Otud5 promotes end-joining of deprotected telomeres by promoting atm-dependent phosphorylation of kap1s824

ABSTRACT Appropriate repair of damaged DNA and the suppression of DNA damage responses at telomeres are essential to preserve genome stability. DNA damage response (DDR) signaling consists

of cascades of kinase-driven phosphorylation events, fine-tuned by proteolytic and regulatory ubiquitination. It is not fully understood how crosstalk between these two major classes of

post-translational modifications impact DNA repair at deprotected telomeres. Hence, we performed a functional genetic screen to search for ubiquitin system factors that promote KAP1S824

phosphorylation, a robust DDR marker at deprotected telomeres. We identified that the OTU family deubiquitinase (DUB) OTUD5 promotes KAP1S824 phosphorylation by facilitating ATM activation,

through stabilization of the ubiquitin ligase UBR5 that is required for DNA damage-induced ATM activity. Loss of OTUD5 impairs KAP1S824 phosphorylation, which suppresses end-joining mediated

DNA repair at deprotected telomeres and at DNA breaks in heterochromatin. Moreover, we identified an unexpected role for the heterochromatin factor KAP1 in suppressing DNA repair at

telomeres. Altogether our work reveals an important role for OTUD5 and KAP1 in relaying DDR-dependent kinase signaling to the control of DNA repair at telomeres and heterochromatin. SIMILAR

CONTENT BEING VIEWED BY OTHERS UBE2D3 FACILITATES NHEJ BY ORCHESTRATING ATM SIGNALLING THROUGH MULTI-LEVEL CONTROL OF RNF168 Article Open access 12 June 2024 REGULATION OF ALT-ASSOCIATED

HOMOLOGY-DIRECTED REPAIR BY POLYADP-RIBOSYLATION Article 12 October 2020 CHROMOSOME END PROTECTION BY RAP1-MEDIATED INHIBITION OF DNA-PK Article Open access 16 April 2025 INTRODUCTION Under

constant exposure to both environmental and endogenous sources of DNA damage, efficient repair of DNA lesions is essential to the maintenance of genome integrity1. Paradoxically, in

particular genomic contexts, such as telomeres, activation of DNA repair mechanisms instead leads to chromosomal aberrations and genomic instability2. Telomeres are specialized nucleoprotein

structures located at the natural ends of chromosomes. They consist of kilobases of TTAGGG DNA repeats and serve to counteract the gradual erosion of chromosomal ends upon each cycle of DNA

replication3,4. Telomeres are bound and protected by shelterin, a six-unit protein complex that prevents misrecognition of telomeres as DNA double-strand breaks (DSBs) and unwanted

activation of DNA repair pathways5,6,7,8. Shelterin blocks DDR signaling and DNA repair in large part through promoting the formation of a telomere-loop (t-loop) and inhibiting the

accessibility of telomeric DNA to components of the DDR machinery9. In the absence of shelterin function, deprotected telomeric DNA ends trigger DDR signaling that induces both cellular

senescence and DNA repair reactions that result in chromosome end-to-end fusions8,10. Such fusions are a major threat to chromosomal stability, particularly in cells escaping from

senescence. The signaling pathways involved in the DDR at dysfunctional telomeres share significant similarities with that of DNA DSBs. The DDR is initiated by the recognition of DSBs or

deprotected telomeres by the MRE11-RAD50-NBS1 (MRN) complex, followed by the activation of ATM, the primary kinase responsible for DDR signaling in DSB repair11,12. While the precise

mechanisms underlying ATM activation are still not fully understood, activated ATM phosphorylates several downstream targets, including p53, CHK2, H2A.X, and KAP1 (also known as TRIM28 or

TIF1B)13,14,15,16. These phosphorylation events lead to cell cycle arrest, local chromatin remodeling, and recruitment of essential repair factors, ultimately enabling the repair process. In

particular, phosphorylation of KAP1 has been shown to be functionally indispensable for the repair of DSBs, specifically at heterochromatin17,18. KAP1, in its native, SUMOylated state,

serves as a critical heterochromatin scaffolding factor that promotes transcriptional repression and chromatin condensation via its interaction with various chromatin remodelers. In the

presence of DSBs, the normally SUMOylated KAP1 is phosphorylated by ATM in a manner facilitated by 53BP1, RNF8 and MDC1, and exhibiting a pan-nuclear phosphorylation pattern18.

