# On the edge: how nuclear pore complexes rule genome stability

Marie-Noëlle Simon, Karine Dubrana, Benoit Palancade

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Marie-Noëlle Simon, Karine Dubrana, Benoit Palancade. On the edge: how nuclear pore complexes rule genome stability. Current Opinion in Genetics and Development, 2024, 84, 10.1016/j.gde.2023.102150. hal-04405822

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# On the edge: how nuclear pore complexes rule genome stability

Marie-Noëlle Simon<fnref n="1" />,#,*, Karine Dubrana<fnref n="2" />,<fnref n="3" />,#,* and Benoit Palancade<fnref n="4" />,*

<figure><img src="image_4.png" /></figure>

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Nuclear organization has emerged as a critical layer in the coordination of DNA repair activities. Distinct types of DNA lesions have notably been shown to relocate at the vicinity of nuclear pore complexes (NPCs), where specific repair pathways are favored, ultimately safeguarding genome integrity. Here, we review the most recent progress in this field, notably highlighting the increasingly diverse types of DNA structures undergoing repositioning, and the signaling pathways involved. We further discuss our growing knowledge of the molecular mechanisms underlying the choice of repair pathways at NPCs, and their conservation — or divergences. Intriguingly, a series of recent findings suggest that DNA metabolism may be coupled to NPC biogenesis and specialization, challenging our initial vision of these processes.

## Addresses

Corresponding author: Palancade, Benoit (benoit.palancade@ijm.fr)

## Current Opinion in Genetics & Development 2024, 84:102150

This review comes from a themed issue on **Genome Architecture and Expression**

Edited by **Maya Capelson** and **Eda Yildirim**

Available online xxxx

https://doi.org/10.1016/j.gde.2023.102150

0959-437X/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

Among the cellular components that contribute to nuclear architecture and genome organization are the nuclear pore complexes (NPCs), which are megadalton-sized proteinaceous assemblies inserted into the nuclear

envelope at the points of fusion of the inner and outer nuclear membranes. NPCs are composed of repeated copies of ~30 polypeptides called nucleoporins (Nups) and assembled into structural modules, delineating a three-ring symmetric scaffold flanked by peripheral appendages, namely the cytoplasmic filaments and the nuclear basket (Figure 1). In addition to their well-characterized interactions with transport receptors, which are instrumental for the nucleocytoplasmic exchanges of macromolecules, NPCs and their components also establish contacts with the genome, influencing virtually all DNA-based transactions, thus impacting the maintenance of genetic integrity [1].

Genome stability critically relies on the DNA damage response (DDR), a series of pathways that sense genotoxic insults or impediments to DNA metabolism, and respond through the establishment of dedicated signaling and DNA repair mechanisms. Several layers of spatiotemporal regulations control the activation and outcome of the DDR, from epigenetic modifications and chromatin organization, to chromosome structure and nuclear compartmentalization (reviewed in Refs. [2,3]). Here, we will focus specifically on the increasingly multifaceted role exerted by NPCs in the control of genetic stability, as first observed in yeast and now expanded to several model organisms.

## Multiple damages on the way to the nuclear periphery

Following the observation that mutations in several Nups confer sensitivity to various DNA-damaging agents and lead to DNA damage accumulation [4], the groundbreaking discovery of a function of NPCs in the DDR was the demonstration of the robust recruitment of hard-to-repair double-strand breaks (DSBs) to the nuclear pores in yeast [5]. This observation hinted at the potential for NPCs to serve as a hub for specific molecular transactions at damaged DNA sites, a function independent of the trafficking of repair factors in and out of the nucleus. Together with the subsequent establishment of a role of NPCs in anchoring eroded telomeres in the absence of telomerase and in regulating telomere recombination [6,7], this led to the notion that NPCs could help resolve ‘congested’ DNA intermediates by promoting noncanonical error-prone repair pathways [8]. However, this initial model faced challenges as increasing evidence pointed to various

<footnote n="1">¹Marseille Cancer Research Center (CRCM), U1068 Inserm, UMR7258 CNRS, Institut Paoli-Calmettes, Equipe Labélisée Ligue, Aix Marseille University, Marseille, France</footnote>

<footnote n="2">²Université Paris Cité, Inserm, CEA, Stabilité Génétique Cellules Souches et Radiations, F-92260 Fontenay-aux-Roses, France</footnote>

<footnote n="3">³Université Paris-Saclay, Inserm, CEA, Stabilité Génétique Cellules Souches et Radiations, F-92260 Fontenay-aux-Roses, France</footnote>

<footnote n="4">⁴Université Paris Cité, CNRS, Institut Jacques Monod, F-75013 Paris, France</footnote>

<footnote># These authors contributed equally.</footnote>

<footnote>* Twitter account: @IJMonod, @DubranaLab, @PalancadeLab</footnote>
<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>

An overview of the various DNA structures relocating to NPCs. The different structural modules of the NPC are represented according to the indicated color code. See text for details.

