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Review
DNA damage and transcription stress
Larissa Milano,1,2,* Amit Gautam,1,2,* and Keith W. Caldecott1,2,*
1Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK
2These authors contributed equally
*Correspondence: l.milano@sussex.ac.uk (L.M.), a.gautam@sussex.ac.uk (A.G.), k.w.caldecott@sussex.ac.uk (K.W.C.)
https://doi.org/10.1016/j.molcel.2023.11.014

SUMMARY

Genome damage and transcription are intimately linked. Tens to hundreds of thousands of DNA lesions arise
in each cell each day, many of which can directly or indirectly impede transcription. Conversely, the process
of gene expression is itself a source of endogenous DNA lesions as a result of the susceptibility of single-
stranded DNA to damage, conflicts with the DNA replication machinery, and engagement by cells of topoi-
somerases and base excision repair enzymes to regulate the initiation and progression of gene transcription.
Although such processes are tightly regulated and normally accurate, on occasion, they can become abortive
and leave behind DNA breaks that can drive genome rearrangements, instability, or cell death.

INTRODUCTION the most helix-distorting DNA base lesions are cross-links

Transcription is essential for all forms of life, enabling conversion
of genetic information into mRNA that can be translated into
protein. Transcription is a tightly regulated and highly coordi-
nated process requiring a DNA template that can support the
accurate progression of RNA polymerases (RNAPs). RNA
polymerase pausing and backtracking are likely common occur-
rences during the transcription of genomic DNA, as part of reg-
ulatory mechanisms and/or as a result of secondary DNA struc-
tures and the complexity of chromatin in cellular genomes.1–3
However, RNAPs must also contend with unscheduled breaks
and DNA lesions in genomic DNA, a problem that is particularly
prevalent in very large genes spanning hundreds of kilobases.4,5
Genome damage is very common, with estimates of tens to hun-
dreds of thousands of DNA lesions arising in each cell, every day.
DNA damage arises not only from exposure to environmental
genotoxins, such as polyaromatic hydrocarbons and ultraviolet
(UV) or ionizing radiation, but also endogenously from by-prod-
ucts of cellular metabolism and because of the intrinsic level of
chemical instability of DNA.6 In addition, the process of tran-
scription itself can be a source of DNA damage, as a result of
the formation of single-stranded DNA that is susceptible to dam-
age, the regulation of transcription using base excision repair
(BER) and/or topoisomerases to cut and remodel the genome,
and as a result of conflicts with DNA replication and the forma-
tion of R loops during RNAP progression.7,8 Here, we will review
the relationships between transcription and DNA damage and
how defects in the management of these relationships may un-
derpin a range of human diseases from neurodegeneration to
cancer.
induced by anti-cancer drugs, such as cisplatin, which can
form intrastrand DNA cross-links that prevent the damaged nu-
cleobase from entering the active site of the RNAP and therefore
block RNAP progression.9 Cross-linking agents can also
generate interstrand DNA cross-links, in which bases in both
strands of the DNA double helix are covalently linked, likely
thereby preventing DNA strand separation and blocking tran-
scription.10 Other environmental sources of bulky DNA lesions
that block RNA progression include carcinogenic monoadducts
formed by benzo[a]pyrene (BPDE)11 and 2-acetylaminofluorene
(AAF),12 and DNA-protein cross-links (DPCs) induced by anti-
cancer topoisomerase poisons13 or aldehyde by-products of
alcohol consumption.14 However, arguably the most common
environmental source of bulky base damage is sunlight. UV-
induced photodimers, such as (6-4) pyrimidine-pyrimidone pho-
toproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs),
are potent blocks to RNAP progression.5 Endogenous sources
of bulky DNA lesions include aldehydes15,16 and reactive oxygen
species (ROS), the latter of which can create cyclopurine mono-
adducts.17 Topoisomerase intermediates are also a significant
source of RNAP blockage and/or inhibition18,19 (Figure 1). For
example, topoisomerase 1 (TOP1) removes the DNA supercoils
that are created by RNAPs during transcription, and to do this,
it transiently breaks one strand of a DNA duplex to create a pro-
tein-linked DNA intermediate known as the cleavage complex.20
Although TOP1 normally reseals the DNA single-strand break
(SSB) present in the cleavage complex at the end of the catalytic
cycle, it can fail to do so if located close to a DNA lesion or un-
usual DNA secondary structure, and following collision with an
RNAP, it can create a protein-linked SSB.20–22