Phosphorylation of KAP1 triggers temporary decompaction of heterochromatin in a CHD3-dependent manner, and facilitates DNA repair in an otherwise restrictive environment19. While the DDR

signaling cascade is primarily dictated by the flow of signals in the form of protein phosphorylation, post-translational modifications by ubiquitination streamlines DDR signaling by

orchestrating protein turnover, chromatin remodeling, and recruitment of repair factors20,21,22,23. Accordingly, key roles in the DDR have been assigned to multiple ubiquitin-system factors.

However, the full complexity of how the phosphorylation and ubiquitination systems together orchestrate the DDR, and especially at telomeres, is still unknown. Here we addressed this by

performing a functional genetic screen targeted at identifying ubiquitin system factors that promote DDR activation at deprotected telomeres, using ATM-dependent phosphorylation of KAP1 at

its Ser-824 residue (KAP1S824) as the readout. Through this screen, we identified the OTU-family deubiquitinase (DUB) OTUD5 as a promoter of DDR activation at deprotected telomeres. OTUD5 is

a phospho-activated DUB that preferentially cleaves K48- and K63-linked poly- and di-ubiquitin chains in vitro24. Together with the ubiquitin ligase UBR5, OTUD5 has been linked to the

process of transcriptional suppression at damaged chromatin but has so far not been directly implicated in the regulation of DDR signaling and DNA repair at telomeres25. We find that OTUD5

promotes ATM-mediated phosphorylation of KAP1S824 in response to telomere deprotection, as well as in response to genome-wide, ionizing radiation (IR)-induced DNA damage. This is dependent

on the ability of OTUD5 to stabilize UBR5, previously implicated in ATM activation by alleviating ATMIN-dependent inhibition on ATM in response to DSBs26. In addition to its role in

promoting the signaling axis of the DDR, we found that OTUD5 promotes the efficiency of DNA repair exclusively at telomeres and heterochromatin, but not in general, non-specific chromatin

contexts. Such facilitation is independent of ATM-driven recruitment of 53BP1 and RIF1, as the localization of these NHEJ factors to deprotected telomeres is either unaffected or only

marginally compromised upon loss of OTUD5. Instead, we reveal that defective KAP1S824 phosphorylation is the crucial underpinning step that hinders DNA repair at telomeres in cells lacking

OTUD5, as ectopic expression of phosphomimic KAP1S824D is sufficient to restore telomeric NHEJ in OTUD5-depleted cells. This shows that the residual ATM activity in OTUD5-deficient cells is

inadequate to support sufficient phosphorylation of KAP1S824, a key step in DNA repair in KAP1-rich genomic environments such as the telomeres and heterochromatin, thereby translating into a

repair defect specifically in these regions. Moreover, by genetic inactivation of KAP1, we identify an unanticipated role for KAP1 in telomere protection by restricting NHEJ at deprotected

telomeres. Altogether, our work identifies OTUD5 as a key contributor to DDR signaling and DNA repair at deprotected telomeres and heterochromatin and points at KAP1 as a critical

determinant of end-joining mediated repair efficiency at dysfunctional telomeres. RESULTS A FUNCTIONAL GENETIC SCREEN IDENTIFIES UBIQUITIN SYSTEM FACTORS THAT PROMOTE DDR ACTIVATION AT

DEPROTECTED TELOMERES In mammalian cells, loss of function of TRF2, a core component of the shelterin complex, causes telomere deprotection, triggering DDR signaling at telomeres. A

well-characterized experimental system for studying DDR activities at telomeres is a _Trf2__−/−_;_p53__−/−_ mouse embryonic fibroblast (MEF) cell line that expresses the

temperature-sensitive TRF2I468A mutant (TRF2ts)27. At the permissive temperature (32 °C), TRF2ts functionally complements the loss of endogenous TRF2 and keeps telomeres in a protected

state. However, at non-permissive temperatures (37–39 °C) TRF2ts is inactivated, causing rapid and synchronous telomere deprotection and telomere-specific DDR activation (Fig. 1a). We

exploited this system to gain understanding on the requirements of ubiquitin system factors for efficient DDR activation at deprotected telomeres. We designed a fluorescence-activated cell

sorting (FACS)-based functional genetic screen in TRF2ts MEFs that allows identification of factors with a critical role in the activation of DDR signaling at deprotected telomers, so-called

activators of the telomere damage response (ATD). To circumvent complications to DDR dynamics from cells being in different cell cycle phases, we used TRF2ts MEFs modified with the

Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) system28 to isolate G1 cells for detection of DDR activation. DDR signaling at TRF2-deprotected telomeres is by at large an ATM

kinase-dependent process5. Amongst ATM substrates, pKAP1S824 is strongly induced by telomere deprotection in TRF2ts MEFs27,29. This induction is also readily detectable by FACS, where a

significant upshift in pKAP1S824 intensity could be detected after incubating TRF2ts MEFs at 39 °C for 3 h (Fig. 1b). To validate the specificity of pKAP1S824 detection by FACS, we

pretreated TRF2ts MEFs with an ATM inhibitor or short hairpin RNA (shRNA) against ATM, before subjecting the cells to telomere deprotection (Fig. 1c, d). Indeed, ablation of ATM activity

abolishes induction of pKAP1S824 by telomere deprotection. This reinforces that our FACS detection strategy specifically detects ATM-dependent KAP1S824 phosphorylation at deprotected

telomeres. To conduct the screen, we infected TRF2ts-FUCCI MEFs in triplicate with a lentiviral library of 609 shRNAs (pLKO.1-Puro) that target 123 ubiquitin system factors in mouse (Fig. 

1e). By FACS, we selected the top 5% G1 cells with lowest pKAP1S824 signal after telomere deprotection for 3 h at 39 °C (Fig. 1e, Supplementary Fig. 1a). From this population displaying

impaired pKAP1S824 induction we extracted genomic DNA, recovered shRNA sequences by PCR, and determined the enrichment of shRNAs in this population, relative to the untreated control cell

population, by next generation sequencing and MaGECK-RRA analysis30. Significantly enriched shRNA targets point at gene candidates that are required for DDR activation following telomere

deprotection. The ATD screen identified UBE2D3, UBE2M and OTUD5 as the top three ubiquitin system factors that promote KAP1S824 phosphorylation at deprotected telomeres (Fig. 1f). UBE2D3 is

a ubiquitin-conjugating E2 enzyme of the UBE2D enzyme family that we recently found to affect KAP1S824 phosphorylation in an independent study31, and hence represents an internal positive

control. UBE2M is an E2 enzyme of both the ubiquitination and neddylation system, while OTUD5, also known as DUBA, is a DUB of the OTU (ovarian tumor) deubiquitinase family. For validation,

we individually depleted UBE2D3, UBE2M or OTUD5 in TRF2ts MEFs with multiple independent shRNAs that were included in the screen and subjected them to telomere deprotection. Indeed,

depletion of all three targets significantly impaired KAP1S824 phosphorylation upon telomere deprotection (Fig. 1g, h, Supplementary Fig. 1b, c). Due to the heavy involvement of UBE2M in the

neddylation pathway (being one of only two described E2 enzymes of the neddylation system)32, this study’s focus on ubiquitination system factors, and given that E2 enzymes in general have

more cellular targets than DUBs, we chose to further investigate the role of OTUD5. OTUD5 PROMOTES ATM-DEPENDENT DDR SIGNALING For more specific and robust depletion of OTUD5 in mechanistic

studies we continued using CRISPR/Cas9-mediated gene knockout approaches33. Depletion of OTUD5 in TRF2ts MEFs with 2 independent sgRNAs (g1, g1B) recapitulated the pKAP1S824 defect observed

with shRNA-mediated knockdown of OTUD5 (Fig. 1i, j). To address whether contribution of OTUD5 to DDR activation extends beyond a telomeric context, we challenged OTUD5-depleted human U2OS

cells with ionizing radiation (IR) to induce genome-wide DSBs. Similar to its role in the response to deprotected telomeres, we found that OTUD5 is also required for robust KAP1S824

phosphorylation in response to genome-wide DNA damage, as IR-induced KAP1S824 phosphorylation was significantly reduced in OTUD5-depleted cells (Fig. 2a, b). We then investigated the

mechanism underpinning the requirement of OTUD5 for robust KAP1S824 phosphorylation. We observed that in addition to pKAP1S824, phosphorylation of other ATM kinase substrates, including

pCHK2T68 and γH2A.X, were also reduced upon loss of OTUD5 (Fig. 2c), suggesting that ATM activity is compromised in absence of OTUD5. In line with this, autophosphorylation of ATM on

