB (FIG. 4f). If the TOP1 cleavage site is adjacent
to a short tandem repeat sequence, strand slippage of the
complementary strand mediated by the tandem repeat
can occur, bringing the 5′‑hydroxyl and the 3′-trapped
TOP1 next to each other. Ligation of the remaining nick
releases TOP1, leaving behind an approximately 5 bp
deletion117 (FIG. 4f). Deletions do not normally exceed
5 bps because larger fragments would not be lost by diffusion118. Another possibility involves nucleolytic trimming of DNA ends by yet-to-be-determined nucleases or
end-processing enzymes, followed by realignment and
ligation117,119 (FIG. 4g). Why deletion, in this case, would
be limited to ~5 bps is unclear. Another possibility,
recently emerged from studies in yeast, can be envisaged as the unwinding of DNA from the 5′ side by Srs2
helicase, followed by 5′ end resection by exonuclease 1
(Exo1), then trimming of the 3′ cyclic sugar phosphate
residue by a yet-to-be-discovered activity, followed by
filling in of the missing nucleotides using the complementary DNA strand as a template and finally DNA
ligation, resulting in error-free repair 120 (FIG. 4h).
Support of a role for TOP1 in generating gene deletions came from experiments in rnh201 mutant budding yeast strains lacking RNH201, which encodes
one of the three subunits of RNaseH2, the primary
enzyme involved in ribose removal from DNA. The
rate at which the ~5 bp deletions occurred in rnh201
mutant cells was ~52 times higher than the rate in wildtype cells119. However, additional deletion of TOP1 in
rnh201 mutant cells led to a transcription-driven deletion rate similar to that of wild-type cells, pointing to
a role for Top1 in ribose removal from genomic DNA
in yeast. Yeast Top1 only removes a subset of ribonucleotides in the genome, which is probably determined
by the transcriptional activity of the locus, chromatin status, genomic location, surrounding sequence
context and identity of the ribonucleotides. Similarly,
an analogous ribose-dependent mutagenic role for
PDBs triggered by other topoisomerases seems possible. For example, RNA-containing substrates have
been reported to stimulate binding and most likely the
trapping of TOP2 in vitro121.
Transcription-associated PDBs and chromosomal rearrangement. Inappropriate formation and processing of
PDBs has been associated with tissue-specific malignant transformation, resulting in prostate cancer and
leukaemias.
Prostate cancer is one of the most common tumours
in males and the second most common cause of cancer
death in men122. In 2005, Chinnaiyan and colleagues
discovered the first prostate cancer-associated fusion
gene using a bioinformatics approach coupled with
fluorescence in situ hybridization123. They reported
fusions between the promoter of the androgenregulated gene transmembrane protease serine 2
(TMPRSS2) and the coding sequence of the ETS transcription factors ERG, ETS variant 1 (ETV1), ETV4 and
ETV5. The fusion proteins led to the overexpression
of oncogenic ETS transcription factors, driven by the
transcriptional control of androgen. TMPRSS2–ERG is
the most common fusion, occurring in approximately
50% of prostate cancer cases. Owing to the high prevalence of prostate cancer, TMPRSS2–ERG is considered
to be the most common chromosomal fusion observed
in any cancer and is a hallmark of aggressive tumours
with poor prognosis123–126.
TMPRSS2 and ERG are located on chromosome 21,
and the fusion occurs either by internal deletion of
approximately 3 megabase pairs (Mb) or by chromosomal translocation. The high prevalence of TMPRSS2–
ERG suggests a controlled underlying mechanism. Until
recently, the role of topoisomerases in transcriptional
regulation was restricted to resolving topological problems. In 2006, Rosenfeld and colleagues provided evidence implicating oestrogen-driven, TOP2β‑induced
PDBs in transcriptional activation of many genes, such
as those encoding nuclear receptors, transcription factors and DNA repair factors109. Consequently, it has
been shown that androgen co‑recruits androgen receptor (AR) and TOP2β to breakpoint sites of TMPRSS2
and ERG to resolve topological problems associated
with the transcription of androgen-responsive genes124.
