ll

OPEN ACCESS

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: [email protected] (L.M.), [email protected] (A.G.), [email protected] (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

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

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

Molecular Cell 84, January 4, 2024 71

 ll
OPEN ACCESS Review

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

72 Molecular Cell 84, January 4, 2024

 ll
Review OPEN ACCESS

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
decatenate and untangle chromosomes and to facilitate short- DNA glycosylase (TDG)-dependent BER and therefore are
and long-range chromosome movements during transcriptional excised and replaced with cytosine.80,81 This mechanism of
regulation. TOP2 is located at chromosome loop anchors, and gene regulation underpins the recent discovery of high levels
its activity can help regulate RNA pausing and release at pro- of programmed DNA single-strand breakage and repair in
moters, splice sites, and enhancers within active genes.57,66,67 neuronal enhancers78,82 and, most likely, in other cell types
Similar to TOP1, TOP2 activity is particularly important for expres- during cell fate determination and maintenance.77 The pro-
sion of very large genes (>200 kb), including numerous genes grammed introduction of SSBs in this way exploits the coordi-
required for normal neuronal function.68 Also, similar to TOP1, nation, rapidity, and accuracy of the BER process. However,
TOP2 cleavage complexes harbor protein-linked DNA breaks similar to the use of topoisomerases to regulate transcription,
that are obligate intermediates of TOP2 activity.65 Although this approach risks the occasional occurrence of failed or
such breaks are normally resealed at the end of each catalytic cy- erroneous BER, resulting in DNA strand breaks or mutations
cle, the cleavage complexes can occasionally become abortive, with potentially pathogenic consequences, including neuro-
resulting in bona fide SSBs and/or DSBs that require DNA- logical disease7 (Figure 3). Intriguingly, failed or erroneous
strand-break repair pathways for the removal59,60 (Figure 3). If BER events are deliberately promoted at sites of transcrip-
such TOP2-induced DSBs are not repaired rapidly and accu- tion-associated cytosine deamination in immunoglobulin
rately, they can lead to oncogenic translocations55,69,70 or neuro- genes in B cells, to induce DNA strand breaks and mutations
logical disease.71 It has been suggested that the induction of to increase antibody diversity.83 However, spurious engage-
bona fide DSBs by TOP2 is required to regulate gene expres- ment of this pathway outside of immunoglobulin genes can
sion.72,73 However, it seems more likely that such DSBs are sim- lead to genome instability.84
ply a consequence of TOP2 activity occasionally becoming
abortive. TRANSCRIPTION-DNA REPLICATION CONFLICTS

TRANSCRIPTION-INDUCED DNA BREAKAGE BY Collisions between the DNA replication and transcription machin-
DNA BER eries are known as transcription-replication conflicts (TRCs) and
can lead to increased R loop formation, DNA breakage, check-
The formation of single-stranded DNA during gene transcrip- point activation, and genome instability.44,85–87 Notably, TRCs
tion can result in increased rates of DNA breakage and likely contribute to the increased risk of breakage at genomic
genetic mutation, for example, as a result of increased spon- fragile sites.88,89 Encounters between the DNA replication ma-
taneous cytosine deamination and other types of base dam- chinery and RNAPs that are moving in the same direction are co-
age38,74 (Figure 3). DNA base damage is also sometimes directional TRCs (CD-TRCs), whereas encounters between the
introduced deliberately in human cells, during transcriptional DNA replication machinery and RNAPs traveling in opposite direc-
regulation. For example, 5-methycytosine (5mC) is an epige- tions, toward each other, are head-on TRCs (HO-TRCs)44,87,90–92
netic mark that typically suppresses transcriptional activ- (Figure 4). In E. coli and yeast, programmed replication fork

Molecular Cell 84, January 4, 2024 73

 ll
OPEN ACCESS Review

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

74 Molecular Cell 84, January 4, 2024

 ll
Review OPEN ACCESS

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

Molecular Cell 84, January 4, 2024 75

 ll
OPEN ACCESS Review
Figure 6. Transcription-associated DNA
damage and genome instability
Oncogene-induced replication stress, such as that
resulting from HRAS, cyclin E, and MYC over-
expression, can lead to increased transcription-
replication conflicts (TRCs) and R loops that may
contribute to genome instability and cancer.

very often, they are in conflict and therefore threaten genome 6. Lindahl, T. (1993). Instability and decay of the primary structure of DNA.
Nature 362, 709–715.
integrity. Currently, it is unclear to what extent the relationship
between these two processes causes diseases such as cancer 7. Caldecott, K.W., Ward, M.E., and Nussenzweig, A. (2022). The threat of
and neurodegeneration or contributes to normal aging. Howev- programmed DNA damage to neuronal genome integrity and plasticity.
Nat. Genet. 54, 115–120.
er, the discoveries reviewed here point strongly to these possibil-
ities and highlight the likelihood that, with a fuller understanding 8. Brickner, J.R., Garzon, J.L., and Cimprich, K.A. (2022). Walking a tight-
of how DNA damage and transcription are related, we may open rope: the complex balancing act of R-loops in genome stability. Mol.
Cell 82, 2267–2297.
up new avenues for therapeutic intervention.
9. Damsma, G.E., Alt, A., Brueckner, F., Carell, T., and Cramer, P. (2007).
Mechanism of transcriptional stalling at cisplatin-damaged DNA. Nat.
ACKNOWLEDGMENTS Struct. Mol. Biol. 14, 1127–1133.