RNAP BLOCKAGE BY ‘‘BULKY’’ DNA LESIONS RNAP BLOCKAGE BY ‘‘NON-BULKY’’ DNA LESIONS

A major source of transcription stress is RNAP pausing or In addition to bulky DNA base lesions, cellular genomes acquire
blockage by bulky (helix-distorting) DNA base lesions induced many thousands of non-bulky lesions, each day.6 Although
at endogenous and environmental genotoxins (Figure 1). Among some of these can be bypassed by RNAPs, at the expense of

70 Molecular Cell 84, January 4, 2024 ª 2023 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Figure 1. RNA polymerase blockage by DNA lesions

Common DNA damage sources of RNA polymerase pausing/blockage (shown in blue). Left, bulky (helix-distorting) DNA lesions such as those induced by solar
radiation and endogenous/environmental genotoxins (see text for details). Middle, abortive cleavage complex intermediates of TOP1 (shown) or topoisomerase II
(TOP2) arising endogenously during transcription or induced by chemotherapeutic topoisomerase poisons. Abortive topoisomerase cleavage complexes can
lead to bona fide SSBs (TOP1/TOP2) and DSBs (TOP2) during transcription. Right, SSBs arising from attack of deoxyribose or nucleobases by reactive oxygen/
nitrogen species, ionizing radiation, endogenous/environmental genotoxins, or during programmed base excision repair events (e.g., epigenetic reprogramming).
Note that only SSBs with aberrant 50 and/or 30 termini (red circles), such as 30 -phosphate termini, appear to strongly pause/block RNA polymerase progression.

increased risk of forming mutant transcripts,23 others perturb the
progression of RNAPs. For example, in vitro biochemical experi-
ments suggest that mammalian RNA polymerase II (RNAPII) is
able to bypass the major non-bulky oxidative base lesion
8-oxoguanine (8-oxoG) even if it is present in the template DNA
strand but is unable to efficiently bypass an abasic site.24,25 Sur-
prisingly, DNA nicks and single-stranded gaps harboring ligatable
termini are also bypassed to a significant extent by a variety of
prokaryotic, viral, and human RNAPs in vitro, albeit at the risk of
mutant transcripts.26–29
In contrast to ligatable SSBs, SSBs with aberrant 30 termini,
such as 30 -phosphate termini, are potent blocks to RNAP pro-
gression, including human RNAPII24,26,28 (Figure 1). This is impor-
tant because DNA breaks with 30 -phosphate termini are likely to
be present at a large fraction of endogenous lesions, such as
SSBs induced by oxidative stress, TOP1-induced SSBs, and
some intermediates of BER.30 RNAP blockage by non-ligatable
30 termini may result from displacement of the SSB termini from
the RNAP active site, and/or because such termini prevent loop-
ing out of the non-transcribed strand.28,29 Notably, SSBs with
aberrant 30 and/or 50 termini likely accumulate in human neurolog-
ical diseases in which SSB repair (SSBR) is defective30–32
because all such diseases identified to date result from mutations
in DNA end processing enzymes, such as tyrosyl DNA phospho-
diesterase 1 (TDP1), polynucleotide kinase phosphatase (PNKP),
aprataxin (APTX), or in the molecular scaffold protein X-ray repair
cross-complementing protein 1 (XRCC1) that interacts with and
coordinates these enzymes.32 For example, 30 -TOP1 linked and
30 -phosphate termini are repaired by TDP1 and PNKP, respec-
tively, and therefore likely impede RNAP progression in the hu-
man neurological diseases in which these proteins are
mutated.33–35
Recently, it has been suggested that SSBs present in a non-
template strand may also block progression of bacteria and
yeast RNAPII, if they are located upstream and close to a pro-
moter, by stabilizing intranucleosomal single-strand loops in
which the RNAP is ‘‘trapped’’ between flanking histone-DNA
contacts with the nucleosome.36,37 Whether this is true for
SSBs in non-template DNA strands in human cells remains to
be determined.
RNAP BLOCKAGE AND R LOOP FORMATION
In addition to DNA damage being a potent source of RNAP
blockage, the process of transcription can itself induce DNA
damage, mutation, and genome instability.7,38 RNAP blockage
at DNA lesions or secondary structures can promote the un-
scheduled formation of R loops, which are RNA-DNA hybrids
containing a displaced single-stranded DNA loop8,39,40 (Figure 2).
R loops fulfil a number of normal cellular roles such as transcrip-
tional regulation and termination, and as such, their formation
and removal are regulated by a variety of nucleic-acid-process-
ing enzymes, including RNA-processing factors, transcription
elongation factors, and TOP140–42 (Figure 2A). In addition, the