TOP2β‑induced PDBs at TOP2β preferential binding
sites trigger a recombinogenic event, causing de novo
TMPRSS2–ERG fusion. The question of why translocation is specific to TMPRSS2 and ERG has been addressed
by two independent studies, which showed the androgen induces proximity between TMPRSS2 and ERG126,127.
Both genes might migrate into the same transcription
factories or hubs where transcription takes place, and
gene–gene interaction at the transcription factories
could facilitate translocation126,127 (FIG. 5a). The dependence of prostate tissue on androgen-driven transcription
might explain why TMPRSS2–ERG fusions are restricted
to prostate cancer. Inspection of the translocation breakpoint junctions reveals microhomologous sequences
and short non-template insertions, indicative of NHEJ
as a causal repair process that accounts for the fusion
event 128. Consistent with this, TMPRSS2–ERG fusion
is suppressed by depletion of components of the NHEJ
pathway and enhanced by depletion of components of
the HR pathway 124,126.
Is there a pre-set threshold of PDBs above which
androgen can drive gene fusions and prostate cancer?
Prolonged androgen treatment induces TMPRSS2–ERG
fusion in both malignant and non-malignant prostate epithelial cells, but at remarkably different rates;
24 hours versus 5 months, respectively 129. In addition,
transient androgen treatment did not induce TMPRSS2–
ERG fusion in non-malignant prostate epithelial cells126.
These observations suggest the presence of a checkpoint
barrier that inhibits PDB-driven genetic instability in
non-malignant prostate epithelial tissue and is lost or
attenuated in prostate cancer tissue. The checkpoint barrier is probably orchestrated by ATM, which is partially
activated in prostate cancer cells130. Consistent with this,
inactivation of ATM in the presence of androgen induces
TMPRSS2–ERG fusion in non-malignant prostate epithelial cells131. ATM may prevent chromosomal translocations by facilitating the repair of androgen-driven PDBs.
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a
1
b
2
3
1
2
3
4
SCI
9
ERG
10
SCII
11
12
MLL
TMPRSS2
TOP2β
RNA
polymerase
Translocation event
MLL translocation
partner
• Loss of
checkpoint
activation
• PDB repair
defect?
• Illegitimate
repair by NHEJ
Transcription
factory
TMPRSS2
ERG
AF4, AF9, ENL, AF10, etc.
MLL
PTEN
Second hit
Second hit
ERG
AKT
Mutations in
TP53, ATM,
RAS genes, etc.
HOX genes,
MEIS1, etc.
Prostate cancer
t-AML or infant leukaemia
c
Cell death
Topoisomerases
PDBs
• Chemotherapy: topoisomerase
poisons and alkylating agents
• Radiotherapy
• Endogenous DNA breaks
• Natural products in fruit and vegetables
PBD repair
?
Chromosomal rearrangements
Prostate cancer
Leukaemia
Nature Reviews | Cancer
Figure 5 | Protein-linked DNA breaks and chromosomal rearrangement. Models for chromosomal translocations
mediated by topoisomerase IIβ (TOP2β) in prostate cancer (a) and leukaemia (b). Both models share common players,
including TOP2β and the generation of protein-linked DNA breaks (PDBs), the proximity of the translocation partners
hosted by a common transcription factory, a possible defect in PDB repair, and the requirement for secondary mutations.
