Chromosoma

https://doi.org/10.1007/s00412-023-00807-5

REVIEW

Genome maintenance meets mechanobiology

Vincent Spegg1 · Matthias Altmeyer1

Received: 4 April 2023 / Revised: 20 June 2023 / Accepted: 26 July 2023

© The Author(s) 2023

Abstract
Genome stability is key for healthy cells in healthy organisms, and deregulated maintenance of genome integrity is a hallmark
of aging and of age-associated diseases including cancer and neurodegeneration. To maintain a stable genome, genome
surveillance and repair pathways are closely intertwined with cell cycle regulation and with DNA transactions that occur
during transcription and DNA replication. Coordination of these processes across different time and length scales involves
dynamic changes of chromatin topology, clustering of fragile genomic regions and repair factors into nuclear repair centers,
mobilization of the nuclear cytoskeleton, and activation of cell cycle checkpoints. Here, we provide a general overview of
cell cycle regulation and of the processes involved in genome duplication in human cells, followed by an introduction to
replication stress and to the cellular responses elicited by perturbed DNA synthesis. We discuss fragile genomic regions
that experience high levels of replication stress, with a particular focus on telomere fragility caused by replication stress
at the ends of linear chromosomes. Using alternative lengthening of telomeres (ALT) in cancer cells and ALT-associated
PML bodies (APBs) as examples of replication stress-associated clustered DNA damage, we discuss compartmentalization
of DNA repair reactions and the role of protein properties implicated in phase separation. Finally, we highlight emerging
connections between DNA repair and mechanobiology and discuss how biomolecular condensates, components of the
nuclear cytoskeleton, and interfaces between membrane-bound organelles and membraneless macromolecular condensates
may cooperate to coordinate genome maintenance in space and time.

Keywords Genome stability · Replication stress · DNA repair · Biomolecular condensates · Telomere maintenance ·
Mechanobiology

Introduction to cell cycle regulation for cellular metabolism and to prepare for later cell cycle
phases. G1 length varies depending on growth conditions
The eukaryotic cell cycle comprises a series of tightly con- and intracellular and extracellular cues. In unfavorable con-
trolled events that culminate in cell division and in the gener- ditions, cells may exit the cell cycle in G1 and enter a non-
ation of two new daughter cells (Figure 1). It can be divided proliferative state known as quiescence or Gap 0 (G0). Fol-
into the two main stages mitosis (M-phase) and interphase. lowing S-phase commitment in favorable growth conditions,
While mitosis refers to the process of chromosome segre- cells duplicate their genome by DNA synthesis. After two
gation followed by cell division, interphase separates two identical copies of the genetic material have been generated
M-phases and provides the time needed for genome dupli- through semi-conservative DNA replication, the ensuing
cation and to prepare for cell division. Interphase can be G2-phase serves as an additional gap phase for protein syn-
subdivided further into three consecutive cell cycle phases, thesis and cell growth in preparation for mitosis. Finally, in
which are called Gap 1 (G1), Synthesis (S), and Gap 2 (G2). M-phase, the two sets of chromosomes are first condensed
During G1, cells grow and produce proteins and organelles and aligned at the equatorial metaphase plate and then seg-
regated as cells divide into two newly emerging daughter
cells. M-phase can be subdivided morphologically and func-
* Matthias Altmeyer tionally into prophase, prometaphase, metaphase, anaphase,
matthias.altmeyer@uzh.ch telophase, and cytokinesis (Figure 1).
https://orcid.org/0000-0003-3780-1170
The central regulators of the cell cycle are cyclin-depend-
1
Department of Molecular Mechanisms of Disease, ent kinases (CDKs). CDKs are serin/threonine kinases
University of Zurich, Zurich, Switzerland

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generating a positive feedback loop (Figure 1). Inactivation

of the APC/C ubiquitin ligase complex at the G1/S
transition allows CyclinA-CDK2 activity to rise and initiate
DNA replication in S-phase. Following DNA replication,
accumulation of CyclinA/B-CDK1 in G2 eventually drives
mitotic entry and allows APC/C reactivation, which is
required for targeted degradation of S/G2-phase cyclins and
mitotic exit to complete the cell cycle (Matthews et al. 2022;
Pennycook & Barr 2020).
Several cell cycle checkpoints have evolved to ensure error-
free progression through the cell cycle (Elledge 1996; Hartwell
& Weinert 1989; Kastan & Bartek 2004). These checkpoints
serve to monitor cell cycle progression and to actively slow
down or halt the cell cycle upon encountering problems (Fig-
ure 1). Cell cycle checkpoint activation can also trigger cell
death upon persistent perturbation and thereby guard against
cellular transformation. DNA replication stress during S-phase
progression and replication stress-induced DNA damage are
Fig. 1  Overview of the eukaryotic cell cycle. Illustration of the differ- important sources for cell cycle checkpoint activation (Gaillard
ent cell cycle phases during interphase (G1, S, G2) and mitosis (M; et al. 2015; Nyberg et al. 2002). In the following paragraphs we
subdivided into prophase, prometaphase, metaphase, anaphase, telo- summarize the processes that are central for DNA replication
phase, and cytokinesis). The associated Cyclin-CDK activities that
and for the cellular response to replication stress.
drive cell cycle progression and the main cell cycle checkpoints are
indicated. Cell cycle exit into quiescence (G0) occurs under unfavora-
ble growth condition. Upon mitogen stimulation, phosphorylation of
the negative regulator Rb by CyclinD-CDK4/6 in G1 activates a posi-
tive feedback loop centered around the transcription factor E2F and DNA replication in eukaryotes
E2F-dependent CyclinE-CDK2 activation. G1, Gap 1; S, S-phase;
G2, Gap 2; G0, Gap 0; M, mitosis; Rb, retinoblastoma protein; P, DNA replication in S-phase is tightly regulated, ensuring
phosphate group; E2F, E2 promoter binding factor; CDK, cyclin-
dependent kinase that the genome is fully copied once and only once in every
cell cycle. The process of semi-conservative DNA replica-
tion, after which each of the two generated DNA copies con-
tains parental and newly synthesized strands, can be divided
whose activation state determines cell cycle entry, progres-
into four distinct phases: licensing, initiation, elongation,
sion, and completion (Barnum & O’Connell 2014; Basu
and termination (Figure 2).
et al. 2022; Matthews et al. 2022). Small proteins called
cyclins are needed as regulatory subunits of CDKs to stimu-
late their kinase activity. Cyclins accumulate during differ- Licensing
ent stages of the cell cycle in a manner that is controlled by
their cell cycle-dependent expression and targeted proteaso- DNA replication begins at defined sites in the genome,
mal degradation. A regulatory feed-forward loop between called origins of replication. In human cells, DNA rep-
cyclin-dependent CDK activity and CDK-driven cell cycle lication is initiated from multiple origins of replication
progression ensures that cell cycle transitions occur in a and an estimated 30,000–50,000 origins are active in each
unidirectional and sequential manner (Novak et al. 2007; cell cycle of a replicating cell (Leonard & Mechali 2013).
Pennycook & Barr 2020). These origins are not all initiated simultaneously at the
After cell division, accumulation of CyclinD-CDK4/6 beginning of S-phase, instead they are activated sequen-
upon exposure to mitogenic growth factors promotes cell tially during S-phase progression (Boos & Ferreira 2019;
cycle commitment and prevents quiescence. CDK4/6 activity Vouzas & Gilbert 2021). A defined consensus sequence
fuels E2F-dependent gene expression, which in turn leads that determines replication origins and their temporal
to the accumulation of CyclinE and promotes progression activation program in mammals has remained elusive, and
through G1 and towards the G1/S transition (Rubin et al. it seems that replication initiation is influenced by DNA
2020). CyclinE-CDK2 activity further stimulates E2F- topology, chromatin organization and transcriptional activ-
dependent transcription by phosphorylating and inactivating ity (Emerson et al. 2022; Ganier et al. 2019; Giles et al.
the transcription inhibitor retinoblastoma protein (Rb), 2022; Limas & Cook 2019; Marchal et al. 2019; Prioleau
thereby amplifying the E2F transcriptional signal and & MacAlpine 2016).