10. Enoiu, M., Jiricny, J., and Scha€rer, O.D. (2012). Repair of cisplatin-
The authors apologize to colleagues for the absence of many excellent publi-
induced DNA interstrand crosslinks by a replication-independent
cations due to length constraints. The Caldecott lab is funded by program pathway involving transcription-coupled repair and translesion synthe-
grants from the MRC (MR/W024128/1) and CRUK (C6563/A27322). Diagrams sis. Nucleic Acids Res. 40, 8953–8964.
were created using Biorender.com. Data statement: no data is associated with
this review. 11. Perlow, R.A., Kolbanovskii, A., Hingerty, B.E., Geacintov, N.E., Broyde,
S., and Scicchitano, D.A. (2002). DNA adducts from a tumorigenic
metabolite of benzo[a]pyrene block human RNA polymerase II elongation
DECLARATION OF INTERESTS in a sequence- and stereochemistry-dependent manner. J. Mol. Biol.
321, 29–47.
The authors declare no competing interests.
12. Donahue, B.A., Fuchs, R.P.P., Reines, D., and Hanawalt, P.C. (1996). Ef-
fects of aminofluorene and acetylaminofluorene DNA adducts on tran-
scriptional elongation by RNA polymerase II. J. Biol. Chem. 271,
REFERENCES
10588–10594.

1. Saponaro, M., Kantidakis, T., Mitter, R., Kelly, G.P., Heron, M., Williams, 13. Solier, S., Ryan, M.C., Martin, S.E., Varma, S., Kohn, K.W., Liu, H., Zee-
H., Söding, J., Stewart, A., and Svejstrup, J.Q. (2014). RECQL5 controls berg, B.R., and Pommier, Y. (2013). Transcription poisoning by topo-
transcript elongation and suppresses genome instability associated with isomerase I is controlled by gene length, splice sites, and miR-142-3p.
transcription stress. Cell 157, 1037–1049. Cancer Res. 73, 4830–4839.

2. Jonkers, I., and Lis, J.T. (2015). Getting up to speed with transcription 14. Mulderrig, L., Garaycoechea, J.I., Tuong, Z.K., Millington, C.L., Dingler,
elongation by RNA polymerase II. Nat. Rev. Mol. Cell Biol. 16, 167–177. F.A., Ferdinand, J.R., Gaul, L., Tadross, J.A., Arends, M.J., O’rahilly,
S., et al. (2021). Aldehyde-driven transcriptional stress triggers an
3. Mayer, A., Landry, H.M., and Churchman, L.S. (2017). Pause & go: from anorexic DNA damage response. Nature 600, 158–163.
the discovery of RNA polymerase pausing to its functional implications.
Curr. Opin. Cell Biol. 46, 72–80. 15. Voulgaridou, G.P., Anestopoulos, I., Franco, R., Panayiotidis, M.I., and
Pappa, A. (2011). DNA damage induced by endogenous aldehydes: cur-
4. Gregersen, L.H., and Svejstrup, J.Q. (2018). The cellular response to rent state of knowledge. Mutat. Res. 711, 13–27.
transcription-blocking DNA damage. Trends Biochem. Sci. 43, 327–341.
16. Garaycoechea, J.I., Crossan, G.P., Langevin, F., Mulderrig, L., Louzada,
5. Lans, H., Hoeijmakers, J.H.J., Vermeulen, W., and Marteijn, J.A. (2019). S., Yang, F., Guilbaud, G., Park, N., Roerink, S., Nik-Zainal, S., et al.
The DNA damage response to transcription stress. Nat. Rev. Mol. Cell (2018). Alcohol and endogenous aldehydes damage chromosomes and
Biol. 20, 766–784. mutate stem cells. Nature 553, 171–177.

76 Molecular Cell 84, January 4, 2024

 ll
Review OPEN ACCESS

17. Brooks, P.J. (2008). The 8,50 -cyclopurine-20 -deoxynucleosides: candi- 36. Pestov, N.A., Gerasimova, N.S., Kulaeva, O.I., and Studitsky, V.M.
date neurodegenerative DNA lesions in xeroderma pigmentosum, and (2015). Structure of transcribed chromatin is a sensor of DNA damage.
unique probes of transcription and nucleotide excision repair. DNA Sci. Adv. 1, e1500021.
Repair (Amst) 7, 1168–1179.
37. Gerasimova, N.S., Pestov, N.A., and Studitsky, V.M. (2023). Role of his-
18. Pourquier, P., and Pommier, Y. (2001). Topoisomerase I-mediated DNA tone tails and single strand DNA breaks in nucleosomal arrest of RNA po-
damage. Adv. Cancer Res. 80, 189–216. lymerase. Int. J. Mol. Sci. 24, 2295.