DNA structures relocating to NPCs with distinct con-
sequences (Figure 1). Notably, replication forks stalled at
several natural or model replication barriers were shown to
relocate to the nuclear pores. These include structures
such as triplet-nucleotide repeats (TNR) [9], tightly DNA-
bound proteins at telomeres [10,11], rDNA [12], or a site-
specific model barrier [13]. Beyond DNA replication, re-
cent research also unveiled the role of NPCs in responding
to R-loops, which are genotoxic structures formed when
nascent RNAs hybridize back to their DNA templates. R-
loops also act as a trigger for relocation to NPCs, yet in-
dependently of damage formation [14]. Importantly,
DNA–NPC interactions extend beyond chromosomes.
Extrachromosomal DNA circles (ecDNA), which are by-
products of recombination events between repeated se-
quences (e.g. at ribosomal DNA or telomeres), also relocate
to yeast NPCs [15,16]. Given their relevance in cancer cells
and potential involvement in mutagenesis [17], ecDNA
relocation to NPCs represents an intriguing avenue of
exploration. In line with this, anchoring of telomeric C-
circles to NPCs in yeast potentially contributes to re-
combination-based alternative telomere lengthening,
driving survival in the absence of telomerase [16]. Thus,
the relocation to the NPCs concerns a wide variety of
chromatin and DNA structures and is not restricted to
pathological situations (Figure 1).

Despite these significant findings, the shared features
dictating the relocation of various DNA structures to
NPCs remain unclear (Table 1). The detection of da-
maged DNA is largely associated with a wave of post-

translational modifications (PTMs) targeting several
DNA-binding proteins and repair factors, notably
through SUMOylation, which relies on the covalent
coupling of the small ubiquitin-like modifier (SUMO)
polypeptide to its targets. Although the specific SU-
MOylated proteins might vary depending on the type of
damage, the common thread is their recognition by
NPC-associated SUMO-targeted ubiquitin ligases
(STUbLs), driving the relocation to nuclear pores (re-
viewed in Ref. [18]). Extensive accumulation of RPA
.replication protein A)-bound single-stranded DNA is
the critical signal for the SUMOylation wave [19] and
RPA interacts with the STUbL component Slx5 in yeast
[7]. Accordingly, RPA appears as a key factor in NPC
relocation [14,20] although its poly-SUMOylation might
be instrumental only when resection is limited [21]. Of
note, ecDNA seems to use a distinct and less-under-
stood pathway relying on factors involved in the cou-
pling between transcription and mRNA export [15,16].

Among the intriguing facets of DNA damage relocation
to NPCs is the role of the cytoskeleton and motor pro-
teins. Nuclear actin filaments participate in DNA da-
mage relocation to the nuclear periphery and/or NPCs,
with Arp2/3 actin nucleators having been shown to
mediate repositioning of heterochromatic DSBs in
*Drosophila* [22] and replication-stressed telomeres in
human cells [11]. Accordingly, F-actin promotes the
mobility of stressed replication foci and DNA breaks
[23,24]. Increased mobility of damaged DNA also in-
volves changes in nuclear microtubule dynamics and
<table><caption>**Table 1**

Factors involved in repositioning at NPCs and associated DNA repair events, as inferred in yeast models.</caption><thead><tr><th rowspan="2">DNA structures</th><th colspan="3">Factors involved</th><th colspan="3">Functional consequences</th></tr><tr><th>DDR</th><th>Chromatin</th><th>SUMO pathway</th><th>NPC</th><th>Repair pathway affected</th><th>[Effect]</th><th>Loss-of-function assessed</th><th>Gain-of-function assessed</th></tr></thead><tbody><tr><td>DSBs [4,5,25,32–34]</td><td>Checkpoint: Mec1 [5]</td><td>SWR1/ Htz1 [33]</td><td>Poly-SUMOylation: SUMO E3 ligases: Siz2, Mms21 [32]; STUbLs: Sbx5–Sbx8 [32]</td><td>Nup84 complex [5]</td><td>NHEJ [4,25,32]</td><td>[+]</td><td>Nup84 complex<sup>(a)</sup>, -</td><td></td></tr><tr><td rowspan="2">Stalled replication forks [5,9,13,20,55]</td><td rowspan="2">Resection: Sgs1, Exo1, and Mre11 [20]; fork remodeling: Rad51 [13]<sup>(b)</sup>, PCNA loading: Ctf18–RFC? [55]</td><td rowspan="2">Smc5/6 complex [20]</td><td rowspan="2">Mono-SUMOylation: Rad52, Rad59, and RPA [20]; SUMO E3 ligases: ScMms21 [20], SpPI1 [13]; STUbLs: ScSbx5–Sbx8 [20], SpRrp1–Rrp2/Sbx8 [13]</td><td rowspan="2">Nup84 complex [9,13], Nup1 [10]</td><td>Homologous recombination (HR) [5]</td><td>[+]</td><td>Nup60, and Ulp1</td><td>LexA–Nup84</td></tr><tr><td>BIR [25,32]</td><td>[+]</td><td>Nup84 complex, Sbx8</td><td></td></tr><tr><td rowspan="2">Eroded telomeres [7,10]</td><td rowspan="2">Checkpoint: Rad9, Rad24 [7]</td><td rowspan="2">?</td><td rowspan="2">Mono-SUMOylation: SUMO E3 ligases: Siz1–Siz2 [7]; STUbLs: Sbx5–Sbx8 [7]</td><td rowspan="2">Nup1 [10]</td><td>Sister chromatid recombination (SCR) [34]</td><td>[+]</td><td>Nup84 complex</td><td></td></tr><tr><td>BIR (type II survivors) [7]</td><td>[-]</td><td>Nup84 complex, Sbx5–Sbx8, and Nup1</td><td></td></tr><tr><td rowspan="2">R-loops [14,35]</td><td rowspan="2">ssDNA coating: RPA [14]</td><td rowspan="2">?</td><td rowspan="2">Mono-SUMOylation: RPA [14]; SUMO E3 ligase: Mms21 [14]; STUbLs: Sbx5–Sbx8 [14]</td><td rowspan="2">(c)</td><td>Unequal sister chromatid recombination [10]</td><td>[-]</td><td>Nup1</td><td></td></tr><tr><td>BIR (type II survivors) [7]</td><td>[+]</td><td>Mip1, Mlp2, Ulp1, LexA–Ulp1C Siz1–Siz2, and Sbx5–Sbx8</td><td></td></tr><tr><td>Telomeric C-circles ecDNA circles [15,16]</td><td>?</td><td>SAGA [15,16]</td><td>TREX2 [15,16]</td><td>TREX2 [15,16]</td><td>BIR (type II survivors) [16] detoxification?Prevention of aging?</td><td>[+]</td><td>TREX2</td><td></td></tr></tbody></table>