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pre-replication complex (pre-RC). Two MCM complexes

are loaded onto the chromatin head-to-head and form the
core of the replicative helicase (Bleichert 2019; Deegan &
Diffley 2016; Evrin et al. 2009; Miller et al. 2019; Remus
et al. 2009).
Cells load an excess number of pre-RCs and only a
subset of licensed origins is used for replication initiation
(Masai et al. 2010). Origins have therefore been classified
into constitutive, flexible, and dormant origins (Blow et al.
2011; Fragkos et al. 2015). The excess of licensed origins
can serve as a backup to complete DNA replication under
conditions of replication stress (Blow et al. 2011; Courtot
et al. 2018; Fragkos et al. 2015).
The restricted time window for origin licensing in late
M and early G1 prevents re-licensing during S-phase. Re-
licensing in S-phase could cause re-replication of already
copied DNA, leading to amplification of DNA sequences
and increased genome instability (Blow & Gillespie 2008;
Fragkos et al. 2015; Limas & Cook 2019; Neelsen et al.
2013). The negative regulator of origin licensing Gemi-
nin, which is destabilized in G1/M by the APC/C ubiqui-
tin ligase complex, accumulates after APC/C inactivation
at the G1/S transition and prevents re-licensing (Machida
et al. 2005; Petropoulos et al. 2019). Geminin interacts with
the licensing factor CDT1 and blocks loading of the MCM
complex, thereby inhibiting the formation of new pre-RCs in
S-phase (Lee et al. 2004; Wohlschlegel et al. 2000; Yanagi
Fig. 2  Overview of DNA replication. Indicated are the four phases
of DNA replication and their timing during the cell cycle. (a) Origin et al. 2002). A second way of controlling pre-RC formation
licensing in late M and early G1. Formation of the pre-replication is mediated by the ubiquitin ligases SCF-Skp2 and DDB1-
complex (pre-RC) by the assembly of ORC1-6, CDC6, CDT1, and Cul4, which ubiquitinate CDT1 and ORC1 in S-phase, lead-
MCM2-7 at the origin of replication. (b) Replication initiation at the
ing to their proteasomal degradation (Li et al. 2003; Méndez
G1/S transition. CDK- and DDK-mediated phosphorylation of the
MCM complex leads to the recruitment of CDC45 and the GINS et al. 2002; Nishitani et al. 2006). Finally, in G2 and early
complex, which together with the MCM proteins form the CDC45- M-phase, high CDK activity inactivates pre-RC components,
MCM-GINS (CMG) helicase, thereby setting up the pre-initiation ensuring that licensing of new origins occurs only after chro-
complex (pre-IC). (c) Elongation during S-phase progression.
mosome segregation (Machida et al. 2005; Petropoulos et al.
Recruitment of additional replication proteins, including PCNA,
RPA, and DNA polymerases (DNA Pol), leads to the transition from 2019).
the pre-IC into two active replisomes moving in opposite directions.
(d) Termination of replication in S/G2 when two replisomes Initiation
converge, followed by C­ RL2Lrr1-dependent ubiquitylation of MCM7
and p97-mediated extraction from the DNA. M, mitosis: G1, Gap 1;
S, S-phase; G2, Gap 2; CDK, cyclin-dependent kinase; DDK, DBF4- Licensed origins with pre-RCs are converted to pre-initiation
dependent kinase; Ub, ubiquitin complexes (pre-ICs) upon S-phase entry. This step is regu-
lated by the recruitment of CyclinE-CDK2, DDK (DBF4/
CDC7), and CyclinB-CDK1 (Suski et al. 2022), which phos-
Origin licensing (Figure 2a) occurs before S-phase in late phorylate the MCM complex (Figure 2b). MCM phospho-
M and early G1 phase of the cell cycle, when the origin rylation leads to the recruitment of CDC45 and the GINS
recognition complex (ORC), a hexameric protein complex protein complex, thereby forming the CDC45-MCM-GINS
consisting of the subunits ORC1-6, binds to replication ori- (CMG) helicase (Lewis et al. 2022). CMG complex forma-
gins (Masai et al. 2010). ORC binding leads to the recruit- tion is a prerequisite to activate the MCM helicase activity
ment of the licensing factors CDC6 and CDT1, which facili- (Ilves et al. 2010; Zou & Stillman 2000). The protein TopBP1
tates the recruitment of inactive hexameric MCM protein and its interaction partner Treslin are also part of the pre-IC
complexes consisting of the proteins MCM2-7 (Nishitani and bind to the MCM complex in a CDK phosphorylation-
& Lygerou 2002). The origin-associated multiprotein com- dependent manner. Recruitment of TopBP1-Treslin to the
plex formed by ORC, CDC6, CDT1, and MCM2-7 is called MCM complex is required for activation of the CMG helicase

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(Kumagai et al. 2010, 2011). As a result, two inactive MCM ubiquitin-selective segregase p97 after polyubiquitylation
complexes get remodeled into two active CMG complexes, of MCM7 by ­CRL2Lrr1 (Dewar & Walter 2017; Fan et al.
which start to unwind the parental DNA in a bidirectional 2021). A backup mechanism seems to exist in mitosis to
manner, thereby allowing the recruitment of additional rep- trigger global replisome disassembly through MCM7 poly-
lication proteins, including replication factor c (RFC), pro- ubiquitylation by the ubiquitin E3 ligase TRAIP, followed
liferating cell nuclear antigen (PCNA), replication protein a by p97-mediated extraction from the chromatin (Deng et al.
(RPA), and DNA polymerases (Limas & Cook 2019; Parker 2019; Priego Moreno et al. 2019; Sonneville et al. 2019;
et al. 2017). Together they mediate the transition from a pre- Villa et al. 2021). Failure to terminate replication, e.g., due
IC into two active replisomes that move in opposite direc- to obstacles that impair replication fork speed, can undermine
tions and generate the replication bubble (Douglas et al. the faithful propagation of the genetic information to the next
2018; Fragkos et al. 2015). cell generation and is associated with DNA replication stress.

Elongation
Replication stress
Once DNA replication has been initiated by origin firing,
replisomes move away from the replication origin to During the process of DNA replication, the replication
copy the parental DNA (Figure 2c). Replicative DNA machinery is confronted with a variety of obstacles that can
polymerases are incapable of initiating DNA synthesis interfere with DNA synthesis and jeopardize timely comple-
de novo, but instead need a start site, or primer, to begin tion of genome duplication (Mazouzi et al. 2014; Saxena &
DNA synthesis. This primer is synthesized by DNA Polα- Zou 2022). Conditions that lead to replication fork slowing
primase, which generates a short stretch of ribonucleic acid or stalling and perturb DNA synthesis are generally referred
(RNA) (Arezi & Kuchta 2000). The primer is recognized to as replication stress (Gaillard et al. 2015; Zeman & Cim-
by RFC, which loads the replication sliding clamp PCNA prich 2014). Replication stress can be caused by exogenous
and displaces DNA Polα-primase with replicative DNA sources such as DNA-modifying chemicals and alterations
Polδ/ε (Moldovan et al. 2007; O'Donnell et al. 2013). of the DNA structure, e.g., through ionizing radiation (IR)
As new deoxyribonucleotides can only be added at the or ultraviolet (UV) light (Figure 3a). Endogenous sources
5’-phosphate ends of nascent DNA strands, one of the two of replication stress include depleted deoxyribonucleotide
daughter strands is synthesized in a continuous manner in pools, ribonucleotide incorporation into DNA, DNA lesions
the same direction as the moving replication fork (leading caused by metabolic byproducts such as reactive oxygen spe-
strand synthesis), while the opposite daughter strand must cies (ROS), interstrand crosslinks (ICLs), DNA secondary
be synthesized away from the replication fork and hence in a structures such as hairpins and G4-quadruplexes, repetitive
discontinuous manner (lagging strand synthesis). For leading DNA sequences, transcription-replication conflicts, and
strand synthesis DNA Polε needs only one primer formed at RNA-DNA hybrids (Brickner et al. 2022; Garcia-Muse &
the origin, whereas for lagging strand synthesis the process Aguilera 2016; Petermann et al. 2022; Saxena & Zou 2022;
of primer synthesis followed by extension must be repeated Zeman & Cimprich 2014). Replication stress has emerged
periodically. The newly synthesized fragments of the lagging as major cause of genome instability and is a hallmark of
strand are termed Okazaki fragments (Okazaki et al. 1968). most cancers (Macheret & Halazonetis 2015). Consider-
Through leading and lagging strand synthesis in conjunction ing the elevated levels of replication stress in cancer cells,
with Okazaki fragment ligation, both parental DNA strands enzymes involved in the response to replication stress are
are copied, and continuous stretches of newly synthesized promising targets for cancer therapy (Cybulla & Vindigni
DNA are formed within each replication unit. 2023; da Costa et al. 2023; Dobbelstein & Sorensen 2015).