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Figure 2. Transcription stress and R loops

(A) The formation of unscheduled R loops behind RNA polymerase is suppressed by engagement of nascent mRNA by RNA processing and splicing factors, by
controlled/processive RNA polymerase elongation, and by the removal of DNA supercoils by TOP1.
(B) Pathogenic processing or attack of R loops by nucleases, TOP1, or reactive oxygen species can result in cleavage of the displaced non-transcribed strand,
creating a SSB or possibly DSB if there is also a break in the transcribed strand ahead of the RNAP (e.g., as shown here).
(C) Alternatively, unscheduled R loops and associated DNA breaks can be processed and repaired by nucleolytic degradation of the nascent mRNA, and removal
of any associated DNA breaks by DNA-strand-break repair.
(D) Unscheduled R loops can be unwound and removed by the activity of helicases, some examples of which are shown.

tumor suppressor breast cancer gene 2 (BRCA2) may suppress
excessive R loop formation by regulating the release of
paused RNAPII during transcription initiation,43 and the DNA
helicase RecQ-like helicase 5 (RECQ5) may do so by suppress-
ing RNAP stalling during transcription elongation.1 However,
unscheduled R loops can arise and are potentially pathogenic,
leading to additional DNA breakage and the activation
of ataxia -telangiectasia-mutated (ATM) and ataxia telangiecta-
sia and Rad3-related (ATR) DNA damage checkpoints.44 For
example, abortive TOP1 activity, nuclease activity, or free
radical-induced breakage of the displaced non-transcribed
strand within the R loop can create a SSB, or even a DNA dou-
ble-strand break (DSB) if the R loop is already associated with
a SSB in the template strand45,46 (Figure 2B). Consequently, cells
possess a number of helicases and repair pathways by which
unscheduled R loops and any associated DNA breaks can be
removed and repaired.39,41,47 (Figures 2C and 2D). For example,
unwinding of R loops can be conducted by numerous helicases,
examples of which are senataxin (SETX), DExH-box helicase 9
(DHX9), DExD-box helicase 39B (UAP56/DDX39B), and regu-
lator of telomerase elongation helicase (RTEL)48–52. In addition,
the nucleolytic degradation of RNA within R loops can be con-
ducted by ribonucleases H1 or H2 (RNases H1/H2),53 or by
DICER,54 and any associated DNA breaks repaired by canonical
DNA-strand-break repair pathways.
TRANSCRIPTION-INDUCED DNA BREAKAGE BY
DNA TOP1
Transcriptional activation has been linked strongly with DNA-
strand-break induction and oncogenic translocations as a result
of, for example, increased cytosine deamination in single-
stranded DNA and abortive DNA topoisomerase activity.38,55,56
Topoisomerases remove torsional stress, catenanes, and chro-
mosome entanglements during transcription and genome dupli-
cation.57,58 The primary role of TOP1 is to remove positive and
negative supercoils ahead and behind of DNA and RNAPs.57,58
Mostly, topoisomerase activity is not a threat to genome stability
because the DNA breaks that these enzymes create are typically
transient intermediates of their catalytic activity. However, as
indicated above, topoisomerase activity can become ‘‘abortive’’
in the presence of nearby DNA lesions, unusual DNA secondary
structure, or collision with DNA or RNAPs18 (Figure 3). The pro-
tein-linked SSBs and DSBs arising from abortive topoisomerase
activity then require dedicated DNA repair pathways for their
removal.59,60 Abortive TOP1 activity may contribute to nucleo-
tide repeat instability61 and may also be prevalent at sites of
genomic ribonucleotides.62 TOP1 activity at the latter leads to
ribonucleotide excision, but potentially at the expense of
inducing short (2–5 bp) deletions.63 Indeed, abortive TOP1 activ-
ity at genomic ribonucleotides accounts for the mutational signa-
ture ID4, which is detected among both cancer and germline mu-
tations.64 Importantly, abortive TOP1 activity may also be a
common source of RNAP blockage and DNA breakage at R
loops.45,47
TRANSCRIPTION-INDUCED DNA BREAKAGE BY
DNA TOP2
TOP2 has a broad array of functions during transcription,
conferred by its ability to create a transient DSB.57,58,65 This al-
lows TOP2 to not only remove DNA supercoils but also to