19. Capranico, G., Ferri, F., Fogli, M.V., Russo, A., Lotito, L., and Baranello, L. 38. Kim, N., and Jinks-Robertson, S. (2012). Transcription as a source of
(2007). The effects of camptothecin on RNA polymerase II transcription: genome instability. Nat. Rev. Genet. 13, 204–214.
roles of DNA topoisomerase I. Biochimie 89, 482–489.
39. Petermann, E., Lan, L., and Zou, L. (2022). Sources, resolution and phys-
20. Pommier, Y., Sun, Y., Huang, S.N., and Nitiss, J.L. (2016). Roles of eu- iological relevance of R-loops and RNA–DNA hybrids. Nat. Rev. Mol. Cell
karyotic topoisomerases in transcription, replication and genomic stabil- Biol. 23, 521–540.
ity. Nat. Rev. Mol. Cell Biol. 17, 703–721.
40. Garcı́a-Muse, T., and Aguilera, A. (2019). R loops: from physiological to
21. Bendixen, C., Thomsen, B., Alsner, J., and Westergaard, O. (1990). pathological roles. Cell 179, 604–618.
Camptothecin-stabilized topoisomerase I-DNA adducts cause prema-
ture termination of transcription. Biochemistry 29, 5613–5619. 41. Crossley, M.P., Bocek, M., and Cimprich, K.A. (2019). R-loops as cellular
regulators and genomic threats. Mol. Cell 73, 398–411.
22. Wu, J., and Liu, L.F. (1997). Processing of topoisomerase I cleavable
complexes into DNA damage by transcription. Nucleic Acids Res. 25, 42. Patel, P.S., Krishnan, R., and Hakem, R. (2022). Emerging roles of DNA
4181–4186. topoisomerases in the regulation of R-loops. Mutat. Res. Genet. Toxicol.
Environ. Mutagen. 876–877, 503450.
23. Brégeon, D., and Doetsch, P.W. (2011). Transcriptional mutagenesis:
causes and involvement in tumour development. Nat. Rev. Cancer 11, 43. Shivji, M.K.K., Renaudin, X., Williams, Ç.H., and Venkitaraman, A.R.
218–227. (2018). BRCA2 regulates transcription elongation by RNA polymerase II
to prevent R-loop accumulation. Cell Rep. 22, 1031–1039.
24. Kathe, S.D., Shen, G.P., and Wallace, S.S. (2004). Single-stranded
breaks in DNA but not oxidative DNA base damages block transcriptional 44. Hamperl, S., Bocek, M.J., Saldivar, J.C., Swigut, T., and Cimprich, K.A.
elongation by RNA polymerase II in HeLa cell nuclear extracts. J. Biol. (2017). Transcription-replication conflict orientation modulates R-loop
Chem. 279, 18511–18520. levels and activates distinct DNA damage responses. Cell 170, 774–
786.e19.
25. Tornaletti, S., Maeda, L.S., and Hanawalt, P.C. (2006). Transcription ar-
rest at an abasic site in the transcribed strand of template DNA. Chem. 45. Cristini, A., Ricci, G., Britton, S., Salimbeni, S., Huang, S.-Y.N., Marinello,
Res. Toxicol. 19, 1215–1220. J., Calsou, P., Pommier, Y., Favre, G., Capranico, G., et al. (2019). Dual
processing of R-loops and topoisomerase I induces transcription-
dependent DNA double-strand breaks. Cell Rep. 28, 3167–3181.e6.
26. Neil, A.J., Belotserkovskii, B.P., and Hanawalt, P.C. (2012). Transcription
blockage by bulky end termini at single-strand breaks in the DNA tem-
46. Sordet, O., Nakamura, A.J., Redon, C.E., and Pommier, Y. (2010). DNA
plate: differential effects of 50 and 30 adducts. Biochemistry 51,
double-strand breaks and ATM activation by transcription-blocking
8964–8970.
DNA lesions. Cell Cycle 9, 274–278.
27. Zhou, W., and Doetsch, P.W. (1993). Effects of abasic sites and DNA sin-
47. Sordet, O., Redon, C.E., Guirouilh-Barbat, J., Smith, S., Solier, S.,
gle-strand breaks on prokaryotic RNA polymerases. Proc. Natl. Acad.
Douarre, C., Conti, C., Nakamura, A.J., Das, B.B., Nicolas, E., et al.
Sci. USA 90, 6601–6605.
(2009). Ataxia telangiectasia mutated activation by transcription- and
topoisomerase I-induced DNA double-strand breaks. EMBO Rep. 10,
28. Zhou, W., and Doetsch, P.W. (1994). Transcription bypass or blockage at 887–893.
single-strand breaks on the DNA template strand: effect of different 30
and 50 flanking groups on the T7 RNA polymerase elongation complex. 48. Aiello, U., Challal, D., Wentzinger, G., Lengronne, A., Appanah, R., Pa-
Biochemistry 33, 14926–14934. sero, P., Palancade, B., and Libri, D. (2022). Sen1 is a key regulator of
transcription-driven conflicts. Mol. Cell 82, 2952–2966.e6.
29. Zhou, W., Reines, D., and Doetsch, P.W. (1995). T7 RNA polymerase
bypass of large gaps on the template strand reveals a critical role of 49. Chakraborty, P., and Grosse, F. (2011). Human DHX9 helicase preferen-
the nontemplate strand in elongation. Cell 82, 577–585. tially unwinds RNA-containing displacement loops (R-loops) and
G-quadruplexes. DNA Repair (Amst) 10, 654–665.
30. Caldecott, K.W. (2008). Single-strand break repair and genetic disease.
Nat. Rev. Genet. 9, 619–631. 50. Pérez-Calero, C., Bayona-Feliu, A., Xue, X., Barroso, S.I., Muñoz, S.,
González-Basallote, V.M., Sung, P., and Aguilera, A. (2020). UAP56/
31. McKinnon, P.J. (2017). Genome integrity and disease prevention in the DDX39B is a major cotranscriptional RNA–DNA helicase that unwinds
nervous system. Genes Dev. 31, 1180–1194. harmful R loops genome-wide. Genes Dev. 34, 898–912.