Termination Replication stress response

DNA replication is terminated when converging replication Obstacles that lead to stalling of DNA polymerases on the
forks coming from two replication units encounter each other lagging strand are generally well tolerated. Due to the discon-
(Figure 2d). When converging forks meet, the replisomes tinuous nature of Okazaki fragment synthesis, such lesions
disassemble leaving a ssDNA gap between the 3’-end of the can be efficiently bypassed, leaving behind short ssDNA
leading strand and the downstream Okazaki fragment of the gaps, which are repaired post-replicatively (Berti & Vindigni
opposing fork. The remaining gap is filled by extension of 2016; Marians 2018; Yeeles et al. 2013). On the other hand,
the leading strand resulting in a continuous DNA molecule stalling of DNA polymerases during leading strand synthesis
(Dewar & Walter 2017). Unloading of the CMG helicase can cause helicase-polymerase uncoupling and challenge fork
from termination sites was recently shown to require the stability (Taylor & Yeeles 2018; Taylor & Yeeles 2019).

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Fig. 3  Replication stress and fragile genomic regions. (a) Sources of a protective mechanism against replication stress. Indicated is the
replication stress that can lead to replication fork stalling and fork dynamic process of fork reversal and re-reversal for fork restart, as
collapse. Indicated are exogenous sources of replication stress (e.g., well as main regulators of fork reversal and of reversed fork protec-
UV light, irradiation) and endogenous sources of replication stress tion. (c) Fragile genomic regions that are prone to experiencing repli-
(e.g., repetitive sequences, secondary structures such as G4-quad- cation stress. Indicated are common fragile sites (CFSs), centromeric
ruplexes and R-loops, reactive oxygen species (ROS), interstrand and pericentromeric regions, ribosomal DNA, rare fragile sites of tri-
crosslinks (ICLs), dNTP depletion). (b) Replication fork reversal as plet repeat expansion, and telomere repeats

Replication fork uncoupling can lead to extensive One way to stabilize stalled replication forks is a pro-
stretches of RPA-bound ssDNA, which recruits the central cess known as fork reversal, also referred to as fork regres-
replication stress response kinase ATR through its interac- sion (Figure 3b). During fork reversal, standard three-way
tion partner ATRIP (MacDougall et al. 2007; Zou & Elledge replication forks are converted into four-way junctions by
2003). Once recruited, ATR gets activated through two spe- unwinding of the newly synthesized DNA strands and sub-
cific activator proteins, namely TopBP1 and ETAA1 (Bass sequent annealing of the two nascent strands and reannealing
et al. 2016; Haahr et al. 2016; Kumagai et al. 2006; Lee et al. of the parental strands (Berti, Cortez, & Lopes 2020). Fork
2016). The ssDNA-bound RPA also serves as a recogni- reversal can fine-tune fork speed and pause DNA synthe-
tion site for the Timeless-Tipin complex, which stimulates sis in response to a variety of genotoxic treatments, upon
claspin-mediated phosphorylation of checkpoint kinase 1 oncogene-induced replication stress, and when endogenous
(CHK1) by ATR (Kemp et al. 2010). ATR/CHK1 activa- replication obstacles are encountered (Berti et al. 2013;
tion leads to fork stabilization and inhibits origin firing, Berti, Cortez, & Lopes 2020; Follonier et al. 2013; Neelsen
thereby controlling the number of active replisomes. This & Lopes 2015; Quinet et al. 2017). After fork uncoupling,
is important to protect already active forks against irrevers- RPA-coated ssDNA leads to the recruitment of RAD51,
ible breakage, which can occur upon exhaustion of limiting which initiates fork reversal (Bhat & Cortez 2018). Differ-
replication factors such as dNTPs and RPA (Buisson et al. ent from the known functions of RAD51 in DNA double-
2015; Toledo et al. 2013). On the other hand, dormant ori- strand break (DSB) repair, RAD51-mediated fork remod-
gins in the vicinity of fork-stalling lesions escape from ATR/ eling does not require its classical loading factor BRCA2,
CHK1-mediated suppression and can rescue stalled forks nor the formation of stable RAD51 filaments (Mijic et al.
to ensure completion of DNA synthesis (Ge & Blow 2010; 2017; Scully et al. 2019). RAD51-mediated fork reversal
Saldivar et al. 2017). is regulated by RADX and the homologous recombination
(HR) co-factors RAD51B, RAD51C, RAD51D, XRCC2,
and XRCC3 (Berti et al. 2020; Bhat et al. 2018; Halder et al.
Fork protection and restart 2022; Krishnamoorthy et al. 2021). Other proteins involved
in fork reversal include SMARCAL1, HLTF, and ZRANB3
To resolve replication stress-inducing problems, multiple (Bai et al. 2020; Kolinjivadi et al. 2017; Poole & Cortez
mechanisms have evolved to stabilize stalled replication 2017; Taglialatela et al. 2017). SMARCAL1 catalyzes fork
forks and promote their later restart. The choice of fork regression and Holliday junction migration, thereby promot-
remodeling and repair pathways is dependent on the type ing efficient fork repair (Bétous et al. 2012). HLTF is a DNA
of problem that triggered replication stress as well as the translocase and ubiquitin E3 ligase that gets recruited to the
duration of the replication block itself (Berti et al. 2020; 3’-ssDNA-end of the leading strand where it polyubiqui-
Panagopoulos & Altmeyer 2021). tylates PCNA (Bai et al. 2020; Kile et al. 2015). ZRANB3