The factors involved in the relocation of the indicated structures to the NPCs are indicated and classified according to their biological function: DNA metabolism and the DDR; chromatin and chromatin-associated factors; SUMOylation pathway (type of modification, targets, and enzymes); NPC components. The repair pathways and the genetic situations assessed are indicated. Increased or decreased activities of the indicated pathways at the NPC are indicated by [+] or [-], respectively. Sc, S. cerevisiae; Sp, S. pombe.

(a) *nup84Δ* does not affect NHEJ in Ref. [25] and affects only imprecise NHEJ in Ref. [32]. (b) Rad51 associates to TNR-stalled forks following their relocation to NPCs in S. cerevisiae [20], while its enzymatic activity is required for the repositioning of a replication fork barrier in S. pombe [13]. (c) TREX2 is not required for R-loop repositioning to NPCs [14]. (d) Sbx5 is not required for ecDNA circle association with NPCs [15].
partners, which might promote repair by relocating the
damage to specific nuclear domains including NPCs
[25–28]. Conversely, microtubules can substantially alter
nuclear morphology (reviewed in Ref. [29]), forming
nuclear membrane invaginations (including lamina,
inner nuclear membrane proteins, and NPCs) that bring
the nuclear envelope to DSBs upon damage induction
[30]. It remains to be uncovered whether other types of
DNA damages similarly interact with NPC components
away from the nuclear periphery, notably in view of the
existence of nucleoplasmic pools of Nups [31].

Nuclear pore complex association: a double-
edged sword for damaged loci

The impact of DNA damage-NPC association on DNA
repair and genome stability has been extensively in-
vestigated in various distinct DNA damage scenarios, uti-
lizing dedicated assays and loss- or gain-of-function
experiments (as outlined in Table 1). Based on these ap-
proaches, NPCs have been proposed to promote non-
homologous end joining (NHEJ [4,32]), recombination
with the sister chromatid [33,34], replication-dependent
fork restart [13,20], or mutagenic break-induced replication
(BIR) repair [25,32]. At eroded telomeres, NPCs similarly
facilitate a BIR-type of repair aiding in maintaining telo-
mere function by generating type II survivors [7]. Con-
versely, NPC association has been shown to reduce R-loop
levels [35] and R-loop-induced recombination on tran-
scribed genes [14]. Yet, despite these efforts, the precise
role of NPCs in repair pathway choice or fork protection
still remains enigmatic (Table 1). Indeed, it is likely that
the outcome of NPC association varies depending on the
damage type and during cell cycle progression. For in-
stance, in the context of replication-associated stresses, the
exact sequence of events leading to fork restart and the
repair proteins under regulation appear to differ depending
on the replication stalling event [18]. In the case of tran-
scription-associated stresses, NPC association in G1, as
observed upon R-loop formation, may preserve genome
stability [14,35]. However, relocation in S phase could turn
detrimental, as highlighted by the increased replication
stress and DNA damage scored in situations of checkpoint
or pre-mRNA processing deficiencies associated with ex-
tended NPC binding [36,37].

Beyond the limitations of repair assays performed on
asynchronous populations, it is also important to ac-
knowledge the biases introduced by loss-/gain-of-function
experiments. Loss-of-function assays can lead to indirect
effects, particularly in NPC mutants that could affect cell
fitness, macromolecule import/export (e.g. [38]), or SU-
MOylation processes, away from the NPC [4]. Similarly,
gain-of-function experiments, which rely on the targeting
to damaged chromatin of a Nup fused to a DNA-binding
domain, could lead to misleading outcomes if Nup
function and/or expression are affected by tagging [10]. In

the future, novel experimental strategies with increased
spatial and temporal resolution will thus be required to
determine whether and how NPCs shield cells against
DSB-induced genetic instability, or foster last-resort re-
pair attempts at the expense of genome integrity.