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decatenate and untangle chromosomes and to facilitate short-
and long-range chromosome movements during transcriptional
regulation. TOP2 is located at chromosome loop anchors, and
its activity can help regulate RNA pausing and release at pro-
moters, splice sites, and enhancers within active genes.57,66,67
Similar to TOP1, TOP2 activity is particularly important for expres-
sion of very large genes (>200 kb), including numerous genes
required for normal neuronal function.68 Also, similar to TOP1,
TOP2 cleavage complexes harbor protein-linked DNA breaks
that are obligate intermediates of TOP2 activity.65 Although
such breaks are normally resealed at the end of each catalytic cy-
cle, the cleavage complexes can occasionally become abortive,
resulting in bona fide SSBs and/or DSBs that require DNA-
strand-break repair pathways for the removal59,60 (Figure 3). If
such TOP2-induced DSBs are not repaired rapidly and accu-
rately, they can lead to oncogenic translocations55,69,70 or neuro-
logical disease.71 It has been suggested that the induction of
bona fide DSBs by TOP2 is required to regulate gene expres-
sion.72,73 However, it seems more likely that such DSBs are sim-
ply a consequence of TOP2 activity occasionally becoming
abortive.
TRANSCRIPTION-INDUCED DNA BREAKAGE BY
DNA BER
The formation of single-stranded DNA during gene transcrip-
tion can result in increased rates of DNA breakage and
genetic mutation, for example, as a result of increased spon-
taneous cytosine deamination and other types of base dam-
age38,74 (Figure 3). DNA base damage is also sometimes
introduced deliberately in human cells, during transcriptional
regulation. For example, 5-methycytosine (5mC) is an epige-
netic mark that typically suppresses transcriptional activ-
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Figure 3. Transcription-induced DNA lesions
The regulation of gene expression is associated with
topoisomerase activity that regulates chromosome
topology, movement, and DNA torsional stress during
transcription initiation and elongation. Abortive in-
termediates of TOP1 or TOP2 activity can arise during
these processes, creating SSBs (TOP1/TOP2) or
DSBs (TOP2) that further impede transcription and
threaten genome integrity and stability. Similarly,
elevated cytosine deamination in single-stranded
DNA during transcription elongation, or programmed
BER reactions employed to demethylate cytosine
during epigenetic (re)programming and gene
activation, can lead to persistent DNA strand breaks
and/or potentially mutagenic intermediates if BER is
not completed rapidly or accurately.
ity,75,76 and the ‘‘programmed’’ excision
of 5mC by BER can be employed to
trigger the expression of the genes
needed to determine cell fate in brain
and other tissues.77,78 In this process,
5mC is converted to 5-formylcytosine
and 5-carboxylcytosine by iterative cy-
cles of hydroxylation/oxidation,79,80
which are then substrates for thymine
DNA glycosylase (TDG)-dependent BER and therefore are
excised and replaced with cytosine.80,81 This mechanism of
gene regulation underpins the recent discovery of high levels
of programmed DNA single-strand breakage and repair in
neuronal enhancers78,82 and, most likely, in other cell types
during cell fate determination and maintenance.77 The pro-
grammed introduction of SSBs in this way exploits the coordi-
nation, rapidity, and accuracy of the BER process. However,
similar to the use of topoisomerases to regulate transcription,
this approach risks the occasional occurrence of failed or
erroneous BER, resulting in DNA strand breaks or mutations
with potentially pathogenic consequences, including neuro-
logical disease7 (Figure 3). Intriguingly, failed or erroneous
BER events are deliberately promoted at sites of transcrip-
tion-associated cytosine deamination in immunoglobulin
genes in B cells, to induce DNA strand breaks and mutations
to increase antibody diversity.83 However, spurious engage-
ment of this pathway outside of immunoglobulin genes can
lead to genome instability.84
TRANSCRIPTION-DNA REPLICATION CONFLICTS
Collisions between the DNA replication and transcription machin-
eries are known as transcription-replication conflicts (TRCs) and
can lead to increased R loop formation, DNA breakage, check-
point activation, and genome instability.44,85–87 Notably, TRCs
likely contribute to the increased risk of breakage at genomic
fragile sites.88,89 Encounters between the DNA replication ma-
chinery and RNAPs that are moving in the same direction are co-
directional TRCs (CD-TRCs), whereas encounters between the
DNA replication machinery and RNAPs traveling in opposite direc-
tions, toward each other, are head-on TRCs (HO-TRCs)44,87,90–92
(Figure 4). In E. coli and yeast, programmed replication fork
Molecular Cell 84, January 4, 2024 73
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Figure 4. Transcription-DNA replication conflicts and R loops