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interacts with polyubiquitylated PCNA and assists replica- 2021). These difficult-to-replicate regions include chromo-
tion fork remodeling through its DNA translocase activity somal fragile sites as well as repetitive sequences at riboso-
(Ciccia et al. 2012; Vujanovic et al. 2017; Weston et al. mal DNA, centromeres, and telomeres (Figure 3c). Fragile
2012). While SMARCAL1 and ZRANB3 guide the ini- sites are prone to form gaps and breaks visible on metaphase
tial annealing of the displaced daughter strands, ZRANB3 chromosomes, often referred to as fragile site expression
and HLTF catalyze branch migration (Halder et al. 2022). (Özer & Hickson 2018). Rare fragile sites are caused by
Upon reversal, the regressed fork resembles a one-ended pathological expansion of trinucleotide repeat sequences and
DSB that must be protected from nucleolytic degradation. are only present in a small percentage of the human popula-
Important factors mediating this protection include BRCA1 tion. One example is CGG triplet expansion in the fragile X
and RAD51, whose function in fork protection, contrary to messenger ribonucleoprotein 1 (FMR1) gene causing frag-
the role in initiating fork reversal, is dependent on BRCA2 ile X syndrome (Zhou et al. 2016). Common fragile sites
(Berti, Cortez, & Lopes 2020; Lemacon et al. 2017; Mijic (CFSs) on the other hand are present in all individuals, e.g.,
et al. 2017; Tarsounas & Sung 2020). the fragile sites FRA16D and FRA3B, which harbor the tumor
Stalled and reversed forks can be restarted in multi- suppressor genes fragile histidine triad diadenosine triphos-
ple ways. Fork restart can be mediated by the helicase phatase (FHIT) and WW domain containing oxidoreductase
RECQ1, which promotes branch migration and converts (WWOX), respectively (Durkin & Glover 2007; Özer & Hick-
four-way junctions back into replication-competent three- son 2018). CFSs in cancer cells are often associated with
way junctions. The activity of RECQ1 is regulated by breakpoints of genomic rearrangements, micro-deletions, and
PARP1-dependent poly(ADP-ribosyl)ation (PARylation), copy number variations (Glover et al. 2017; Sarni & Kerem
which ensures that forks restart only upon repaired dam- 2016). The sensitivity of CFSs to replication stress is caused
age (Berti et al. 2013). Additionally, re-establishment of by their tendency to have AT-rich sequences, which are prone
three-way junctions can be mediated through fork pro- to form secondary structures, sparsity of replication origins,
cessing by the Werner syndrome helicase WRN and the association with very long genes that can take more than
DNA2 nuclease (Datta et al. 2021; Thangavel et al. 2015). one cell cycle to be fully transcribed, and their late replica-
Replication obstacles on the leading strand can also be tion timing, typically being the last regions of the genome
overcome by fork repriming (Bianchi et al. 2013; García- to be replicated (Brison et al. 2019; Kaushal et al. 2019).
Gómez et al. 2013; Guilliam et al. 2017; Quinet et al. These features challenge faithful and complete replication
2021). For repriming, new primers are placed downstream during S-phase and can lead to under-replicated DNA and
of the obstacles to continue DNA replication, at the cost CFS instability (Debatisse & Rosselli 2019).
of leaving behind unreplicated ssDNA gaps that have to Ribosomal DNA (rDNA) consists of DNA tandem repeats
be repaired after replication (Mourón et al. 2013). The that encode ribosomal RNAs (rRNAs) required for ribosome
key enzyme involved in repriming is primase and DNA- biosynthesis. Their high rate of transcription makes repli-
directed polymerase (PrimPol), which gets recruited to cation-transcription conflicts almost inevitable. Although
ssDNA via direct interaction with RPA (González-Acosta replication fork barriers positioned within each rDNA unit
et al. 2021; Guilliam et al. 2017). PrimPol not only has were shown to coordinate progression of replication with
primase activity, but also acts as DNA polymerase with transcription in eukaryotic cells (Akamatsu & Kobayashi
low processivity and fidelity (Bianchi et al. 2013; García- 2015; Gadaleta & Noguchi 2017), R-loops form at tran-
Gómez et al. 2013; Guilliam & Doherty 2017; Tirman scribed rDNA repeats and cause replication-transcription
et al. 2021). Gaps that are left behind the fork can be conflicts in the nucleolus that undermine rDNA stability (El
repaired post-replicatively by either DNA translesion syn- Hage et al. 2010; Lezaja & Altmeyer 2021; Özer & Hickson
thesis (TLS), in which specific TLS polymerases medi- 2018; Salvi et al. 2014; Tsekrekou et al. 2017; Warmerdam &
ate replication across the lesion, or by template switching Wolthuis 2019). Additionally, non-transcribed rDNA repeats
(TS), a process in which the intact sister strand is used for cluster in heterochromatic regions at the nucleolar periphery
homologous recombination repair (Piberger et al. 2020; and show, unlike actively transcribed repeats, late replication
Tirman et al. 2021; Wong et al. 2021). timing, which makes them prone to form under-replicated
DNA and breaks late in the cell cycle (Lezaja & Altmeyer
2021; Warmerdam & Wolthuis 2019).
Fragile sites in the human genome Centromeres are chromosomal domains needed for faith-
ful transmission of duplicated chromosomes to daughter
Certain regions in the genome are particularly vulnerable cells during cell division by assembling the kinetochore and
to endogenous replication stress due to their inherent diffi- mitotic spindle microtubules for sister chromatid separation.
culty to be replicated (Glover et al. 2017; Lezaja & Altmeyer They are composed of a series of 171 nt long AT-rich DNA
tandem repeats, named alpha satellites (Barra & Fachinetti

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2018). The surrounding pericentromeric heterochromatin is chromosome ends and unwanted DNA repair reactions that
also organized in short tandem repeats. Due to the repetitive could lead to telomere fusions (Kratz & de Lange 2018). A
nature of centromeric and pericentromeric DNA, secondary telomeric long non-coding RNA termed telomeric repeat-
structures like DNA loops and catenates are being formed, containing RNA (TERRA) comprised of G-rich telomere
giving rise to target sites for DNA topoisomerases and the repeats is important for the regulation of telomeric chromatin
DNA recombination machinery (Barra & Fachinetti 2018). structure and telomere stability (Azzalin et al. 2007).
These secondary structures, together with the heterochro- Binding of TERRA to telomeric DNA leads to the formation
matic environment and the late replication timing, contribute R-loops by displacement of the G-rich DNA strand, which
to the fragility of centromeric and pericentromeric repeats are stabilized when telomeres experience elevated levels
(Lezaja & Altmeyer 2021; Mitrentsi et al. 2020), and make of replication stress (Feretzaki et al. 2020; Fernandes et al.
centromeres hotspots of DNA damage and recombination 2021; Lu & Pickett 2022; Niehrs & Luke 2020).
(Saayman et al. 2023; Yilmaz et al. 2021).
A fourth important class of fragile regions is represented
by telomeres, constitutive heterochromatic regions at Telomere elongation in cancer
chromosome ends that determine replicative (im)mortality.
Replication stress at telomeres is primarily driven by Due to the end-replication problem associated with telo-
their composition of terminal tracts of tandem repeats, meric lagging strand DNA synthesis, telomeres shorten in
the presence of secondary DNA structures including each cell cycle (Figure 4a). In somatic cells, when critically
G-quadruplexes, R-loops, and telomere loops (t-loops) short telomeres accumulate, cellular senescence, apopto-
formed by the G-rich 3’-telomeric ssDNA overhang, and sis, or a permanent cell cycle arrest is triggered (D'Souza
the fact that stalled replication forks downstream of the most et al. 2013; Koliada et al. 2015). In contrast to most somatic
distal origin cannot be rescued by dormant origin firing cells, stem cells and progenitor cells express low levels of
(Lezaja & Altmeyer 2021; Lu & Pickett 2022). Telomeres the enzyme telomerase, which extends telomere repeats and
are protected by the shelterin complex, consisting of the contributes to prolonged proliferative capacity. Telomerase
subunits TRF1, TRF2, RAP1, POT1, TPP1, and TIN2 (de is a ribonucleoprotein complex consisting of the enzyme
Lange 2005). At intact telomeres, the shelterin complex telomerase reverse transcriptase (TERT) and the telomere-
competes with RPA and promotes t-loop formation, sequence containing non-coding human telomerase RNA,
thereby suppressing DNA damage response signaling from which binds to the telomeric 3’-ssDNA overhang (Roake &

Fig. 4  Telomere maintenance in cancer. (a) End-replication prob- 3’-ssDNA overhang. Successive shortening of telomeres caused by
lem and successive telomere shortening in somatic cells with inacti- the end-replication problem is countered by telomerase activity. (c)
vated telomerase. Terminal gaps at the lagging strand cause telomere Alternative lengthening of telomeres (ALT) in telomerase-negative
shortening, which can lead to senescence or cell death when telom- cancer cells. ALT-positive cancer cells use recombination-based
eres become critically short. (b) Reactivation of telomerase in cancer mechanisms for telomere elongation. For simplicity, productive BTR
cells enables replicative immortality. The ribonucleoprotein com- complex-mediated D-loop dissolution is indicated, although unpro-
plex telomerase uses its reverse transcriptase activity and a telomere ductive non-crossover and crossover resolution leading to aborted tel-
sequence-containing non-coding RNA for elongation of the telomeric omere extension can also occur