Amid the diverse repair outcomes, common themes
emerge regarding the molecular mechanisms through
which NPCs could regulate DNA repair and fork restart.
NPCs could serve as anchor points, facilitating en-
counters between damaged DNA and its homologous
sequences used for repair. This concept is especially
relevant for damaged telomeres, which naturally localize
to the nuclear periphery, at the vicinity of nuclear pores
[39]. Supporting this notion, the absence of the Nup84
complex reduces DSB-induced BIR between sub-
telomeric repeats [25]. Similarly, telomeric C-circles,
proposed as BIR templates for elongating eroded telo-
meres, concentrate at NPCs at the time of type II sur-
vivor formation, potentially enhancing their interaction
with telomeres and promoting telomere elongation [16].
Intriguingly, NPCs, with their ability to selectively ex-
port RNAs, might enhance spatial proximity between
DNA damage and RNA molecules, which have the po-
tential to serve as templates for recombination [40,41].
Alternatively, NPCs might sequester damaged DNA
away from unwanted recombination substrates, pre-
venting deleterious recombination events, a phenom-
enon proposed for rDNA in yeast [12] and
heterochromatin in Drosophila [42]. By facilitating
mRNA export and eviction from transcription sites, the
vicinity to NPCs may similarly disfavor encounters be-
tween RNA molecules and their DNA templates, alle-
viating R-loop formation [14,35].

Furthermore, NPCs associate with and locally con-
centrate specific factors, in particular the enzymes of
the SUMO pathway, creating functional compartments
that might regulate repair processes. Within these
NPC-bound factors, the STUBL Slx5-Slx8 and the
SUMO protease Ulp1/SENP1-2 have been implicated
in the anchoring of DNA damages to NPCs and their
resolution, respectively. A broad model posits that
DNA repair proteins are regulated through modulation
of SUMOylation and ubiquitinylation at NPCs, po-
tentially leading to proteasome-mediated degradation,
as previously proposed ([8], discussed in Refs. [18,43]).
Importantly, factors needed for targeting DNA damage
to NPCs may not necessarily be the same as those
modulating repair in the NPC environment. For in-
stance, Ulp1 is essential for restarting stalled replica-
tion forks but not for their targeting to NPC in S. pombe
[13]. NPCs could also concentrate other factors directly
involved in damage processing. A recent example is
NUMEN, a membrane-bound endo-/exonuclease pro-
cessing DSBs with 3'-overhangs, which localizes at the
nuclear periphery and interacts with NPCs [44]. In this
<figure><img src="image_6.png" /><figcaption>Figure 2</figcaption></figure>

NPC variants and their interactions with DNA structures. The different structural modules of the NPC are represented as indicated. PTMs are illustrated by stars (P, phosphorylation; S, SUMOylation). *DNA pol*, DNA polymerase; *RNA pol*, RNA polymerase.

context, removal of 3'-ssDNA overhangs by NUMEN is thought to favor NHEJ, particularly, though not exclusively, at perinuclear heterochromatic DSBs. Last, NPCs interact with RNA-binding proteins, which are increasingly implicated in DNA repair [41], possibly facilitating the recruitment of these proteins to sites of DNA damage, an aspect that will deserve further exploration.

**Are specialized nuclear pore complexes involved in DNA-related transactions?**

A growing body of evidence underscores the coexistence of structurally distinct NPC variants within a single nucleus (reviewed in Ref. [45]), sparking questions about their potential roles in the specific genomic processes described above. A first level of spatiotemporal variations among individual NPCs is provided by a spectrum of
PTMs targeting nucleoporins in response to various stimuli, including genotoxic stress. For instance, during replicative stress, nuclear basket subunits undergo phosphorylation by the Mec1ATR replication checkpoint kinase and its downstream effector Rad53CHK1 [36,46]. These modifications have been proposed to directly trigger the release of transcribed genes from the NPC, thus minimizing the topological constraints that would lead to fork reversal [36]. Recent findings also reveal that the DNA replication checkpoint triggers the eviction of RNA polymerases from their templates [47], impacting their transcription-dependent association to NPCs during regular or challenged replication [46]. Other DNA damage-induced PTMs, such as Nup60 ubiquitylation [48] or SUMOylation [49], might further influence NPC-chromatin association, warranting further investigation.