Replication (purple) and transcription (blue) machineries present on the same DNA template can lead to transcription-replication conflicts (TRCs), R loop for-
mation, and to DNA breakage and genome rearrangement. TRCs can be codirectional TRCs (CD-TRCs) if the DNA replication machinery and RNAPs are moving
in the same direction or head-on TRCs (HO-TRCs) if RNAP and the DNA replication machinery are traveling in opposite directions toward each other. The cellular
response to TRCs includes the activation of checkpoints to prevent unnecessary replication fork cleavage, the clearance of blocked RNAP and/or the replisome,
the removal of associated R loops, and the restart of DNA replication. Some examples of the factors implicated in these processes are shown.

barriers (RFBs) act in specific regions of the genome to prevent
HO-TRCs.93,94 In mammalian cells, there is little evidence for
RFBs for preventing HO-TRC, although asymmetric poly(AT)
tracts may play such a role in murine cells.95 However, the
complexity of DNA replication and transcription in mammalian ge-
nomes offers additional opportunities for temporal and spatial
regulation. For example, the initiation of DNA replication upstream
of active genes may favor codirectional movement of the DNA
replication and transcription machineries, thereby reducing HO-
TRCs.96 In addition, DNA replication may be spatially separated
from transcription by DNA loop extrusion during the latter97,98
and/or by temporally separating the two processes in different
S-phase territories.99
A major component of the cellular response to TRCs is the pro-
tection and restoration of replication fork progression.87,100 In
addition to the proper processing of R loops described above,
and the established canonical responses to DNA replication
stress,101,102 cells possess specific mechanisms to promote
fork progression at TRCs.100 For example, MUS81 structure-
specific endonuclease and RECQ5 helicase can promote fork
breakage and restart at TRC-associated R loops,103 and the acti-
vation of ATR-Chk1 by MUS81 triggers a negative feedback loop
that protects reversed forks from excessive cleavage.104 The
DNA polymerase clamp (PCNA) unloader ATPase family AAA
domain containing 5 (ATAD5) can resolve R loops at stalled
forks, by recruiting DEAD/DDX RNA helicases,105 and the ubiq-
uitin E3 ligase TRAIP can unload the stalled replication and/or
transcription machinery at TRCs, most likely in conjunction
with p97 segregase.106 In addition, PP1 nuclear targeting subunit
(PNUTS), and the E3 ubiquitin ligase tripartite-motif-containing
28 (TRIM28), in conjunction with RECQ5, can remove RNAPII
from chromatin, thereby suppressing TRCs.107,108
TRANSCRIPTIONAL STRESS AND NEUROLOGICAL
DISEASE
The interrelationship between transcription and DNA damage
has major implications for human health and may underpin a
variety of human diseases (Figure 5). In particular, unrepaired