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Artandi 2020). The telomeric 3’-ssDNA overhang is then telomere maintenance, either directly or indirectly (Cho
extended by the reverse transcriptase activity of TERT, using et al. 2014; Dilley et al. 2016; Lu & Pickett 2022; Min et al.
the telomerase RNA as template (Figure 4b). While TERT 2019; Zhang et al. 2019). ALT utilizes break-induced replica-
is usually silenced in somatic cells, most cancer cells show tion (BIR), a recombination process initiated by one-ended
reconstituted expression of the enzyme, thereby achieving DSBs, which results in sequence extension by conservative
replicative immortality (Shay & Wright 2019). DNA replication using a homologous template (Anand et al.
Although expression of telomerase was long seen 2013; Kramara et al. 2018; Zhang & Zou 2020). Resection
as a general cancer marker, around 10–15% of tumors, of one-ended DSBs might be involved, mediated by BLM
predominantly of mesenchymal origin, lack this enzyme and DNA2/EXO1 (Nimonkar et al. 2011; Sturzenegger et al.
(Bhargava et al. 2022; Lu & Pickett 2022; Shay & Wright 2014), which can lead to the formation of longer 3’-ssDNA
2019). These cancer cells use a homologous recombination- overhangs to facilitate strand invasion of homologous tem-
based pathway to extend and maintain their telomeres plates. The ssDNA overhang is first bound by RPA before
(Figure 4c), known as alternative lengthening of telomeres being handed over to RAD52, which promotes annealing of
(ALT) (Barthel et al. 2017; Dilley & Greenberg 2015; the broken telomere end with a homologous template to form
Heaphy et al. 2011). Typically, they display several a D-loop (Verma et al. 2019; Zhang et al. 2019). Extension
hallmarks of ALT, including long and heterogeneous of the D-loop is then mediated by DNA Polδ and its subu-
telomere length, abundant extrachromosomal circular nits POLD3 and POLD4 (O’Rourke et al. 2019; Zhou et al.
telomere repeats (ECTR), incorporation of non-telomeric 2012). A RAD52-independent ALT pathway seems to exist
sequences, high levels of telomere sister chromatid as well, because RAD52 loss leads to BLM- and POLD3/4-
exchange, and the formation of ALT-associated PML bodies dependent ALT DNA synthesis associated with increased
(APBs) (Bhargava et al. 2022; Bryan et al. 1995; Cesare & c-circle formation (Epum & Haber 2022; Zhang et al. 2019).
Griffith 2004; Londoño-Vallejo et al. 2004; Silva et al. 2019; A central positive regulator of ALT is the DNA helicase
Yeager et al. 1999a; Zhang & Zou 2020). BLM, which functions in a complex together with TOP3A
APBs are membraneless nuclear condensates that contain and RMI1/2, thereby forming the BLM-TOP3A-RMI1/2
the promyelocytic leukemia (PML) protein, clustered (BTR) complex (Bhargava, Lynskey, & O’Sullivan 2022;
telomeres, and several proteins involved in DNA repair, Manthei & Keck 2013). BLM is critical for ALT-associated
recombination, and replication, thereby generating a local DNA synthesis upon telomere clustering, for mitotic DNA
hub for telomere recombination and telomere DNA synthesis synthesis (MiDAS) at telomeres, and for functional APB
(Draskovic et al. 2009; Grobelny et al. 2000; Nabetani et al. formation (Min et al. 2019; O'Sullivan et al. 2014; Shorrocks
2004; Stavropoulos et al. 2002; Wu et al. 2000; Yeager et al. 2021; Sobinoff et al. 2017; Stavropoulos et al. 2002;
et al. 1999b). APB formation was suggested to involve Zhang et al. 2019). The BLM-containing BTR complex pro-
phase separation properties of APB-associated proteins, cesses recombination intermediates formed during strand
including multivalent SUMO-SIM interactions (Min et al. invasion and initiates POLD3/4-dependent telomere synthe-
2019; Spegg & Altmeyer 2021; Zhang et al. 2020). Of note, sis. Upon completion of the replicative process, the BTR
the shelterin complex components TRF1 and TRF2 also complex dissolves Holliday junctions, hence its alias dis-
show features of phase separation and form condensates at solvasome, thereby preventing the exchange of telomeric
telomere repeats (Jack et al. 2022; Soranno et al. 2022). The sequences between sister chromatids (Sobinoff et al. 2017).
interplay between TRF1/2-driven telomere condensates and RAD51 associated protein 1 (RAD51AP1) was also
APB condensates in ALT-positive cancer cells remains to shown to be important for telomere clustering and break-
be determined. induced telomere synthesis (BITS). Loss of RAD51AP1
ALT is induced at stalled and collapsed replication forks, leads to decreased ALT activity, reduction of APBs, defec-
suggesting that replication stress at telomeres and the associ- tive recruitment of RAD52 and POLD3 to telomeres, and
ated telomere fragility are a driving force behind this process causes telomere shortening (Barroso-González et al. 2019;
(Amato et al. 2020; Pan et al. 2019; Silva et al. 2021; Turkalo Kaminski et al. 2022; Yadav et al. 2022).
et al. 2023). The ALT pathway was initially characterized Negative regulators of ALT include the ATRX/DAXX
in budding yeast mutants lacking functional telomerase, histone chaperone complex and the histone variant H3.3,
where two distinct subpathways were described: Rad51- and which are often mutated in ALT-positive cancers (Hea-
Rad52-mediated amplification of repetitive subtelomeric phy et al. 2011; Kannan et al. 2012; Minasi et al. 2021;
sequences, and Rad52-dependent but Rad51-independent Schwartzentruber et al. 2012). H3.3 mutations deregulate
telomere maintenance by expanding telomeric repeats H3K9 methylation and heterochromatin formation at tel-
(Kockler et al. 2021; Le et al. 1999; McEachern & Haber omeres (Udugama et al. 2022). The ATRX/DAXX histone
2006; Zhang & Zou 2020). Also in human cancer cells both chaperone complex is involved in H3.3 deposition and chro-
RAD51 and RAD52 seem to be involved in ALT-dependent matin compaction, thereby regulating expression of TERRA