Additional layers of structural variations among individual pores encompass differences in (i) subunit composition, with the notable absence of the nuclear basket in a subset of NPCs in fungi [50,51], (ii) stoichiometry, as revealed by the detection of NPC isoforms containing either a single or a double nucleoplasmic outer ring in S. cerevisiae [52], and (iii) conformation, as reflected by varying dimensions of the inner ring-delivered channel [53,54] (Figure 2). In this respect, ‘basketless’ NPCs were recently proposed to interact with different DNA metabolism intermediates in budding yeast. On the one hand, the Ctf18-containing clamp loader replication factor C (RFC$^{Ctf18}$) associates with a subset of NPCs devoid of the basket subunits Mlp1 and Mlp2, especially during S phase and replicative stress [55]. While this interaction could contribute to the re-localization of RFC$^{Ctf18}$-associated stalled forks to the NPC, it may also reflect the existence of specialized NPCs, which could promote the PCNA loading and checkpoint activities of RFC$^{Ctf18}$ at challenged replicosomes, as supported by the decline in PCNA chromatin levels scored in *nup170Δ* mutants. Another example of pore specialization is provided by the detection of basketless NPCs in interaction with ecDNA circles [56]. These NPC species would contribute to the proper retention of such toxic DNA molecules in the mother cell during mitosis, although their specific composition is also likely to impact nucleocytoplasmic transport, as suggested by their decreased association with several mRNA export factors.

Which mechanisms could generally specify the association of distinct DNA intermediates with particular NPC variants (Figure 2)? On the one hand, chromatin or DNA-binding interfaces could be accessible in certain NPCs while masked in others, as already suggested [57]. Supporting this hypothesis, the chromatin-binding ac-

tivity of Nup155 was found to be suppressed in the presence of Nup62 in flies [58]. Alternatively, the association with DNA structures may disrupt NPC organization, displacing individual Nups or Nup modules, as proposed in the case of DNA circles [56]. Finally, DNA loci could associate with assembling NPCs, locking them into an immature state, a worth-considering scenario since basketless pores represent a natural intermediate in interphasic NPC assembly in yeast [59].

A more intricate coupling between DNA metabolism and NPC assembly emerges from reported interactions between DNA replication factors and Elys, a chromatin-binding Nup pioneering postmitotic NPC assembly in metazoans (reviewed in Ref. [60]). Elys reportedly associates with distinct components of the origin licensing and replication initiation machinery, such as MCM2-7 helicases in *Xenopus* extracts [61] and the origin recognition complex in flies [62]. Elys contributes to the loading of both complexes onto chromatin [61–63], and prevents replication stress-associated genetic instability, possibly by enabling additional dormant origin licensing events [62]. Reciprocally, MCM2-7 is required for optimal Elys recruitment onto chromatin, NPC assembly, and nuclear growth [61]. Interactions between Nups and factors involved in DNA metabolism are thus likely to reciprocally influence both NPC assembly and genome integrity.

## Concluding remarks

The last years of research have revealed an unsuspected versatility of NPCs in DNA-related processes, notably regarding the type of DNA lesions or intermediates locally engaged, the mechanisms ensuring their recruitment, and the repair pathways affected. In the future, refining our experimental strategies will be instrumental to further disentangle the specific functions of NPC components in DNA metabolism from their more canonical roles in nuclear structure and homeostasis, and to pinpoint their regulation according to the physiological or pathological status of the cell, as exemplified by the recently-reported circadian control of DSB association to the nuclear envelope [64]. The potential for NPC structural variants, as observed in the same nucleus or between species [45], to recruit and handle distinct types of DNA lesions, represents an additional, exciting hypothesis that will deserve careful investigation, at the crossroads of several research areas and communities.

## Data Availability

No data were used for the research described in the article.
**Declaration of Competing Interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Acknowledgements**

Work in the authors' labs is supported by Agence Nationale pour la Recherche (ANR-18-CE12-0003 to B.P. and K.D, ANR-19-CE12-0023 to M.N.S., and ANR-23-CE12-0007 to M.N.S and B.P.), Fondation ARC pour la recherche contre le Cancer (project ARC to B.P.), Ligue Nationale contre le Cancer (comité de Paris, to B.P.) and the Université Paris Cité IdEx (ANR-18-IDEX-0001 to B.P.).

**References and recommended reading**

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

1. Raices M, D'Angelo MA: Structure, maintenance, and regulation of nuclear pore complexes: the gatekeepers of the eukaryotic genome. Cold Spring Harb Perspect Biol 2022, 14:a040691.

2. Mitrentsi I, Yilmaz D, Soutoglou E: How to maintain the genome in nuclear space. Curr Opin Cell Biol 2020, 64:58-66.

3. Stanic M, Mekhail K: Integration of DNA damage responses with dynamic spatial genome organization. Trends Genet TIG 2022, 38:290-304.

4. Palancade B, Liu X, Garcia-Rubio M, Aguilera A, Zhao X, Doye V: Nucleoporins prevent DNA damage accumulation by modulating Ulp1-dependent sumoylation processes. Mol Biol Cell 2007, 18:2912-2923.

5. Nagai S, Dubrana K, Tsai-Pflugfelder M, Davidson MB, Roberts TM, Brown GW, Varela E, Hediger F, Gasser SM, Krogan NJ: Functional targeting of DNA damage to a nuclear pore-associated SUMO-dependent ubiquitin ligase. Science 2008, 322:597-602.

6. Khadaroo B, Teixeira MT, Luciano P, Eckert-Boulet N, Germann SM, Simon MN, Gallina I, Abdallah P, Gilson E, Geli V, et al.: The DNA damage response at eroded telomeres and tethering to the nuclear pore complex. Nat Cell Biol 2009, 11:980-987.