a | Androgen signalling brings transmembrane protease serine 2 (TMPRSS2) and ERG into close proximity and is likely to
trigger the association of these two genes within the same transcription factory. Androgen also induces the recruitment of
TOP2β and androgen receptor (not shown) to the sites of genomic breakpoints. TOP2β induces recombinogenic PDBs,
leading to TMPRSS2–ERG translocation and the overexpression of the oncogenic ERG transcription factor. Carcinogenesis
is likely to be triggered by a subsequent hit, possibly mutations in PTEN, which encodes the negative regulator of the
PI3K–AKT survival pathway175,176. b | Chromosomal breakpoints in mixed lineage leukaemia (MLL) fall within an 8 kilobase
pair (Kb) breakpoint cluster region (BCR) encompassing two recombination BCR subclusters (SCI and SCII). Breakpoints
from therapy-related acute myeloid leukaemia (t‑AML) and infant leukaemia cluster in SCII, which is DNase I
hypersensitive and colocalizes with a cryptic promoter. TOP2β induces recombinogenic PDBs, leading to translocation
between MLL and more than 80 partner genes (for example, AF4, AF9, ENL and AF10), which are likely to be relocalized to
the same transcription factory. MLL fusion proteins contribute to carcinogenesis by inducing high expression of HOX
genes and MEIS1, thereby inhibiting differentiation and promoting survival of haematopoietic cells136. Similarly to
TMPRSS2–ERG translocation in prostate cancer, the MLL translocation is not sufficient to induce leukaemia, which requires
another mutation, possibly in TP53, ATM or RAS genes. c | A diagram summarizing the origin and consequences of PDBs.
PDBs can arise by poisoning, or ‘trapping’, cellular enzymes on DNA, such as poly(ADP-ribose) polymerases (not shown nor
discussed here) and DNA or RNA topoisomerases. Poisoning both classes of enzymes has been widely exploited in cancer
therapy. Trapping of topoisomerases can also arise endogenously by the nearby presence of DNA breaks, such as
single-strand DNA breaks and abasic sites. Thus, a number of cancer therapy protocols that induce ‘conventional’ DNA
breaks can also induce PDBs, such as radiotherapy and alkylating agents (for example, temozolomide). If not repaired,
PDBs can interfere with cellular replication and transcription, causing cell death. Conversely, persistence of PDBs, in
specific tissues, can trigger chromosomal rearrangements and carcinogenesis, such as leukaemia and prostate cancer.
The repair of PDBs involves tight coordination of signalling and repair factors. Suppression of PDB repair is predicted to
enhance the cancer-killing activity of PDB targeting therapies. A fine balance, however, seems to be in place to tightly
control cellular PDB repair levels; too much or too little, possibly in conjunction with other genetic defects, may promote
carcinogenesis. NHEJ, non-homologous end-joining.
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Alternative NHEJ
Repair of DNA double-strand
breaks by an error-prone
non-homologous end-joining
(NHEJ) pathway that is
independent of DNA ligase IV.
If this is the case, one would predict increased levels of
TMPRSS2–ERG fusions and increased prostate cancer
predisposition in mouse models in which Atm and
canonical PDB repair factors such as Tdp2 are deleted.
TDP2 activity may reduce chromosomal translocations
in prostate tissue by ensuring rapid and accurate resolution of TOP2‑mediated PDBs. Consistent with this,
depletion of TDP2 in prostate cancer cell lines reduced
transcriptional induction of androgen-responsive
genes as a result of abortive TOP2 activity 132. The DNA
termini left after TDP2‑mediated PDB repair are readily ligatable (FIG. 2a), so excessive TDP2 activity may,
conversely, promote erroneous DNA ligation and translocation77. In support of this, proteasomal degradation
of full-length TOP2, which is required to generate PDB
substrates for TDP2, is required to induce TOP2‑poisoninduced chromosomal translocations80,133,134. Thus, it
seems that a fine balance is in place to tightly control
cellular TDP2 levels; too much or too little, possibly in
conjunction with other genetic defects, may promote
carcinogenesis. What controls this balance and to what
extent this control differs in prostate tissue compared to
other tissues are important questions to be addressed.