Ser-1981 (pATMS1981), indicative of active ATM kinase, was significantly reduced in OTUD5-depleted U2OS cells and TRF2ts MEFs, while total ATM levels remained unaltered (Fig. 2c, d,

Supplementary Fig. 2a). This suggests that OTUD5 promotes ATM activation, and thereby supports phosphorylation of its downstream targets, including KAP1, in the DDR signaling cascade. The

Ser-177 and Cys-224 residues of OTUD5 are critical to its phospho-activation and catalytic activity, respectively24. To assess the importance of these functional aspects of OTUD5 in

promoting KAP1S824 phosphorylation, we performed complementation experiments with ectopic expression of OTUD5WT, OTUD5S177A or OTUD5C224S. While expression of sgRNA-resistant,

codon-optimized OTUD5WT cDNA restored KAP1S824 phosphorylation in OTUD5-depleted cells, both OTUD5S177A and OTUD5C224S expression failed to do so (Fig. 2e, f, Supplementary Fig. 2b). Thus,

both the DUB activity and the phospho-activation of OTUD5 are required for ATM-dependent DDR signaling. OTUD5 PROMOTES END-JOINING MEDIATED FUSION OF DYSFUNCTIONAL TELOMERES Loss of

shelterin factor TRF2 results in processing of the deprotected telomeres by ligase IV- and Ku-dependent classical non-homologous end-joining (NHEJ), giving rise to chromosome-type telomere

fusions (abbreviated here as chromosome fusions)34,35. These fusions are generated primarily in G1 phase and appear in metaphase spreads as end-to-end fusions of both sister chromatids of

one chromosome to the sister chromatids of another chromosome. As OTUD5 is required for robust DDR activation at deprotected telomeres, we assessed whether telomeric NHEJ also requires

OTUD5. Indeed, depletion of OTUD5 significantly reduced the frequency of NHEJ-mediated chromosome end-to-end fusions upon telomere deprotection in TRF2ts MEFs, albeit to a lesser extent than

direct inhibition of ATM with an ATM inhibitor (KU-55963) (Fig. 3a, b). This indicates that OTUD5 is required for efficient NHEJ at telomeres. To address whether OTUD5 is required for NHEJ

at deprotected telomeres similarly in human cells, we depleted OTUD5 in HeLa cells harboring a doxycycline-inducible shRNA against TRF2, where telomere deprotection can be induced by

doxycycline treatment36. Consistent with our results in TRF2ts MEFs, ablation of OTUD5 in these HeLa cells significantly reduced both telomere fusions and KAP1S824 phosphorylation upon

doxycycline-induced TRF2 loss (Fig. 3c, Supplementary Fig. 3a, b). Furthermore, the defect in telomeric NHEJ observed in OTUD5-depleted cells is not attributable to aberrant cell cycle

progression, as OTUD5 depletion did not significantly perturb cell cycle distribution under unchallenged or telomere deprotection conditions (Supplementary Figs. 3c and 4a). Besides via

classical NHEJ, deprotected telomeres can also be fused by microhomology mediated end-joining (MMEJ). This is apparent in the absence of the core NHEJ factor Ku70 and, in particular, when

not only the function of TRF2 is lost but also that of TRF110. To assess the impact of OTUD5 on telomeric MMEJ, we depleted OTUD5 in

_Trf1__F/F__;Trf2__F/F__;Ku70__−/−__;p53__−/−__;Cre-ER__T2_ MEFs, and induced telomere deprotection by Cre-mediated deletion of TRF1 and TRF2. Although to a lesser extent than NHEJ-mediated

fusions, also MMEJ-mediated chromosome fusions were significantly reduced in OTUD5-deficient cells (Fig. 3d, e, Supplementary Fig. 3d, e). Taken together, we conclude that OTUD5 is required

both for efficient NHEJ and for efficient MMEJ at deprotected telomeres. Having established that OTUD5 promotes DNA repair by NHEJ and MMEJ at telomeres, we assessed if the role of OTUD5 in

DNA repair extends beyond a telomeric context, especially since we found OTUD5 to promote both telomere-specific and genome-wide DDR activation in mouse and human cells. To determine the

requirement of OTUD5 for end-joining mediated DNA repair in a genome-wide context, we assessed its impact on the NHEJ-dependent integration of a linearized plasmid, randomly into the genome

of U2OS cells37. Unlike loss of MAD2L2, a well-established factor that promotes NHEJ both at telomeres and genome-wide29, loss of OTUD5 did not significantly compromise NHEJ in this assay