32. Caldecott, K.W. (2022). DNA single-strand break repair and human ge- 51. Cristini, A., Groh, M., Kristiansen, M.S., and Gromak, N. (2018). RNA/
netic disease. Trends Cell Biol. 32, 733–745. DNA hybrid interactome identifies DXH9 as a molecular player in tran-
scriptional termination and R-loop-associated DNA damage. Cell Rep.
33. Kalasova, I., Hailstone, R., Bublitz, J., Bogantes, J., Hofmann, W., Leal, 23, 1891–1905.
A., Hanzlikova, H., and Caldecott, K.W. (2020). Pathological mutations
in PNKP trigger defects in DNA single-strand break repair but not DNA 52. Kotsantis, P., Segura-Bayona, S., Margalef, P., Marzec, P., Ruis, P., He-
double-strand break repair. Nucleic Acids Res. 48, 6672–6684. witt, G., Bellelli, R., Patel, H., Goldstone, R., Poetsch, A.R., et al. (2020).
RTEL1 regulates G4/R-loops to avert replication-transcription collisions.
34. Katyal, S., Lee, Y., Nitiss, K.C., Downing, S.M., Li, Y., Shimada, M., Zhao, Cell Rep. 33, 108546.
J., Russell, H.R., Petrini, J.H.J., Nitiss, J.L., et al. (2014). Aberrant
topoisomerase-1 DNA lesions are pathogenic in neurodegenerative 53. Lockhart, A., Pires, V.B., Bento, F., Kellner, V., Luke-Glaser, S., Yakoub,
genome instability syndromes. Nat. Neurosci. 17, 813–821. G., Ulrich, H.D., and Luke, B. (2019). RNase H1 and H2 are differentially
regulated to process RNA-DNA hybrids. Cell Rep. 29, 2890–2900.e5.
35. El-Khamisy, S.F., Saifi, G.M., Weinfeld, M., Johansson, F., Helleday, T.,
Lupski, J.R., and Caldecott, K.W. (2005). Defective DNA single-strand 54. Camino, L.P., Dutta, A., Barroso, S., Pérez-Calero, C., Katz, J.N., Garcı́a-
break repair in spinocerebellar ataxia with axonal neuropathy-1. Nature Rubio, M., Sung, P., Gómez-González, B., and Aguilera, A. (2023). DICER
434, 108–113. ribonuclease removes harmful R-loops. Mol. Cell 83, 3707–3719.e5.