Therapy-induced secondary malignancy. One of the
most common secondary side effects of TOP2 poisons is
the development of secondary malignancies. Aggressive
acute leukaemias, both lymphoblastic and myeloid, are
associated with translocations of mixed-lineage leukaemia
(MLL; also known as KMT2A). The product of MLL is a
histone methyltransferase that is implicated in the global
regulation of transcription. MLL translocations are the
most frequent types of gene rearrangements in leukaemias that arise following treatment with TOP2 poisons
and are a hallmark of therapy-associated acute myeloid
leukaemias (t‑AMLs)135,136. Chromosomal breakpoints fall
within an 8 kilobase pair (Kb) breakpoint cluster region
(BCR) of MLL. Within this region, two recombination
BCR subclusters were identified: centromeric subcluster I (SCI) and telomeric subcluster II (SCII). Breakpoints
reported from t‑AML and infant acute leukaemia (discussed below) seem to cluster in the telomeric portion
of the MLL BCR (FIG. 5b), suggesting the involvement of
a common pathway in their production137,138. Subsequent
studies revealed the SCII subcluster to be a DNase I hypersensitive site, which may explain the susceptibility of this
region to illegitimate recombination139. This was further
supported by the colocalization of SCII with a cryptic promoter element that encodes a truncated version of MLL140
and, more recently, by the Encyclopedia of DNA Elements
(ENCODE) DNase I hypersensitivity data141.
Some evidence suggests that higher-order chromatin fragmentation triggered by TOP2 poison-induced
apoptosis is responsible for chromosomal translocation
in MLL142. However, apoptotic nucleases fail to colocalize at MLL hotspots143–145. Furthermore, introduction
of an artificial non-PDB strand break by zinc finger
nucleases within the BCR of MLL is not sufficient to
induce rearrangements or translocations146. Irrespective
of the nature and source of translocation, MLL and its
translocation partner must be in close proximity to
allow illegitimate repair (FIG. 5a,b). As discussed above,
transcription factories could induce gene proximity,
thereby facilitating translocation events147,148. Austin and
colleagues recently reported a higher association frequency between MLL and its translocation partners
within the same transcription factory compared to
other genes that do not feature translocation events
with MLL149. The authors proposed that TOP2‑induced
PDBs during transcription, coupled with the proximity
of the translocation partner within the same transcription factory may account for the translocation event. If
true, then PDBs generated in any gene pair undergoing
transcription might provide an opportunity for inappropriate recombination leading to translocation if the
two genes reside within a common transcription factory.
MLL translocation breakpoint junctions display characteristic features of NHEJ repair, including short insertions or deletions and the presence of short sequence
microhomology in MLL and its partner genes150. PARP1
inhibition reduced etoposide-induced translocations,
pointing to a role for PARP1‑driven alternative NHEJ in
this process151. Whether this role is due to PARP1 activity during alternative NHEJ or poisoning of PARP1 on
DNA, which would shield DNA ends from illegitimate
recombination, is yet to be determined. Nevertheless,
these observations suggest the potential utility of PARP1
inhibitors in preventing oncogenic translocations driven
by TOP2‑targeting therapies151.
Some of the most important evidence for PDBinduced oncogenic translocations came from studies of
therapy-related acute promyelocytic leukaemia (t‑APL),
which is typified by a fusion between promyelocytic leukaemia (PML) and retinoic acid receptor‑α (RARA)152,153.
The fusion results from translocation t (15:17), which is
one of the most frequent secondary cancers arising from
the treatment of breast cancer with TOP2‑targeting
therapies154,155. Intriguingly, t‑APL has been reported following the treatment of patients with multiple sclerosis
with the TOP2 poison mitoxantrone, supporting a causative role for TOP2 poisons in carcinogenic trans­location
rather than an indirect role by selecting cells with preexisting translocations156. In contrast to MLL rearrangements, in which TOP2‑mediated cleavage sites do not
coincide with the translocation hotspots, breakpoints
in t‑APL seem to cluster within an 8 bp hotspot in PML
intron 6, which is a preferential mitoxantrone-induced
TOP2 cleavage hotspot 152,157. NHEJ probably mediates the translocation because small microhomologous
sequences at the junction between PML and RARA have
been reported158.