(Fig. 3f, g). In addition, we assessed the contribution of OTUD5 to NHEJ outside of a telomeric context in HEK293T cells harboring a NHEJ-HR dual reporter system, in which the efficiency of

NHEJ can be measured by FACS38. Again, loss of OTUD5 did not significantly reduce NHEJ efficiency, in agreement with the results of the random plasmid integration assay (Fig. 3h,

Supplementary Figs. 3f and 4b). Altogether, this indicates that OTUD5 is not required for NHEJ in a general, nonspecific chromatin context, but promotes end-joining mediated DNA repair

pathways (both NHEJ and MMEJ), in a context-specific manner, at telomeres. OTUD5 PROMOTES ATM-DEPENDENT DDR ACTIVATION BY REGULATING THE UBR5-ATMIN AXIS After establishing a role for OTUD5

in promoting ATM-dependent DDR and DNA repair at deprotected telomeres, we sought to further understand the underlying regulatory circuit. OTUD5 was previously reported to play a role in

transcriptional repression at damaged chromatin by stabilizing the E3 ubiquitin ligase UBR525. Independently, UBR5 has been reported to promote ATM activation by inhibiting ATMIN, a negative

regulator of DSB-induced ATM activity, through non-degradative ubiquitination26. These previously established links prompted us to hypothesize that OTUD5 promotes ATM-dependent DDR and

telomeric DNA repair in a UBR5 and ATMIN-dependent manner. To test our hypothesis, we first assessed whether there is any detectable interaction between OTUD5, UBR5, ATMIN and ATM. We

ectopically expressed GFP-tagged OTUD5 in HEK293T cells and performed co-immunoprecipitation. Despite our expectation that OTUD5 interacts directly only with UBR5 and interacts transiently

and indirectly with ATMIN and/or ATM, thereby posing a challenge for detection, we readily detected an interaction of OTUD5 with all three endogenous proteins (UBR5, ATMIN and ATM) (Fig. 

4a). Next, we assessed the potential functional involvement of UBR5 in OTUD5-promoted KAP1S824 phosphorylation, by depleting OTUD5, UBR5, or both in U2OS cells. In line with previous

findings in the context of transcriptional repression, depletion of OTUD5 led to reduced levels of endogenous UBR5 protein, both in the absence and presence of IR (Fig. 4b). Upon IR, U2OS

cells lacking either OTUD5 or UBR5 displayed reduced pKAP1S824 (Fig. 4b), revealing that not only OTUD5, but also UBR5 is important for efficient KAP1S824 phosphorylation upon DNA damage.

Furthermore, depletion of OTUD5 in UBR5-deficient cells did not further aggravate the pKAP1S824 defect of these cells, indicating that OTUD5 and UBR5 act in an epistatic manner to promote

KAP1S824 phosphorylation, by OTUD5 acting upstream of UBR5 to stabilize it. In further support of this, depletion of UBR5 with independent sgRNAs in TRF2ts MEFs recapitulated the KAP1S824

phosphorylation defect displayed by OTUD5-depleted TRF2ts MEFs (Fig. 4c). Moreover, loss of UBR5 also significantly reduced telomere fusions upon telomere deprotection (Fig. 4d, e,

Supplementary Fig. 5a). Of note, we observed that protein abundance of OTUD5 is elevated in UBR5-deficient cells (Fig. 4b, c). This could indicate some form of internal homeostasis in

ubiquitination-mediated regulation of protein stability between OTUD5 and UBR5. UBR5 is known to promote DSB-induced DDR by counteracting ATMIN-mediated inhibition on ATM26. If OTUD5 indeed

promotes ATM-dependent DDR and telomeric DNA repair through the UBR5-ATMIN axis, ablation of ATMIN is expected to reverse the DDR defects exhibited by OTUD5-depleted cells. To test this, we

inhibited ATMIN expression in OTUD5-depleted TRF2ts MEFs by using 3 independent shRNAs (#5, #24, #84) (Supplementary Fig. 5b). In line with our hypothesis, depletion of ATMIN strongly and

significantly rescued both the KAP1S824 phosphorylation defect and the telomere fusion defect of OTUD5-depleted cells (Fig. 4f, g, Supplementary Fig. 5c). Furthermore, depletion of OTUD5 did

not further aggravate the telomere fusion defect of ATM inhibitor-treated cells (Fig. 4h, Supplementary Fig. 5d). Taken together, these results indicate that the functional role of OTUD5 in