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Chromosoma

and the formation of TERRA R-loops (Bhargava, Lynskey, prevent excessive telomere damage and promote a process
& O’Sullivan 2022; Clynes et al. 2015; Flynn et al. 2015; termed post-mitotic DNA synthesis (post-MiDAS) in G1
Law et al. 2010). The annealing helicase SMARCAL1 was cells (Lezaja et al. 2021). Thus, telomere maintenance by
also found to harbor inactivating mutations in ALT-positive ALT seems uncoupled from the general cell cycle-embed-
cancers (Brosnan-Cashman et al. 2021; Diplas et al. 2018). ded principle of temporally separating genome duplication
SMARCAL1 counteracts replication stress at telomeres by and maintenance from cell division. Clustering of telomere
promoting fork reversal and fork restart, thereby suppressing repeats from multiple chromosomes may allow telomere
ALT (Bétous et al. 2012; Cox et al. 2016; Poole et al. 2015). recombination irrespective of cell cycle phase, and in the
The endonuclease SLX4, in complex with SLX1 and next paragraphs we discuss emerging principles of telomere
ERCC4, plays an opposing role to the BTR complex. clustering in ALT-positive cancer cells in connection with
While the complex around BLM supports non-crossover RPA condensation.
dissolution of replication intermediates and productive
telomere extension, the complex around SLX4 counteracts
dissolution by crossover and non-crossover resolution with DNA repair condensates and ALT
aborted telomere extension (Sobinoff et al. 2017). Therefore,
a tightly regulated balance between BLM and SLX4 seems Biomolecular condensates that support DNA repair reactions,
to determine ALT productivity and telomere maintenance. their molecular compositions, mechanisms of assembly, and
An important regulator of this balance is the SLX4 functions are manifold (Alghoul et al. 2023; Dall'Agnese
interacting protein SLX4IP. SLX4IP favors SLX4-mediated et al. 2023; Laflamme & Mekhail 2020; Mine-Hattab et al.
resolution by antagonizing BLM’s dissolution activity. Loss 2022; Spegg & Altmeyer 2021). Their formation typically
of SLX4IP leads to an increase in ALT-related phenotypes, follows a multi-step process, in which several types of asso-
and in conjunction with loss of SLX4 to a synthetic growth ciative interactions cooperate to build functional compart-
defect (Panier et al. 2019). While overexpression of SLX4 ments (Spegg & Altmeyer 2021). This multi-step process,
reduces APB formation, its depletion leads to an increase in once initiated through specific interactions at sites of DNA
APBs, elevated c-circles and ALT telomere extension, and damage, can be seen as a self-perpetuating assembly process,
reduces telomeric MiDAS (Özer et al. 2018; Sobinoff et al. promoted in part by self-association of the recruiting factors.
2017). Combined depletion of SLX4 and RAD52 results in In addition to such feed-forward amplification, negative feed-
increased telomere loss, unresolved telomere recombination back regulation is typically also involved to avoid excessive
intermediates, and mitotic infidelity, representing a synthetic (in space and/or time) recruitment (Altmeyer & Lukas 2013).
lethal effect (Panier et al. 2019; Verma et al. 2019). Interestingly, ALT itself is a self-perpetuating process: ALT
Two proteins of the Fanconi anemia pathway were also activity promotes replication stress, which in turn induces
shown to control ALT activity: FANCD2 counteracts BLM- a BIR-driven feedforward loop of SUMO-dependent repair
mediated resection and strand exchange, which promotes protein recruitment and ALT telomere synthesis in APB con-
intramolecular resolution of stalled replication forks during densates (Zhang et al. 2021). Disruption of this feedforward
ALT. Loss of FANCD2, similar to the loss of SLX4IP, leads loop results in reduced replication stress at telomeres and
to hyperactivation of ALT with increased extrachromosomal reduced RPA recruitment. RPA protects telomeric ssDNA in
telomeric DNA and recombinational byproducts (Root et al. ALT-positive cancer cells not only in S/G2 but also at post-
2016). The ATPase and DNA translocase FANCM controls MiDAS sites in G1 (Lezaja et al. 2021). Despite its ultra-high
ALT at multiple levels: Similar to SMARCAL1, FANCM affinity for ssDNA, the RPA complex readily phase separates
promotes remodeling of stalled replication forks and fork in solution through associative interactions to form ssDNA-
reversal, thereby counteracting replication stress at telomeres containing liquid droplets (Spegg et al. 2023). An excess of
(Gari et al. 2008). FANCM also counteracts replication free RPA was previously shown to facilitate rapid exchange
stress prior to fork stalling by controlling TERRA levels and of RPA on ssDNA (Gibb et al. 2014; Ma et al. 2016). Con-
regulating telomeric R-loop formation (Silva et al. 2019). sistently, sub-stoichiometric amounts of ssDNA were most
Additionally, FANCM interacts with the BTR complex and effective in triggering dynamic RPA condensates (Spegg
regulates its branch migration activity (Lu et al. 2019; Silva et al. 2023). Taken together, these findings suggest that RPA
et al. 2019). condensation generates a reservoir of highly concentrated
Telomeric DNA synthesis during ALT occurs in S/G2 and RPA around ssDNA to promote rapid exchange between
in mitosis, indicating that telomere elongation and mainte- the free and bound state and allow handover to downstream
nance are not completed during S-phase. Recent findings ssDNA-binding proteins such as RAD51/RAD52 (Spegg
suggest that telomere replication and recombination inter- et al. 2023). This model implies non-stoichiometric assembly
mediates in ALT-positive cancer cells are even transmitted of RPA on ssDNA, with the surplus of RPA around ssDNA
to the next cell cycle, where they are shielded by RPA to facilitating continuous RPA exchange. RPA condensation

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 Chromosoma

properties are modulated by phosphorylation-induced nega- actin filaments and transport cargo (Minozzo & Rassier 2013;
tive charges on an intrinsically disordered region (IDR), and Woolner & Bement 2009).
phosphomimetic mutants of RPA fail to form liquid drop- The actinomyosin network is not only involved in cyto-
lets in vitro and light-induced condensates in cells (Spegg plasmic transport processes, but was more recently also
et al. 2023). Charge blockiness, rather than specific target shown to participate in nuclear processes such as chroma-
site phosphorylation, was recently shown to regulate cell tin decondensation and nuclear volume expansion after cell
cycle-specific phase separation (Yamazaki et al. 2022) and, division, initiation of DNA replication, and in the regulation
consistently, multisite phosphorylation of the IDR in RPA of transcription by enhancing RNA polymerase II cluster-
cooperatively affects RPA clustering (Spegg et al. 2023). ing (Baarlink et al. 2017; Krippner et al. 2020; Parisis et al.
Cells expressing phosphomimetic RPA show altered ALT 2017; Plessner & Grosse 2019; Ulferts et al. 2021; Wei et al.
activity, with reduced telomere clustering, elevated ssDNA 2020). Intriguingly, nuclear actin filaments were also shown
at telomeres, impaired RAD52 recruitment, and increased to form upon treatment with different DNA damaging agents
telomere loss (Spegg et al. 2023). As telomere clustering is including UV-radiation, methylmethanosulfonate (MMS),
a hallmark of ALT, defective clustering may cause unpro- and neocarzinostatin (NCS), and are increasingly recognized
ductive telomere synthesis and exacerbated telomeric DNA to play a role in DNA repair (Andrin et al. 2012; Belin et al.
damage. Although the exact mechanism of impaired telomere 2015; Hurst et al. 2019). The actin regulating ARP2/3 pro-
clustering in RPA phosphomimetic mutant cells remains to tein complex and its associated factor WASP were found to
be fully elucidated, several observations suggest an emerging localize to sites of DNA damage in mammalian cells where
connection between RPA condensation at fragile genomic they nucleate actin filament formation. This promotes DSB
regions, including ALT telomeres, and activation of the mobility and clustering for repair by HR (Schrank et al. 2018;
nuclear cytoskeleton for enhanced chromatin mobilization. Schrank & Gautier 2019). Consistently, nuclear actin polym-
erization and myosin are required for the directed movement
of DSBs within heterochromatin towards the nuclear periph-
ery for error-free HR repair in Drosophila and mammalian
Emerging links between repair condensates cells (Caridi et al. 2018; Caridi et al. 2019; Merigliano &
and the nuclear cytoskeleton Chiolo 2021; Rawal et al. 2019), and damaged rDNA relo-
calizes to the nucleolar periphery in an ATM-, ARP2/3-,
Using optogenetic tools for controlled light-inducible Cry2- and myosin-dependent manner (Harding et al. 2015; Mar-
dependent protein condensation (Kilic et al. 2019; Shin et al. nef et al. 2019). Similarly, rDNA breaks in yeast transiently
2017) coupled to sensitive TurboID proximity labeling prot- move to extranucleolar regions for recombinational repair
eomics (Alghoul et al. 2021; Frattini et al. 2021) revealed that (Torres-Rosell et al. 2007), and breaks in pericentric hetero-
RPA condensation not only results in selective partitioning chromatin of mouse cells relocate to the periphery of hetero-
of RAD52 and the ALT-promoting BTR complex, but also in chromatin domains after resection (Tsouroula et al. 2016).
the selective enrichment of several components of the actin- More recently, nuclear F-actin was also found to play a role
and myosin network (Spegg et al. 2023). Considering that in response to replication stress in mammalian cells, where
the light-induced clustering of RPA and the simultaneous it counteracts nuclear deformation and promotes myosin-
TurboID-mediated proximity labeling were performed for a dependent re-localization of stressed replication forks to the
comparatively short duration of only 15 minutes, the iden- nuclear periphery in an ATR- and WASP-ARP2/3-dependent
tified proteins likely represent the first responders to RPA manner (Lamm et al. 2020; Lamm et al. 2021). Interestingly,
condensation. Among them were ACTN4, MYO1C, and WASP associates with RPA at stressed replication forks and
MYH9, which have known nuclear functions in chromatin promotes RPA binding to ssDNA (Han et al. 2022), and actin
organization, transcription, and post-mitotic nuclear expan- nucleators regulate RPA availability under conditions of rep-
sion (Almuzzaini et al. 2015; Krippner et al. 2020; Kumeta lication stress (Nieminuszczy 2023).
et al. 2010; Sarshad et al. 2013; Ye et al. 2020). As central Reciprocally, RPA condensates may concentrate mono-
component of the cytoskeleton, actin plays fundamental roles meric G-actin and thereby trigger nucleation and growth of
in cell division, cell movement, cell signaling, and organelle actin filaments at sites of DNA damage (Figure 5a). Previous
transport across species (Boldogh et al. 2001; Chakrabarti work has shown that self-assembled polypeptide condensates
et al. 2018; Grosse et al. 2003). Actin exists in a monomeric serve as hub for actin enrichment and polymerization in vitro
globular form (G-actin) or as polymerized multimers form- (Graham et al. 2023; McCall et al. 2018). Furthermore, con-
ing the filamentous actin (F-actin) network (Dominguez & densation of actin by intrinsically disordered regions of actin-
Holmes 2011; Gunning et al. 2015). Myosins, on the other associated proteins was proposed as general mechanism for
hand, are ATP-dependent motor proteins that move along actin network organization (Billault-Chaumartin et al. 2022).