Molecular Cell 84, January 4, 2024 77

 ll
OPEN ACCESS Review
55. Gómez-Herreros, F. (2019). DNA double strand breaks and chromo- 73. Ju, B.-G., Lunyak, V.V., Perissi, V., Garcia-Bassets, I., Rose, D.W., Glass,
somal translocations induced by DNA topoisomerase II. Front. Mol. Bio- C.K., and Rosenfeld, M.G. (2006). A topoisomerase IIbeta-mediated
sci. 6, 141. dsDNA break required for regulated transcription. Science 312,
1798–1802.
56. Canela, A., Maman, Y., Huang, S.N., Wutz, G., Tang, W., Zagnoli-Vieira,
G., Callen, E., Wong, N., Day, A., Peters, J.-M., et al. (2019). Topoisom- 74. Jinks-Robertson, S., and Bhagwat, A.S. (2014). Transcription-associated
erase II-induced chromosome breakage and translocation is determined mutagenesis. Annu. Rev. Genet. 48, 341–359.
by chromosome architecture and transcriptional activity. Mol. Cell 75,
252–266.e8. 75. Schu€beler, D. (2015). Function and information content of DNA methyl-
ation. Nature 517, 321–326.
57. Baranello, L., Kouzine, F., and Levens, D. (2013). DNA topoisomerases
beyond the standard role. Transcription 4, 232–237. 76. Hill, P.W.S., Amouroux, R., and Hajkova, P. (2014). DNA demethylation,
Tet proteins and 5-hydroxymethylcytosine in epigenetic reprogramming:
58. Pommier, Y., Nussenzweig, A., Takeda, S., and Austin, C. (2022). Human an emerging complex story. Genomics 104, 324–333.
topoisomerases and their roles in genome stability and organization. Nat.
Rev. Mol. Cell Biol. 23, 407–427. 77. Wang, D., Wu, W., Callen, E., Pavani, R., Zolnerowich, N., Kodali, S.,
Zong, D., Wong, N., Noriega, S., Nathan, W.J., et al. (2022). Active
59. Zagnoli-Vieira, G., and Caldecott, K.W. (2020). Untangling trapped topoi- DNA demethylation promotes cell fate specification and the DNA dam-
somerases with tyrosyl-DNA phosphodiesterases. DNA Repair (Amst) age response. Science 378, 983–989.
94, 102900.
78. Wu, W., Hill, S.E., Nathan, W.J., Paiano, J., Callen, E., Wang, D., Shinoda,
60. Pommier, Y., Huang, S.Y., Gao, R., Das, B.B., Murai, J., and Marchand, K., Wietmarschen, N. van, Colón-Mercado, J.M., Zong, D., et al. (2021).
C. (2014). Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2). DNA Neuronal enhancers are hotspots for DNA single-strand break repair. Na-
Repair (Amst) 19, 114–129. ture 593, 440–444.
61. Hubert, L., Lin, Y., Dion, V., and Wilson, J.H. (2011). Topoisomerase 1 79. Tahiliani, M., Koh, K.P., Shen, Y., Pastor, W.A., Bandukwala, H., Brudno,
and single-strand break repair modulate transcription-induced CAG Y., Agarwal, S., Iyer, L.M., Liu, D.R., Aravind, L., et al. (2009). Conversion
repeat contraction in human cells. Mol. Cell. Biol. 31, 3105–3112. of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by
MLL partner TET1. Science 324, 930–935.
62. Kim, N., Huang, S.N., Williams, J.S., Li, Y.C., Clark, A.B., Cho, J.-E., Kun-
kel, T.A., Pommier, Y., and Jinks-Robertson, S. (2011). Mutagenic pro- 80. He, Y.-F., Li, B.-Z., Li, Z., Liu, P., Wang, Y., Tang, Q., Ding, J., Jia, Y.,
cessing of ribonucleotides in DNA by yeast topoisomerase I. Science Chen, Z., Li, L., et al. (2011). Tet-mediated formation of
332, 1561–1564. 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science
333, 1303–1307.
63. Williams, J.S., Smith, D.J., Marjavaara, L., Lujan, S.A., Chabes, A., and
Kunkel, T.A. (2013). Topoisomerase 1-mediated removal of ribonucleo-
81. Maiti, A., and Drohat, A.C. (2011). Thymine DNA glycosylase can rapidly
tides from nascent leading-strand DNA. Mol. Cell 49, 1010–1015.
excise 5-formylcytosine and 5-carboxylcytosine POTENTIAL IMPLICA-
64. Reijns, M.A.M., Parry, D.A., Williams, T.C., Nadeu, F., Hindshaw, R.L., TIONS FOR ACTIVE demethylation OF CpG SITES*. J. Biol. Chem.
Rios Szwed, D.O.R., Nicholson, M.D., Carroll, P., Boyle, S., Royo, R., 286, 35334–35338.
et al. (2022). Signatures of top1 transcription-associated mutagenesis
82. Reid, D.A., Reed, P.J., Schlachetzki, J.C.M., Nitulescu, I.I., Chou, G.,
in cancer and germline. Nature 602, 623–631.
Tsui, E.C., Jones, J.R., Chandran, S., Lu, A.T., McClain, C.A., et al.
65. Nitiss, J.L. (2009). DNA topoisomerase II and its growing repertoire of (2021). Incorporation of a nucleoside analog maps genome repair sites
biological functions. Nat. Rev. Cancer 9, 327–337. in postmitotic human neurons. Science 372, 91–94.