DDR activation and end-joining at dysfunctional telomeres relies on its control of the UBR5-ATMIN-ATM regulatory axis. OTUD5 PROMOTES TELOMERIC END-JOINING BY PROMOTING KAP1S824

PHOSPHORYLATION To further investigate the mechanism underlying the control of end-joining mediated repair by OTUD5, we first considered the possibility that OTUD5 (in part) facilitates

telomeric NHEJ by promoting the stability of the core NHEJ factor Ku80. This, since it has been reported that OTUD5 counteracts proteasomal degradation of Ku80 in certain cell lines39. To

address this, we assessed the protein levels of Ku80 in OTUD5-depleted TRF2ts MEFs and U2OS cells. However, contrary to the previously reported finding in different cell lines, loss of OTUD5

did not lead to a reduction in Ku80 protein levels in MEFs or U2OS cells, regardless of DNA damage induction by IR or telomere deprotection (Supplementary Fig. 6a, b). Thus, OTUD5 does not

appear as a critical regulator of Ku stability in the cell types of our study, in line with that multiple E3s and DUBs have been implicated in controlling Ku stability39,40. Moreover,

destabilization of Ku80 is also unlikely to explain the telomere end-joining defects in OTUD5-depleted cells, as OTUD5-depleted cells also display reduced telomere MMEJ in cells deficient

for TRF1, TRF2 and Ku70, where Ku80 is both dispensable and absent. Additionally, destabilization of a pivotal NHEJ factor such as Ku80 would impair global NHEJ significantly, regardless of

genomic context, which is not what we observed for OTUD5-deficient cells. Hence, we conclude that the impact of OTUD5 on telomeric end-joining in the cells and conditions of our assays, is

not mediated via changes in the level of Ku70/80. At deprotected telomeres, ATM-dependent recruitment of 53BP1 is a critical determinant of NHEJ efficiency41. As OTUD5-depleted cells display

impaired ATM activation and ATM-dependent DDR, it reasonably follows that 53BP1 recruitment could be compromised in these cells, giving rise to defective telomere NHEJ. To address this, we

measured co-localization of 53BP1 foci with telomeres by IF-FISH upon TRF2 inactivation in TRF2ts cells. While treatment with an ATM inhibitor completely abolished the appearance of 53BP1

telomere dysfunction induced foci (TIFs), OTUD5 depletion did not hinder 53BP1 accumulation at deprotected telomeres (Fig. 5a, Supplementary Fig. 6d). This indicates that the residual ATM

activity in OTUD5-depleted cells is sufficient to support efficient 53BP1 recruitment to dysfunctional telomeres, and that the telomere end-joining defect in OTUD5-depleted cells is not

caused by failed 53BP1 recruitment. We next assessed the recruitment of RIF1 in response to telomere deprotection in OTUD5-depleted cells, by immunofluorescence. RIF1 is a key NHEJ factor

that is recruited to DSBs and dysfunctional telomeres in an ATM and 53BP1-dependent manner and promotes end-joining by restricting DNA end resection42,43,44,45. In line with previous

findings, ATM inhibitor treatment fully abolished RIF1 foci formation upon telomere deprotection. On the other hand, RIF1 foci formation was only mildly impaired in OTUD5-depleted cells,

reaching statistical significance for only one of the two sgRNAs (sgOTUD5 g1B) used (Fig. 5b, Supplementary Fig. 6e), thereby recapitulating only a fraction of the effect of ATM inhibitor

treatment. Similar to RIF1 foci, formation of γH2A.X foci upon telomere deprotection was marginally compromised in cells treated with sgOTUD5 g1B and not significantly changed with the other

sgRNA (Supplementary Fig. 6c, f). Despite that ATM activity was not compromised to an extent that abolishes 53BP1 and RIF1 recruitment, the impairment in ATM-dependent DDR signaling,

especially that of KAP1S824 phosphorylation, is substantial. Hence, we next considered the possibility that KAP1, the central factor of our screening approach, is itself of key importance

for how OTUD5 promotes efficient DNA repair activity at telomeres. KAP1 is known to be a heterochromatin scaffold protein that orchestrates the formation and maintenance of

heterochromatin46,47. At heterochromatic DSBs, KAP1 imposes a strong hindrance on DNA repair by maintenance of a compact, non-permissive chromatin state that deters DNA repair