13
Chromosoma

Fig. 5  Emerging links between biomolecular condensates and acti- RPA and associated proteins. (b) Model of actin filament formation
vation of the nucleoskeleton. (a) Model of actin filament formation through G-actin concentration on the surface of DNA repair conden-
through G-actin concentration in DNA repair condensates formed by sates formed by RPA and associated proteins

Consistently, during oocyte development, actomyosin cor- (Figure 6a). A conceptually related mechanism seems to be
tex activation is promoted by the emergence of thousands of at work during neuronal long-distance transport of RNAs,
short-lived protein condensates enriched in actin, WASP, and where phase-separated RNA granules hitchhike on lys-
ARP2/3 forming an active micro-emulsion (Yan et al. 2022). osomes through a low complexity domain-containing tether
Phase separation of actin regulatory factors was shown to protein (Liao et al. 2019).
increase the dwell time of nucleators to initiate F-actin for- Both in yeast and in human cells, stressed telomeres
mation, as demonstrated for WASP and ARP2/3 at the cell relocalize to the nuclear periphery and this process is driven
membrane (Case et al. 2019). Reducing dynamic interaction by nuclear F-actin polymerization and involves RPA and
landscapes from a 3D environment to a 2D interface may SUMOylation (Churikov et al. 2016; Pinzaru et al. 2020;
generally help to concentrate molecules and promote their Spichal et al. 2016). Interestingly, RPA contains a SIM
activation. Along these lines, actin and actin nucleation fac- motif, through which it can interact with SUMOylated
tors might get selectively enriched on the surface of RPA proteins (Zhu et al. 2023), and RPA itself is SUMOylated
condensates through interfacial affinity, rather than in the when collapsed replication forks are relocated to the nuclear
interior (Figure 5b). Similar interactions have been observed periphery in yeast (Whalen et al. 2020). Whether RPA
between microtubule subunits and stress granules (Böddeker SUMOylation and RPA-SUMO interactions are involved in
et al. 2022). RPA condensation and actin polymerization remains to be
In yeast, Rad52-dependent DNA repair condensates were addressed.
previously shown to induce nuclear microtubule filaments, Similar to actin, also myosin is increasingly recognized to
which is required for moving the damaged DNA compart- play important roles in the nucleus for transcription and in
ment to the nuclear periphery for repair (Oshidari et al. the DNA damage response, and was recently shown to regu-
2020). Microtubule-dependent DNA damage mobility was late phase separation by promoting condensate coalescence
also observed in mouse cells with unprotected, dysfunc- (Cook & Toseland 2021; Feng et al. 2022; Hari-Gupta et al.
tional telomeres (Lottersberger et al. 2015). In human can- 2022). The actinomyosin network appears to be more impor-
cer cells that use ALT-dependent recombination at fragile tant for homology-directed repair and RAD52-mediated sin-
telomeres, nuclear actin filaments might serve as molecular gle-strand annealing (SSA), compared to DNA repair by non-
highways for the directed movement of RPA-enriched repair homologous end-joining (NHEJ) or alternative end-joining
condensates, with the condensate surface or co-condensing (alt-EJ) (Pfitzer et al. 2019), consistent with a predominant
adaptor molecules serving as anchoring points for myosin role during homology-directed repair of fragile repetitive

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 Chromosoma

Fig. 6  DNA repair condensate mobilization and surface wetting at surface wetting-assisted anchoring of DNA repair condensates at
the nuclear envelope. (a) Model of actinomyosin-mediated movement the nuclear periphery. INM, inner nuclear membrane; ONM, outer
of DNA repair condensates through the nuclear space. (b) Model of nuclear membrane

sequences such as telomeres. Moreover, formation of nuclear et al. 2022). Moreover, mechanosensing of cell density by
F-actin in response to genotoxic stress was recently shown to adhesion forces was shown to control cell cycle progression
serve as scaffold for PML nuclear bodies (Cobb et al. 2022). at the G2/M transition through activation of the WEE1
Whether the same holds true for APBs at telomeres remains checkpoint kinase (Donker et al. 2022). Alterations in cell
to be shown. Nevertheless, several connections between tension also affects cell cycle progression from G1 to S-phase
self-assembly features of repair condensates and the nuclear (Perez-Gonzalez et al. 2019), and the mechanosensing
cytoskeleton have started to emerge, spurring considerations Hippo pathway with its mechanotransducers YAP/TAZ and
about the mechanobiology of genome maintenance. LATS1/2 has also been implicated in DNA damage signaling
and repair (Pefani et al. 2014; Pefani & O'Neill 2016).
Nuclear condensates are not only scaffolds for biochemical
Connecting DNA repair condensates reactions, but also mechanic entities with viscoelastic proper-
to mechanobiology ties that generate and respond to mechanical force (Spagnol
et al. 2016). Similarly, the chromatin polymer itself is a main
Cells are exposed to dynamically changing extrinsic factor in defining nuclear mechanical properties (Barbieri
mechanical forces, including shear stress, stretching, et al. 2013; Nicodemi & Pombo 2014; Stephens et al. 2019).
stiffening, and compression, and these forces are Depending on the time and length scale, chromatin behaves
counteracted by cell-intrinsic force generation through the both as an elastic solid and as a viscous liquid (Zidovska
cytoskeleton and through modulation of the viscoelastic 2020), a rheological behavior that is also observed for the
properties of the cellular interior (Bertillot et al. 2022; cytoskeleton (Wiegand & Hyman 2020). The viscoelastic
Dupont & Wickström 2022). With the intensified research properties of the nuclear environment is regulated by several
on material properties of biomolecular condensates, new factors, including external cytoskeletal forces that change
connections between mechanobiology and viscoelastic nuclear morphology, the nuclear lamina, chromatin compac-
polymer networks in membraneless organelles are being tion and structural organization, and the formation and regu-
revealed (Lee et al. 2022; Wiegand & Hyman 2020). For lation of biomolecular condensates (dos Santos & Toseland
instance, recent work demonstrated that cytoplasmic forces 2021). As physical properties and dynamic forces are often
tune nuclear condensate coalescence and molecular kinetics altered in human disease, which can deregulate gene expres-
within condensates (Al Jord et al. 2022). Mechanical force sion programs and challenge genome stability, understanding
experienced by the nucleus modulates permeability across the interplay between genome organization and mechanobiol-
nuclear pores, indicating that transport of proteins through ogy may have clinical implications.
the hydrogel-like meshwork of FG-rich repeats inside Nuclear deformation in migrating immune and meta-
nuclear pore complexes is mechanosensitive (Andreu static cancer cells causes nuclear blebbing and rupture and