7. Churikov D, Charifi F, Eckert-Boulet N, Silva S, Simon MN, Lisby M, Geli V: SUMO-dependent relocalization of eroded telomeres to nuclear pore complexes controls telomere recombination. Cell Rep 2016, 15:1242-1253.

8. Geli V, Lisby M: Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex. Bioessays 2015, 37:1287-1292.

9. Su XA, Dion V, Gasser SM, Freudenreich CH: Regulation of recombination at yeast nuclear pores controls repair and triplet repeat stability. Genes Dev 2015, 29:1006-1017.

10. Aguilera P, Whalen J, Minguet C, Churikov D, Freudenreich C, Simon M-N, Géli V: The nuclear pore complex prevents sister chromatid recombination during replicative senescence. Nat Commun 2020, 11:160.

11. Pinzaru AM, Kareh M, Lamm N, Lazzerini-Denchi E, Cesare AJ, Sfeir A: Replication stress conferred by POT1 dysfunction promotes telomere relocalization to the nuclear pore. Genes Dev 2020, 34:1619-1636.

This study demonstrates that telomeres under replication stress relocate to the nuclear periphery in mammalian cells, expanding a concept hitherto limited to the yeast system.

12. Horigome C, Unozawa E, Ooki T, Kobayashi T: Ribosomal RNA gene repeats associate with the nuclear pore complex for maintenance after DNA damage. PLoS Genet 2019, 15:e1008103.

13. Kramarz K, Schirmeisen K, Boucherit V, Ait Saada A, Lovo C, Palancade B, Freudenreich C, Lambert SAE: The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal. Nat Commun 2020, 11:5643.

14. Penzo A, Dubarry M, Brocas C, Zheng M, Mangione RM, Rougemaille M, Goncalves C, Lautier O, Libri D, Simon M-N, et al.: A R-loop sensing pathway mediates the relocation of transcribed genes to nuclear pore complexes. Nat Commun 2023, 14:5606.

In this report, it is shown that R-loops formed on highly-transcribed loci also act as a signal for NPC repositioning, yet independent of DNA damage or replication impairment.

15. Denoth-Lippuner A, Krzyzanowski MK, Stober C, Barral Y: Role of SAGA in the asymmetric segregation of DNA circles during yeast ageing. eLife 2014, 3:e03790.

16. Aguilera P, Dubarry M, Hardy J, Lisby M, Simon M-N, Géli V: Telomeric C-circles localize at nuclear pore complexes in Saccharomyces cerevisiae. EMBO J 2022, 41:e108736.

This study reveals that DNA circles encompassing telomeric DNA can associate with NPCs, along with eroded telomeres, possibly serving as a substrate for telomere maintenance.

17. Koche RP, Rodriguez-Fos E, Helmsauer K, Burkert M, MacArthur IC, Maag J, Chamorro R, Munoz-Perez N, Puiggrós M, Dorado Garcia H, et al.: Extrachromosomal circular DNA drives oncogenic genome remodeling in neuroblastoma. Nat Genet 2020, 52:29-34.

18. Schirmeisen K, Lambert SAE, Kramarz K: SUMO-based regulation of nuclear positioning to spatially regulate homologous recombination activities at replication stress sites. Genes 2021, 12:2010.

19. Chung I, Zhao X: DNA break-induced sumoylation is enabled by collaboration between a SUMO ligase and the ssDNA-binding complex RPA. Genes Dev 2015, 29:1593-1598.

20. Whalen JM, Dhingra N, Wei L, Zhao X, Freudenreich CH: Relocation of collapsed forks to the nuclear pore complex depends on sumoylation of DNA repair proteins and permits Rad51 association. Cell Rep 2020, 31:107635.

21. Charifi F, Churikov D, Eckert-Boulet N, Minguet C, Jourquin F, Hardy J, Lisby M, Simon M-N, Géli V: Rad52 SUMOylation functions as a molecular switch that determines a balance between the Rad51- and Rad59-dependent survivors. iScience 2021, 24:102231.

22. Caridi CP, D'Agostino C, Ryu T, Zapotoczny G, Delabaere L, Li X, Khodaverdian VY, Amaral N, Lin E, Rau AR, et al.: Nuclear F-actin and myosins drive relocalization of heterochromatic breaks. Nature 2018, 559:54-60.

23. Schrank BR, Aparicio T, Li Y, Chang W, Chait BT, Gundersen GG, Gottesman ME, Gautier J: Nuclear ARP2/3 drives DNA break clustering for homology-directed repair. Nature 2018, 559:61-66.

24. Lamm N, Read MN, Nobis M, Van Ly D, Page SG, Masamsetti VP, Timpson P, Biro M, Cesare AJ: Nuclear F-actin counteracts nuclear deformation and promotes fork repair during replication stress. Nat Cell Biol 2020, 22:1460-1470.

This report establishes a critical role for nuclear actin in handling and repositioning stressed replication forks within the nucleus in mammalian cells.

25. Chung DKC, Chan JNY, Strecker J, Zhang W, Ebrahimi-Ardebili S, Lu T, Abraham KJ, Durocher D, Mekhail K: Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process. Nat Commun 2015, 6:7742.