66. Dellino, G.I., Palluzzi, F., Chiariello, A.M., Piccioni, R., Bianco, S., Furia, 83. Alt, F.W., Zhang, Y., Meng, F.-L., Guo, C., and Schwer, B. (2013). Mech-
L., Conti, G. De, Bouwman, B.A.M., Melloni, G., Guido, D., et al. (2019). anisms of programmed DNA lesions and genomic instability in the im-
Release of paused RNA polymerase II at specific loci favors DNA dou- mune system. Cell 152, 417–429.
ble-strand-break formation and promotes cancer translocations. Nat.
Genet. 51, 1011–1023. 84. Casellas, R., Basu, U., Yewdell, W.T., Chaudhuri, J., Robbiani, D.F., and
Di Noia, J.M. (2016). Mutations, kataegis and translocations in B cells:
67. Canela, A., Maman, Y., Jung, S., Wong, N., Callén, E., Day, A., Kieffer- understanding AID promiscuous activity. Nat. Rev. Immunol. 16,
Kwon, K.R., Pekowska, A., Zhang, H., Rao, S.S.P., et al. (2017). Genome 164–176.
organization drives chromosome fragility. Cell 170, 507–521.e18.
85. Macheret, M., and Halazonetis, T.D. (2018). Intragenic origins due to
68. King, I.F., Yandava, C.N., Mabb, A.M., Hsiao, J.S., Huang, H.S., Pearson, short G1 phases underlie oncogene-induced DNA replication stress. Na-
B.L., Calabrese, J.M., Starmer, J., Parker, J.S., Magnuson, T., et al. ture 7694, 112–116.
(2013). Topoisomerases facilitate transcription of long genes linked to
autism. Nature 501, 58–62. 86. Goehring, L., Huang, T.T., and Smith, D.J. (2023). Transcription-replica-
tion conflicts as a source of genome instability. Annu. Rev. Genet. 13, 21.
69. Lin, C., Yang, L., Tanasa, B., Hutt, K., Ju, B.G., Ohgi, K.A., Zhang, J.,
Rose, D.W., Fu, X.D., Glass, C.K., et al. (2009). Nuclear receptor-induced 87. Gómez-González, B., and Aguilera, A. (2019). Transcription-mediated
chromosomal proximity and DNA breaks underlie specific translocations replication hindrance: a major driver of genome instability. Genes Dev.
in cancer. Cell 139, 1069–1083. 33, 1008–1026.

70. Haffner, M.C., Aryee, M.J., Toubaji, A., Esopi, D.M., Albadine, R., Gurel, 88. Helmrich, A., Ballarino, M., and Tora, L. (2011). Collisions between repli-
B., Isaacs, W.B., Bova, G.S., Liu, W., Xu, J., et al. (2010). Androgen- cation and transcription complexes cause common fragile site instability
induced TOP2B-mediated double-strand breaks and prostate cancer at the longest human genes. Mol. Cell 44, 966–977.
gene rearrangements. Nat. Genet. 42, 668–675.
89. Sanchez, A., de Vivo, A., Tonzi, P., Kim, J., Huang, T.T., and Kee, Y.
71. Gómez-Herreros, F., Schuurs-Hoeijmakers, J.H.M., McCormack, M., (2020). Transcription-replication conflicts as a source of common fragile
Greally, M.T., Rulten, S., Romero-Granados, R., Counihan, T.J., Chaila, site instability caused by BMI1-RNF2 deficiency. PLoS Genet. 16,
E., Conroy, J., Ennis, S., et al. (2014). TDP2 protects transcription from e1008524.
abortive topoisomerase activity and is required for normal neural func-
tion. Nat. Genet. 46, 516–521. 90. Mirkin, E.V., and Mirkin, S.M. (2005). Mechanisms of transcription-repli-
cation collisions in bacteria. Mol. Cell. Biol. 25, 888–895.
72. Madabhushi, R., Gao, F., Pfenning, A.R., Pan, L., Yamakawa, S., Seo, J.,
Rueda, R., Phan, T.X., Yamakawa, H., Pao, P.-C., et al. (2015). Activity- 91. Prado, F., and Aguilera, A. (2005). Impairment of replication fork progres-
induced DNA breaks govern the expression of neuronal early-response sion mediates RNA PolII transcription-associated recombination. EMBO
Genes. Cell 161, 1592–1605. J. 24, 1267–1276.

78 Molecular Cell 84, January 4, 2024

 ll
Review OPEN ACCESS

92. Lang, K.S., and Merrikh, H. (2018). The clash of macromolecular titans: 110. Marteijn, J.A., Lans, H., Vermeulen, W., and Hoeijmakers, J.H.J. (2014).
replication-transcription conflicts in bacteria. Annu. Rev. Microbiol. Understanding nucleotide excision repair and its roles in cancer and
72, 71–88. ageing. Nat. Rev. Mol. Cell Biol. 15, 465–481.