Another example of PDB-induced cancer is infant
leukaemia, which represents ~10% of childhood leukaemias and features a high rate of chromosomal translocation in MLL159. The observations that TOP2 poisons
induce leukaemia with MLL translocations raise the possibility that dietary TOP2 poisons may be causal factors
for infant leukaemia. Some natural products possess
TOP2 poison activity, such as bioflavonoids that are
present in many fruits and vegetables. In addition, quinone metabolites of some drugs and industrial chemicals possess TOP2 poisoning activity 160. Reduction of
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TOP2β expression has been shown to promote resistance to bioflavonoids161,162, and at least two independent studies have shown a possible association between
maternal dietary intake of TOP2 poisons and infant leukaemia163,164. Exposure of human CD34+ haematopoietic
progenitor cells to flavonoids induces MLL translocations in vitro165,166, and prenatal exposure of mice to flavonoids induces MLL rearrangements in the presence
of compromised DNA repair, such as defective ATM167.
Which topoisomerase isoform is implicated in transcription-associated carcinogenesis? Although TOP1 has been
implicated in gene deletion (FIG. 4f,g), its role and that of
TOP3 in carcinogenesis is less clear. This is in contrast
to TOP2: TOP2α is likely to account for the cytotoxicity
of TOP2 poisons, and TOP2β drives oncogenic translocations. The initial support for a role of TOP2β as a cancer driver came from the Liu laboratory: using a mouse
skin carcinogenesis model, Liu and colleagues found that
the incidence of etoposide-induced carcinogenesis was
significantly reduced in mice with a skin-specific Top2b
deletion133. Subsequent studies showed that TOP2β mediates translocation events in prostate cancer and in TOP2
poison-induced secondary leukaemias124,134,149,168,169.
Despite substantial evidence for TOP2β in carcinogenesis, an analogous but distinct role for TOP2α cannot be
excluded. The ability of TOP2α to prevent DNA entanglement at mitosis underpins the tumour suppressor
role of BRG1- or HRBM-associated factor (BAF) chromatin remodelling complexes, components of which are
mutated in more than 20% of all human malignancies170.
Shure, M. & Vinograd, J. The number of superhelical
turns in native virion SV40 DNA and minicol DNA
determined by the band counting method. Cell 8,
215–226 (1976).
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Summary and future directions
Chromosomal PDBs can arise from the inappropriate
activity of DNA and RNA topoisomerases, causing cell
death or chromosomal rearrangements (FIG. 5c). Cell death
has been widely exploited to treat cancer, whereas chromosomal rearrangements can trigger cancer. There are
multiple topoisomerase hotspots throughout the genome,
yet PDB-driven oncogenic translocations are remarkably
tissue specific. What confers specificity? Is it the transcription activity at certain loci? Do translocations that
only confer a survival advantage in appropriate tissues or
cell progenitors result in cancer? Cancer initiation and
response to therapy is probably controlled by variations in
the stoichiometry of PDB repair factors across tissues, differences in transcriptional activity, the frequency at which
repair within a specified locus is initiated and concluded,
and the likelihood of gene–gene interaction at transcription factories. The unexpected abundance of ribonucleotides in genomic DNA makes one wonder how efficient
PDB repair is in tissues, cells or genomic loci enriched
with ribose. What is the genome-wide landscape of ribonucleotides in genomic DNA? Does the enrichment of
ribonucleotides in specific loci or tissues contribute to
the development of cancer or to the response of cancer
cells to therapy? Can we exploit specific transcriptional
programmes that are intrinsic to topoisomerase activity
to selectively target cancer cells? Moving from focussing
on one factor per study to a systems biology approach,
integrating multiple pathways, will help to address some
of these questions with the aim of better understanding
and exploiting chromosomal PDB repair in cancer.
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Acknowledgements
This work is funded by a Wellcome Trust Investigator Award
(103844) and a Lister Institute of Preventative Medicine
Fellowship to S.F.E.‑K. The authors thank P. Jeggo,
A. Goldman, K. Caldecott, A. Lehmann and members of the
El‑Khamisy laboratory for helpful discussions. The authors
apologize to colleagues whose primary research articles are
not cited owing to space restrictions.
Competing interests statement
The authors declare no competing interests.
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