DNA lesions that block transcription are implicated strongly as
a cause of neurodegeneration and aging.5 Currently, this is
best exemplified by unrepaired bulky lesions. The repair of
bulky DNA lesions is conducted by nucleotide excision repair
(NER); a complex pathway comprising two sub-pathways de-
noted global genome NER (GG-NER) and transcription-
coupled NER (TC-NER).5,109,110 Whereas GG-NER interro-
gates the entire genome for bulky lesions, TC-NER is rapidly
triggered on transcribed strands of active genes at sites of
stalled RNAP. Genetic defects in these pathways result in
the diseases xeroderma pigmentosum (XP) and Cockayne
syndrome (CS), respectively.5,109,110 Importantly, mutations
that inactivate only GG-NER (e.g., XP complementation group
C [XPC] and XP complementation group E [XPE]) result pri-
marily in UV hypersensitivity and cancer predisposition,
whereas mutations that inactivate both sub-pathways (e.g.,
XP complementation group A [XPA]) result in XP that is
additionally associated with neurodegeneration.111 In
contrast, mutations that inactivate only TC-NER (e.g., CSA
or CSB) result in CS, a disease associated with UV hypersen-
sitivity, severe neurological defects, and accelerated ag-
ing.111,112 The more severe neuropathology in CS may reflect
that CSA and CSB are specifically required to backtrack and
remove stalled RNAP from the site of a lesion, whereas XPA
is not, leading to a more prolonged RNAP blockage.5,109,110
Consistent with this idea, CSA/CSB mutations that do not

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Figure 5. Transcription-associated DNA damage and neurological disease

(A) In diseases lacking GG-NER, increased UV sensitivity and cancer are observed (e.g., XPC and XPE). In diseases lacking all NER, but in which RNA polymerase
clearance can occur, increased UV sensitivity, cancer, and neurodegeneration are observed (e.g., XPA, XPF, and XPG). In diseases lacking only TC-NER, but in
which RNA polymerase clearance cannot occur, neurodevelopmental defects, neurodegeneration, and premature aging are observed (e.g., CS type A [CSA] and
CS type B [CSB]). In diseases lacking SSBR, in which SSBs with damaged termini persist, neurodevelopmental and/or neurodegenerative disease is observed.
XPA, xeroderma pigmentosum complementation group A; XPC, xeroderma pigmentosum complementation group C; XPG, xeroderma pigmentosum
complementation group G; CS, Cockayne syndrome; SCAN1, spinocerebellar ataxia with axonal neuropathy type 1 (mutated in TDP1); AOA1, ataxia-oculomotor
apraxia type-1 (mutated in APTX); MCSZ, microcephaly with early onset seizures (mutated in PNKP); AOA4, ataxia oculomotor apraxia-4 (mutated in PNKP);
CMT2B2, Charcot-Marie-Tooth disease type 2B2 (mutated in PNKP); SCAR23, spinocerebellar ataxia autosomal recessive 23 (mutated in TDP2); SCAR26,
spinocerebellar ataxia autosomal recessive 26 (mutated in XRCC1).