26. Oshidari R, Strecker J, Chung DKC, Abraham KJ, Chan JNY, Damaren CJ, Mekhail K: Nuclear microtubule filaments mediate
non-linear directional motion of chromatin and promote DNA repair. *Nat Commun* 2018, 9:2567.

27. Zhu S, Paydar M, Wang F, Li Y, Wang L, Barrette B, Bessho T, Kwok BH, Peng A: *Kinesin Kif2C* in regulation of DNA double strand break dynamics and repair. *eLife* 2020, 9:e53402.

28. Oshidari R, Huang R, Medghalchi M, Tse EYW, Ashgriz N, Lee HO, Wyatt H, Mekhail K: *DNA repair by Rad52 liquid droplets*. *Nat Commun* 2020, 11:695.

29. Shokrollahi M, Mekhail K: *Interphase microtubules in nuclear organization and genome maintenance*. *Trends Cell Biol* 2021, 31:721-731.

30. Shokrollahi M, Stanic M, Hundal A, Chan JNY, Urman D, Hakem A, Garcia RE, Hao J, Maass PG, Dickson BC, et al.: DNA double-strand break-capturing nuclear envelope tubules drive DNA repair. *bioRxiv*; 2023. doi.org/10.1101/2023.05.07.539750..

This study demonstrates that in human cells, interactions between
NPCs and damaged chromatin can occur away from the nuclear per-
iphery, involving nuclear envelope invaginations penetrating the nu-
cleus.

31. Hou C, Corces VG: Nups take leave of the nuclear envelope to regulate transcription. *Cell* 2010, 140:306-308.

32. Horigome C, Bustard DE, Marcomini I, Delgoshae N, Tsai-Pflugfelder M, Cobb JA, Gasser SM: *PolySUMOylation by Siz2* and *Mms21* triggers relocation of DNA breaks to nuclear pores through the *Slx5/Slx8* STUbL. *Genes Dev* 2016, 30:931-945.

33. Horigome C, Oma Y, Konishi T, Schmid R, Marcomini I, Hauer MH, Dion V, Harata M, Gasser SM: *SWR1* and *INO80* chromatin remodelers contribute to DNA double-strand break perinuclear anchorage site choice. *Mol Cell* 2014, 55:626-639.

34. Gaillard H, Santos-Pereira JM, Aguilera A: *The Nup84 complex coordinates the DNA damage response to warrant genome integrity*. *Nucleic Acids Res* 2019, 47:4054-4067.

35. Garcia-Benitez F, Gaillard H, Aguilera A: *Physical proximity of chromatin to nuclear pores prevents harmful R loop accumulation contributing to maintain genome stability*. *Proc Natl Acad Sci USA* 2017, 114:10942-10947.

36. Bermejo R, Capra T, Jossen R, Colosio A, Frattini C, Carotenuto W, Cocito A, Doksani Y, Klein H, Gómez-González B, et al.: *The replication checkpoint protects fork stability by releasing transcribed genes from nuclear pores*. *Cell* 2011, 146:233-246.

37. Teloni F, Michelena J, Lezaja A, Kilic S, Ambrosi C, Menon S, Dobrovolna J, Imhof R, Janscak P, Baubec T, et al.: Efficient pre-mRNA cleavage prevents replication-stress-associated genome instability. *Mol Cell* 2019, 73:670-683.e12.

38. Moudry P, Lukas C, Macurek L, Neumann B, Heriche J-K, Pepperkok R, Ellenberg J, Hodny Z, Lukas J, Bartek J: *Nucleoporin NUP153* guards genome integrity by promoting nuclear import of 53BP1. *Cell Death Differ* 2012, 19:798-807.

39. Taddei A, Hediger F, Neumann FR, Bauer C, Gasser SM: Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins. *Embo J* 2004, 23:1301-1312.

40. Meers C, Keskin H, Banyai G, Mazina O, Yang T, Gombolay AL, Mukherjee K, Kaparos EI, Newnam G, Mazin A, et al.: Genetic characterization of three distinct mechanisms supporting RNA-driven DNA repair and modification reveals major role of DNA polymerase ζ. *Mol Cell* 2020, 79:1037-1050.e5.

41. Zong D, Oberdoerffer P, Batista PJ, Nussenzweig A: RNA: a double-edged sword in genome maintenance. *Nat Rev Genet* 2020, 21:651-670.

42. Ryu T, Spatola B, Delabaere L, Bowlin K, Hopp H, Kunitake R, Karpen GH, Chiolo I: *Heterochromatic breaks move to the nuclear periphery to continue recombinational repair*. *Nat Cell Biol* 2015, 17:1401-1411.

43. Whalen JM, Freudenreich CH: Location, location, location: the role of nuclear positioning in the repair of collapsed forks and protection of genome stability. *Genes* 2020, 11:635.

44. Chen B, Ge T, Jian M, Chen L, Fang Z, He Z, Huang C, An Y, Yin S, Xiong Y, et al.: *Transmembrane nuclease NUMEN/ENDOD1*

regulates DNA repair pathway choice at the nuclear periphery.
*Nat Cell Biol* 2023, 25:1004-1016.