93. Hori, Y., Engel, C., and Kobayashi, T. (2023). Regulation of ribosomal 111. Abeti, R., Zeitlberger, A., Peelo, C., Fassihi, H., Sarkany, R.P.E., Leh-
RNA gene copy number, transcription and nucleolus organization in eu- mann, A.R., and Giunti, P. (2019). Xeroderma pigmentosum: overview
karyotes. Nat. Rev. Mol. Cell Biol. 24, 414–429. of pharmacology and novel therapeutic strategies for neurological symp-
toms. Br. J. Pharmacol. 176, 4293–4301.
94. Rothstein, R., Michel, B., and Gangloff, S. (2000). Replication fork
pausing and recombination or ‘‘gimme a break.’’. Genes Dev. 14, 1–10. 112. Nance, M.A., and Berry, S.A. (1992). Cockayne syndrome: review of 140
cases. Am. J. Med. Genet. 42, 68–84.
95. Tubbs, A., Sridharan, S., van Wietmarschen, N., Maman, Y., Callen, E.,
Stanlie, A., Wu, W., Wu, X., Day, A., Wong, N., et al. (2018). Dual roles 113. Crossley, M.P., Song, C., Bocek, M.J., Choi, J.H., Kousorous, J., Sathir-
of poly(dA:dT) tracts in replication initiation and fork collapse. Cell 174, achinda, A., Lin, C., Brickner, J.R., Bai, G., Lans, H., et al. (2023). R-loop-
1127–1142.e19. derived cytoplasmic RNA–DNA hybrids activate an immune response.
Nature 613, 187–194.
96. Chen, Y.H., Keegan, S., Kahli, M., Tonzi, P., Fenyö, D., Huang, T.T., and
Smith, D.J. (2019). Transcription shapes DNA replication initiation and 114. Kwak, Y.D., Shaw, T.I., Downing, S.M., Tewari, A., Jin, H., Li, Y., Dumitr-
termination in human cells. Nat. Struct. Mol. Biol. 26, 67–77. ache, L.C., Katyal, S., Khodakhah, K., Russell, H.R., et al. (2021). Chro-
matin architecture at susceptible gene loci in cerebellar Purkinje cells
97. Pope, B.D., Ryba, T., Dileep, V., Yue, F., Wu, W., Denas, O., Vera, D.L., characterizes DNA damage-induced neurodegeneration. Sci. Adv. 7,
Wang, Y., Hansen, R.S., Canfield, T.K., et al. (2014). Topologically asso- eabg6363.
ciating domains are stable units of replication-timing regulation. Nature
7527, 402–405. 115. Groh, M., Albulescu, L.O., Cristini, A., and Gromak, N. (2017). Senataxin:
genome guardian at the interface of transcription and neurodegenera-
98. Li, Y., Xue, B., Zhang, M., Zhang, L., Hou, Y., Qin, Y., Long, H., Su, Q.P., tion. J. Mol. Biol. 429, 3181–3195.
Wang, Y., Guan, X., et al. (2021). Transcription-coupled structural dy-
namics of topologically associating domains regulate replication origin 116. Hoch, N.C., Hanzlikova, H., Rulten, S.L., Tétreault, M., Komulainen, E.,
efficiency. Genome Biol. 22, 1–29. Ju, L., Hornyak, P., Zeng, Z., Gittens, W., Rey, S.A., et al. (2017).
XRCC1 mutation is associated with PARP1 hyperactivation and cere-
99. Wei, X., Samarabandu, J., Devdhar, R.S., Siegel, A.J., Acharya, R., and bellar ataxia. Nature 541, 87–91.
Berezney, R. (1998). Segregation of transcription and replication sites
into higher order domains. Science 281, 1502–1506. 117. Adamowicz, M., Hailstone, R., Demin, A.A., Komulainen, E., Hanzlikova,
H., Brazina, J., Gautam, A., Wells, S.E., and Caldecott, K.W. (2021).
100. Hamperl, S., and Cimprich, K.A. (2016). Conflict resolution in the XRCC1 protects transcription from toxic PARP1 activity during DNA
genome: how transcription and replication make it work. Cell 167, base excision repair. Nat. Cell Biol. 23, 1287–1298.
1455–1467.
118. Komulainen, E., Badman, J., Rey, S., Rulten, S., Ju, L., Fennell, K., Kala-
101. Quinet, A., Lemaç, D., and Vindigni, A. (2015). Molecular cell minireview sova, I., Ilievova, K., McKinnon, P.J., Hanzlikova, H., et al. (2021). Parp1
replication fork reversal: players and guardians. Mol. Cell 68, 830–833. hyperactivity couples DNA breaks to aberrant neuronal calcium signalling
and lethal seizures. EMBO Rep. 22, e51851.
102. Ait Saada, A.A., Lambert, S.A.E., and Carr, A.M. (2018). Preserving repli-
cation fork integrity and competence via the homologous recombination 119. Lodato, M.A., Rodin, R.E., Bohrson, C.L., Coulter, M.E., Barton, A.R.,