13
Chromosoma

is associated with increased DNA damage, depletion of DNA deformation, driven by Piezo1-triggered reduction of lamina-
repair factors, and cell cycle checkpoint activation (Denais associated H3K9me3-marked heterochromatin to insulate the
et al. 2016; Irianto et al. 2016; Irianto et al. 2017; Isermann genetic material from mechanical force, promotes genome
& Lammerding 2017; Pfeifer et al. 2018; Raab et al. 2016; stability (Nava et al. 2020).
Xia et al. 2018; Xia et al. 2019; Xie et al. 2020). Moreover, The size to which biomolecular condensates can grow in a
external mechanical stimuli from the cytoplasm can modulate viscoelastic environment is limited, because with increasing
processes inside the nucleus by transmitting force through growth more energy is required to deform the surrounding
connections between the cytoskeleton and the nucleoskel- stiffer matrix (Lee et al. 2022; Wiegand & Hyman 2020).
eton (Dupont & Wickström 2022; Goelzer et al. 2021). Such Indeed, the chromatin polymer may mechanically suppress
connections are provided by linker of nucleoskeleton and droplet coalescence and ripening and control condensate num-
cytoskeleton (LINC) complexes (Alam et al. 2016; Crisp ber, size, and positioning (Zhang et al. 2021). Such considera-
et al. 2005; Leno 1992; Mammoto et al. 2012; Wang et al. tions could also apply to DNA repair condensates and might
2018). LINC complexes, embedded in the nuclear envelope, affect their mobility and growth. Possibly, repair condensates
connect cytoplasmic intermediary filaments, microtubules, redirected to the nuclear periphery for error-free repair could
and actin filaments with the nuclear lamina and with silenced wetten the inner nuclear membrane (Mangiarotti et al. 2022;
heterochromatin regions in lamina-associated domains Oshidari et al. 2020; Strom et al. 2023), which might be a
(LADs) (dos Santos & Toseland 2021; Spichal & Fabre mechanism to anchor repair compartments to the nuclear
2017). Defects in the nuclear lamina are associated with dis- envelope by means of adsorption (Figure 6b). Upon mem-
eases like Hutchinson-Gilford progeria, muscular dystrophy, brane wetting, biochemical reactions at the 2D membrane-
and cardiomyopathies, and can lead to changes in chromatin condensate interface may be accelerated compared to the 3D
structure as well as deregulated DNA replication, repair, and volume of a non-membrane-tethered condensate, potentially
gene expression (Cho et al. 2019; dos Santos & Toseland providing an additional advantage for genome repair.
2021; Schreiber & Kennedy 2013). Interestingly, LINC com-
plex components are involved in DNA damage relocaliza-
tion and clustering and promote homologous recombination Conclusions and perspectives
repair (Aymard et al. 2017; Bozec et al. 2023; Lawrence et al.
2016; Lottersberger et al. 2015; Marnef et al. 2019; Swartz Recent work has started to unveil intriguing connections
et al. 2014). Moreover, the LINC complex proteins SUN1 between nuclear condensates involved in the cellular
and SUN2, together with dynamic microtubules and nuclear response to replication stress and DNA damage and the
pore proteins, drive the formation of DSB-capturing nuclear nuclear cytoskeleton. Moreover, material properties and
envelope tubules (dsbNETs) to support repair in the interior the mechanobiology of chromatin, nuclear condensates,
of the nucleus (Shokrollahi et al. 2023). and the nuclear cytoskeleton are receiving increasing
Condensed chromatin is a barrier for the DNA repair attention. While it is becoming clear that genome func-
machinery (Mitrentsi et al. 2022), and the local viscoelasticity tions and cellular responses to stress, including genotoxic
of the nucleus varies by compartment and degree of chromatin stress, are tightly linked to nuclear architecture and to the
condensation (Lee et al. 2022). While nucleosomes and dynamically changing material properties of the nuclear
nucleosome clusters on the nanoscale are mobile and have interior, the varying length- and timescales at which dif-
liquid-/gel-like properties, condensed chromatin polymers ferent nuclear processes and macromolecular assemblies
on the mesoscale seem physically constrained and more occur complicate their analysis and interpretation. Many
immobile, with soluble chromatin-binding proteins open questions remain about whether and how nuclear
coalescing on the solid chromatin scaffold (Hansen et al. condensates sense mechanical stimuli and if they regulate
2021; Strickfaden et al. 2020; Tortora et al. 2022). Chromatin genome functions (e.g. chromatin organization, epigenetic
decompaction is needed to improve the efficiency of DNA states, replication timing, DNA repair) in response to exter-
repair upon damage (Polo & Almouzni 2015), and induced nal forces. Conversely, how changes in chromatin structure
chromatin decompaction reduces nuclear stiffness by and genome stability affect nuclear mechanobiology is cur-
~35–50% (Hobson et al. 2020; Krause et al. 2013; Shimamoto rently not well understood.
et al. 2017; Stephens et al. 2017). The change from a locally Fragile telomeres, which per se exhibit strong subdiffu-
stiffer to a softer chromatin environment may energetically sive motion (Lee et al. 2022) yet become mobilized upon
favor nucleation and growth of DNA repair condensates. telomeric replication stress and DNA damage (Lamm et al.
Nuclear stiffness is indeed reduced upon DNA damage and 2021), may represent a paradigm for emerging connections
the reduction in nuclear tension promotes repair (dos Santos between viscoelastic repair condensates formed around dam-
et al. 2021). Moreover, nuclear softening upon severe nuclear aged genomic regions and nuclear mechanobiology. Further

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insights into the material properties of chromatin domains Al Jord A, Letort G, Chanet S, Tsai FC, Antoniewski C, Eichmuller
and nuclear compartments and how they are linked to mate- A, Da Silva C, Huynh JR, Gov NS, Voituriez R et al (2022)
Cytoplasmic forces functionally reorganize nuclear condensates
rial properties of the cytoskeleton and of the surrounding in oocytes. Nat Commun 13:5070
membranes are going to benefit a mechanistic understand- Alam SG, Zhang Q, Prasad N, Li Y, Chamala S, Kuchibhotla R, Kc B,
ing of genome functioning and its deregulation in disease. Aggarwal V, Shrestha S, Jones AL (2016) The mammalian LINC
Elucidating the interplay between material properties and complex regulates genome transcriptional responses to substrate
rigidity. Sci Rep 6:1–11
biochemical reactions in cells may also enable their targeted Alghoul E, Basbous J, Constantinou A (2021) An optogenetic proxim-
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authors acknowledge the research funding from the Swiss National ment for polymerized actin in DNA double-strand break repair.
Science Foundation (grant 310030_197003) and the European Research Nucleus 3:384–395
Council (ERC) under the European Union’s Horizon 2020 research Arezi B, Kuchta RD (2000) Eukaryotic DNA primase. Trends Biochem
and innovation program (ERC-2016-STG 714326). V.S. acknowledges Sci 25:572–576
additional funding from the University of Zurich Candoc program. Aymard F, Aguirrebengoa M, Guillou E, Javierre BM, Bugler B,
Arnould C, Rocher V, Iacovoni JS, Biernacka A, Skrzypczak
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Telomeric repeat–containing RNA and RNA surveillance factors
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Attribution 4.0 International License, which permits use, sharing, Baarlink C, Plessner M, Sherrard A, Morita K, Misu S, Virant D,
adaptation, distribution and reproduction in any medium or format, Kleinschnitz EM, Harniman R, Alibhai D, Baumeister S et al
as long as you give appropriate credit to the original author(s) and the (2017) A transient pool of nuclear F-actin at mitotic exit controls
source, provide a link to the Creative Commons licence, and indicate chromatin organization. Nat Cell Biol 19:1389–1399
if changes were made. The images or other third party material in this Bai G, Kermi C, Stoy H, Schiltz CJ, Bacal J, Zaino AM, Hadden MK,
article are included in the article's Creative Commons licence, unless Eichman BF, Lopes M, Cimprich KA (2020) HLTF promotes
indicated otherwise in a credit line to the material. If material is not fork reversal, limiting replication stress resistance and preventing
included in the article's Creative Commons licence and your intended multiple mechanisms of unrestrained DNA synthesis. Mol Cell
use is not permitted by statutory regulation or exceeds the permitted 78:1237–1251.e1237
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