This study represents the first evidence of a DNA repair factor specifi-
cally compartmentalized at the nuclear envelope in human cells.

45. Fernandez-Martinez J, Rout MP: One ring to rule them all? *Structural and functional diversity in the nuclear pore complex*. *Trends Biochem Sci* 2021, 46:595-607.

46. Hurst V, Challa K, Jonas F, Forey R, Sack R, Seebacher J, Schmid CD, Barkai N, Shimada K, Gasser SM, et al.: *A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress*. *EMBO J* 2021, 40:e108439.

47. Poli J, Gasser SM, Papamichos-Chronakis M: *The INO80* remodeler in transcription, replication and repair. *Philos Trans R Soc Lond B Biol Sci* 2017, 372:20160290.

48. Niño CA, Guet D, Gay A, Brutus S, Jourquin F, Mendiratta S, Salamero J, Géli V, Dargemont C: *Posttranslational marks control architectural and functional plasticity of the nuclear pore complex basket*. *J Cell Biol* 2016, 212:167-180.

49. Folz H, Niño CA, Taranum S, Caesar S, Latta L, Waharte F, Salamero J, Schlenstedt G, Dargemont C: *SUMOylation of the nuclear pore complex basket is involved in sensing cellular stresses*. *J Cell Sci* 2019, 132:jcs224279.

50. Bensidoun P, Reiter T, Montpetit B, Zenklusen D, Oeffinger M: Nuclear mRNA metabolism drives selective basket assembly on a subset of nuclear pore complexes in budding yeast. *Mol Cell* 2022, 82:3856-3871.e6.

51. Varberg JM, Unruh JR, Bestul AJ, Khan AA, Jaspersen SL: *Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe*. *Life Sci Alliance* 2022, 5:e202201423.

52. Akey CW, Singh D, Ouch C, Echeverria I, Nudelman I, Varberg JM, Yu Z, Fang F, Shi Y, Wang J, et al.: *Comprehensive structure and functional adaptations of the yeast nuclear pore complex*. *Cell* 2022, 185:361-378.e25.

53. Schuller AP, Wojtynek M, Mankus D, Tatli M, Kronenberg-Tenga R, Regmi SG, Dip PV, Lytton-Jean AKR, Brignole EJ, Dasso M, et al.: *The cellular environment shapes the nuclear pore complex architecture*. *Nature* 2021, 598:667-671.

54. Zimmerli CE, Allegretti M, Rantos V, Goetz SK, Obarska-Kosinska A, Zagoriy I, Halavatyi A, Hummer G, Mahamid J, Kosinski J, et al.: *Nuclear pores dilate and constrict in cellulo*. *Science* 2021, 374:eabd9776.

55. Kumar S, Neal ML, Li S, Navare AT, Mast FD, Rout MP, Aitchison JD: *Nuclear pore complexes mediate subtelomeric gene silencing by regulating PCNA levels on chromatin*. *J Cell Biol* 2023, 222:e202207060.

This work provides pioneering evidence for the existence of specialized
NPCs, lacking their nuclear basket, and engaged in interactions with the
replication clamp loader RFC<sup>Ctr18</sup>, impacting PCNA recruitment onto
replicating chromatin.

56. Meinema AC, Marzelliussardottir A, Mirkovic M, Aspert T, Lee SS, Charvin G, Barral Y: *DNA circles promote yeast ageing in part through stimulating the reorganization of nuclear pore complexes*. *eLife* 2022, 11:e71196.

This study reveals that DNA circles associate with NPC variants, suggesting that DNA association could influence their structural organization.

57. Lapetina DL, Ptak C, Roesner UK, Wozniak RW: *Yeast silencing factor Sir4 and a subset of nucleoporins form a complex distinct from nuclear pore complexes*. *J Cell Biol* 2017, 216:3145-3159.

58. Breuer M, Ohkura H: *A negative loop within the nuclear pore complex controls global chromatin organization*. *Genes Dev* 2015, 29:1789-1794.

59. Onischenko E, Noor E, Fischer JS, Gillet L, Wojtynek M, Vallotton P, Weis K: *Maturation kinetics of a multiprotein complex revealed by metabolic labeling*. *Cell* 2020, 183:1785-1800.e26.
60. Otsuka S, Ellenberg J: Mechanisms of nuclear pore complex assembly – two different ways of building one molecular machine. FEBS Lett 2018, 592:475-488.

61. Gillespie PJ, Khoudoli GA, Stewart G, Swedlow JR, Blow JJ: ELYS/MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing. Curr Biol CB 2007, 17:1657-1662.

62. Richards L, Lord CL, Benton ML, Capra JA, Nordman JT: Nucleoporins facilitate ORC loading onto chromatin. Cell Rep 2022, 41:111590.

63. Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M: Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress. PLoS Genet 2008, 4:e1000240.

64. Le Bozec B, Guitton-Sert L, Collins S, Rocher V, Guillou E, Payrault C, Arnould C, Guénolé A, Aguirrebengoa M, Finoux AL, et al.: Circadian PERIOD complex regulates TC-DSB repair through anchoring to the nuclear envelope. bioRxiv 2023, https://doi.org/10.1101/2023.05.11.540338