affect RNAP clearance result in UV-sensitive syndrome
(UVSS), a relatively mild disease without severe neurological
symptoms.109
Alternatively, the severe neurological pathology in CS may
also reflect the loss of transcription-coupled repair of non-bulky
DNA base lesions and/or SSBs.5 Defects in the repair of SSBs
with aberrant termini, which as described above can block
RNAP progression, are strongly implicated in hereditary neuro-
logical disease.30,32 For example, mutations in the PNKP protein
that repairs DNA strand breaks with 30 -phosphate and/or 50 -hy-
droxyl termini are associated with the neurodevelopmental and/
or neurodegenerative diseases microcephaly with early onset
seizures (MCSZ), ataxia-oculomotor apraxia-4 (AOA4), and
Charcot-Marie-Tooth disease type 2B2 (CMT2B2).32 Similarly,
mutations in the TDP1 and TDP2 proteins that repair topoisom-
erase-linked DNA strand breaks are associated with transcrip-
tional disruption and the neurodevelopmental/neurodegenera-
tive diseases spinocerebellar ataxia with axonal neuropathy-1
(SCAN1) and spinocerebellar ataxia autosomal recessive 23
(SCAR23), respectively.59,60
Blocked RNAP progression is likely not the only contributing
factor to SSBR-defective diseases. For example, the inability to
rapidly repair sites of cytosine demethylation during epigenetic
reprogramming may prevent the transcriptional initiation of
genes critical for neuronal function.7 In addition, R loops such
as those formed at sites of blocked RNAP at SSBs also likely
contribute to neurological disease.41,113,114 Consistent with
this, mutations in the R loop helicase SETX result in the neurode-
generative diseases ataxia-oculomotor apraxia type-2 (AOA2)
and amyotrophic lateral sclerosis type 4 (ALS4).115 Finally, hyper-
activation of the SSB sensor protein poly(ADP-ribose) polymer-
ase 1 (PARP1) at unrepaired SSBs is linked with aberrant histone
monoubiquitylation, prolonged transcriptional suppression, and
neurological disease.116–118
TRANSCRIPTIONAL STRESS, GENOME INSTABILITY,
AND CANCER
In addition to acute impacts on gene expression, transcription-
associated DNA damage may result in irreversible genetic
changes and genome instability. For example, deep sequencing
of human neurons has identified an age-related increase in both
somatic point mutations and insertion/deletions in neurons
within gene regulatory elements.119,120 The source of such muta-
tions is currently unknown, but transcription-associated DNA
damage such as that arising stochastically or resulting from
abortive topoisomerase activity and/or cytosine demethylation
are candidates.119 In proliferating cells, transcription-associated
DNA damage is also likely to be a major contributor to cancer.
For example, as discussed above, sites of abortive TOP1 activity
at genomic ribonucleotides are associated with short 2–5 bp de-
letions and account for the cancer mutational signature ID4.64
Moreover, oncogene-induced replication stress is associated
with increased TRCs and R loop formation in multiple cancer
models,121,122 including HRAS overexpression,123 cyclin E over-
expression,124 and MYC overexpression125 (Figure 6). As
described earlier, some fragile sites are linked to TRCs and
correlate strongly with translocations linked to cancer.126,127
FUTURE QUESTIONS AND PERSPECTIVES
The interrelationship between transcription and DNA damage is
intimate and complex. While in some contexts DNA damage and
transcription work synergistically to regulate gene expression,

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very often, they are in conflict and therefore threaten genome
integrity. Currently, it is unclear to what extent the relationship
between these two processes causes diseases such as cancer
and neurodegeneration or contributes to normal aging. Howev-
er, the discoveries reviewed here point strongly to these possibil-
ities and highlight the likelihood that, with a fuller understanding
of how DNA damage and transcription are related, we may open
up new avenues for therapeutic intervention.
ACKNOWLEDGMENTS
The authors apologize to colleagues for the absence of many excellent publi-
cations due to length constraints. The Caldecott lab is funded by program
grants from the MRC (MR/W024128/1) and CRUK (C6563/A27322). Diagrams
were created using Biorender.com. Data statement: no data is associated with
this review.
DECLARATION OF INTERESTS
The authors declare no competing interests.
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