pathway. DNA Repair (Amst) 71, 135–147. Kwon, M., Sherman, M.A., Vitzthum, C.M., Luquette, L.J., Yandava, C.,
et al. (2017). Aging and neurodegeneration are associated with increased
103. Chappidi, N., Nascakova, Z., Boleslavska, B., Zellweger, R., Isik, E., mutations in single human neurons. Science 359, eaao4426.
Andrs, M., Menon, S., Dobrovolna, J., Balbo Pogliano, C., Matos, J.,
et al. (2020). Fork Cleavage-Religation Cycle and Active Transcription 120. Luquette, L.J., Miller, M.B., Zhou, Z., Bohrson, C.L., Zhao, Y., Jin, H., Gul-
Mediate Replication Restart after Fork Stalling at Co-transcriptional R- han, D., Ganz, J., Bizzotto, S., Kirkham, S., et al. (2022). Single-cell
Loops. Mol. Cell 77, 528–541.e8. genome sequencing of human neurons identifies somatic point mutation
and indel enrichment in regulatory elements. Nat. Genet. 54, 1564–1571.
104. Matos, D.A., Zhang, J.-M., Ouyang, J., Nguyen, H.D., Genois, M.-M., and
Zou, L. (2020). ATR protects the genome against R loops through a 121. Bowry, A., Kelly, R.D.W., and Petermann, E. (2021). Hypertranscription
MUS81-triggered feedback loop. Mol. Cell 77, 514–527.e4. and replication stress in cancer. Trends Cancer 7, 863–877.
105. Kim, S., Kang, N., Park, S.H., Wells, J., Hwang, T., Ryu, E., Kim, B.G., 122. Kotsantis, P., Petermann, E., and Boulton, S.J. (2018). Mechanisms of
Hwang, S., Kim, S.J., Kang, S., et al. (2020). ATAD5 restricts R-loop for- oncogene-induced replication stress: jigsaw falling into place. Cancer
mation through PCNA unloading and RNA helicase maintenance at the Discov. 8, 537–555.
replication fork. Nucleic Acids Res. 48, 7218–7238.
123. Kotsantis, P., Silva, L.M., Irmscher, S., Jones, R.M., Folkes, L., Gromak,
106. Scaramuzza, S., Jones, R.M., Sadurni, M.M., Reynolds-Winczura, A., N., and Petermann, E. (2016). Increased global transcription activity as a
Poovathumkadavil, D., Farrell, A., Natsume, T., Rojas, P., Cuesta, C.F., mechanism of replication stress in cancer. Nat. Commun. 7, 13087.
Kanemaki, M.T., et al. (2023). TRAIP resolves DNA replication-transcrip-
tion conflicts during the S-phase of unperturbed cells. Nat. Commun. 124. Jones, R.M., Mortusewicz, O., Afzal, I., Lorvellec, M., Garcı́a, P., Helle-
14, 5071. day, T., and Petermann, E. (2013). Increased replication initiation and
conflicts with transcription underlie cyclin E-induced replication stress.
107. Li, M., Xu, X., Chang, C.W., and Liu, Y. (2020). TRIM28 functions as the Oncogene 32, 3744–3753.
SUMO E3 ligase for PCNA in prevention of transcription induced DNA
breaks. Proc. Natl. Acad. Sci. USA 117, 23588–23596. 125. Dominguez-Sola, D., and Gautier, J. (2014). MYC and the control of DNA
replication. Cold Spring Harb. Perspect. Med. 4, a014423.
108. Landsverk, H.B., Sandquist, L.E., Bay, L.T.E., Steurer, B., Campsteijn, C.,
Landsverk, O.J.B., Marteijn, J.A., Petermann, E., Trinkle-Mulcahy, L., 126. Barlow, J.H., Faryabi, R.B., Callén, E., Wong, N., Malhowski, A., Chen,
and Syljuåsen, R.G. (2020). WDR82/PNUTS-PP1 prevents transcrip- H.T., Gutierrez-Cruz, G., Sun, H.-W., Mckinnon, P., Wright, G., et al.
tion-replication conflicts by promoting RNA polymerase II degradation (2013). Identification of early replicating fragile sites that contribute to
on chromatin. Cell Rep. 33, 108469. genome instability. Cell 152, 620–632.

109. Jia, N., Guo, C., Nakazawa, Y., van den Heuvel, D., Luijsterburg, M.S., 127. Bignell, G.R., Greenman, C.D., Davies, H., Butler, A.P., Edkins, S., An-
and Ogi, T. (2021). Dealing with transcription-blocking DNA damage: drews, J.M., Buck, G., Chen, L., Beare, D., Latimer, C., et al. (2010). Sig-
repair mechanisms, RNA polymerase II processing and human disorders. natures of mutation and selection in the cancer genome. Nature 463,
DNA Repair (Amst) 106, 103192. 893–898.

Molecular Cell 84, January 4, 2024 79