factors17,18,19,48. ATM-dependent phosphorylation of KAP1 is essential to potentiate local chromatin decompaction to enable DNA repair, and ectopic overexpression of a KAP1S824D phosphomimic

mutant is able to fully restore DNA repair efficiency at heterochromatic DSBs that is crippled by ATM dysfunction. Although telomeres are not entirely heterochromatic by consent49,50, they

are enriched in heterochromatin-binding factors KAP1 and HP1 in both mouse and human cells51,52,53, and telomere deprotection strongly triggers KAP1S824 phosphorylation. To address the

significance of KAP1 in telomere NHEJ, we first individually, or in combination, depleted KAP1 and OTUD5 in TRF2ts MEFs and assessed telomere fusions upon telomere deprotection. Strikingly,

not only did depletion of KAP1 drastically enhance telomere fusions, but it also completely abolished the telomere fusion defect of OTUD5-deficient cells, indicating that OTUD5 facilitates

telomeric NHEJ in a KAP1-dependent manner (Fig. 5c, d, Supplementary Fig. 7a). To address the functional significance of KAP1S824 phosphorylation at deprotected telomeres, we ectopically

expressed the phosphomimic KAP1S824D mutant and assessed if this rescues the telomeric NHEJ defect of OTUD5-depleted cells. Indeed, overexpression of KAP1S824D restored telomere fusion

efficiency in OTUD5-depleted cells, while ectopic expression of wild-type (KAP1WT) failed to do so (Fig. 5e, f, Supplementary Fig. 7b, c). From this, we conclude that KAP1 and KAP1S824

phosphorylation play a prominent role in telomere NHEJ, and that OTUD5 facilitates telomere NHEJ by promoting ATM-dependent KAP1S824 phosphorylation. Moreover, ectopic expression of

phosphomimic KAP1S824D also rescued the telomere MMEJ defect in OTUD5-depleted _Trf1__F/F__;Trf2__F/F__;Ku70__−/−__;p53__−/−__;Cre-ER__T2_ MEFs (Fig. 5g, Supplementary Fig. 7d, e). Together,

these results indicate that the phosphorylation status of KAP1S824 is an important functional determinant for end-joining efficiency (both NHEJ and MMEJ) at dysfunctional telomeres and is

the critical node through which OTUD5 facilitates telomeric DNA repair. It has been reported that at DSBs in heterochromatic regions, where KAP1 is enriched, the phosphorylation of KAP1

promotes DNA repair by facilitating the displacement of the nucleosome remodeler CHD3, thereby triggering transient chromatin decompaction19. To test whether KAP1 phosphorylation promotes

telomeric DNA repair in a similar manner as it does at DSBs in heterochromatin, we quantified telomere fusions at deprotected telomeres in TRF2ts MEFs depleted of CHD3. However, unlike

ablation of KAP1, we did not observe an increase in telomere fusions in the absence of CHD3 (Supplementary Fig. 8a, b). Besides indicating that CHD3 is irrelevant to NHEJ of TRF2-deprotected

telomeres, this suggests either that KAP1 functions via a different, potentially redundant, chromatin remodeler at telomeres, or that the phosphorylation of KAP1 promotes NHEJ at telomeres

through a mechanism independent of CHD3 or chromatin decompaction. OTUD5 IS REQUIRED FOR THE EFFICIENT REPAIR OF IR-INDUCED DNA DAMAGE IN HETEROCHROMATIN Given the role of OTUD5 in DNA

repair at dysfunctional telomeres by promoting ATM-dependent KAP1S824 phosphorylation, and the established importance of KAP1S824 phosphorylation for repair of heterochromatic

DSBs17,18,19,48, we investigated whether OTUD5 also facilitates DSB repair at heterochromatin. Hereto we tracked the rate of resolution of IR-induced γH2A.X foci associated with

heterochromatic chromocenters in NIH3T3 cells17. After induction of DSBs by IR, over 90% of induced γH2A.X foci associated with chromocenters were resolved within 24 h (Fig. 6a–d,

Supplementary Fig. 8c). In contrast, and as demonstrated before17, cells pre-treated with ATM inhibitor were inefficient in resolving heterochromatin-associated γH2A.X foci, retaining 70% of

induced foci after 24 h of recovery. OTUD5-depleted cells also showed reduced resolution of γH2A.X foci, with ~40% of heterochromatin-associated foci remaining unresolved at 24 h after IR,

indicating inefficient repair of DSBs at heterochromatic regions. Thus, in addition to telomeres, OTUD5 also plays a role in facilitating DNA repair at heterochromatic regions, in line with

the here uncovered role of OTUD5 in regulating KAP1. DISCUSSION Ubiquitin system factors play a major role in orchestrating responses to DNA damage20. The DUB OTUD5 was previously shown to

suppress transcription at DNA breaks25. Using a novel screening approach, we now revealed OTUD5 as a prominent promotor of ATM activation and ATM-dependent KAP1 phosphorylation, in response

to both telomere deprotection and genome-wide DSBs. Moreover, we found that OTUD5 promotes DNA repair at deprotected telomeres and at heterochromatin. The role of OTUD5 in transcriptional

suppression has been ascribed to its ability to stabilize UBR5. Indeed, we confirm destabilization of UBR5 upon loss of OTUD5. As independently, UBR5 was found to inhibit the interaction

between ATM and ATMIN, a negative regulator of canonical DSB-induced ATM activation26, we hypothesized that OTUD5 may promote ATM-dependent DDR signaling via stabilization of UBR5. Indeed,

we found OTUD5 to promote ATM-dependent DDR signaling in a manner that is epistatic with UBR5, dependent on its catalytic activity and involving ATMIN, supporting that OTUD5 acts via UBR5.

Early biochemical studies performed on OTUD5 suggested that phosphorylation at residue Ser-177 is vital for its DUB activity in vitro24. Consistent with this, we showed that the

phospho-activation of OTUD5 is indispensable to its function in promoting DDR signaling, as phospho-dead OTUD5S177A failed to restore the pKAP1S824 level in OTUD5-depleted cells. This

demonstrates the biological significance of phospho-regulation to the functions of OTUD5 in vivo. To date, little is known about the kinases that phosphorylate OTUD5, apart from recent in

vivo evidence that mTOR kinase promotes OTUD5 stability and activity via direct phosphorylation of multiple sites on OTUD5. This in turns activates mTORC1/2, thereby constituting positive

feedback signaling54,55. It is conceivable that also in the DDR, an upstream kinase phosphorylates OTUD5 to amplify DDR signaling through a similar positive feedback mechanism. Of note,

OTUD5 contains multiple conserved SQ sites (S425, S497, S549) roughly within an SCD domain (≥3 S/TQ sites within 100 amino acids), which are features of ATM/ATR kinase substrates56,57. This

suggests the possibility that OTUD5 is directly phosphorylated by ATM to function as an amplifier of ATM-dependent DDR signaling in a potential positive-feedback circuit, resembling its role

in mTOR signaling. In the context of DNA repair, OTUD5 has been suggested to promote NHEJ by counteracting the degradation of Ku80, a core NHEJ factor, in certain cell lines39. Contrary to

that work, we find in MEFs and U2OS cells that Ku80 stability is not compromised in the absence of OTUD5. This indicates that in these cells OTUD5 is not a critical regulator of Ku stability

and that other DUBs, such as potentially UCHL3, are dominantly responsible for stabilizing Ku40. In line with this, we did not observe a significant defect in genome-wide NHEJ in

OTUD5-depleted cells, which would be expected from Ku70/80-deficient cells. Instead, we propose that OTUD5 facilitates DNA repair exclusively in genomic regions that are particularly

sensitive to ATM activity, such as telomeres and heterochromatin5,17, by promoting full DDR-induced ATM activity via the UBR5-ATMIN axis. Despite impaired ATM activation, recruitment of

53BP1 (and most of RIF1) to deprotected telomeres, an ATM-dependent process5, is unaffected in OTUD5-depleted cells. We reason that the loss of OTUD5 is insufficient to abolish all aspects

of ATM function, and that the residual ATM activity is sufficient to support 53BP1 (and RIF1) recruitment. This indicates also that the cause of the end-joining defect in OTUD5-deficient

cells lies downstream of the end-joining promoting activity of 53BP1. We demonstrate here that this is at the level of KAP1. We show that pKAP1S824, amongst other ATM targets, is an

especially sensitive and functionally important substrate that dictates NHEJ efficiency at telomeres. The sensitivity of KAP1 phosphorylation to disturbance in ATM activity is in line with

previous observations stating that KAP1 phosphorylation requires localized, concentrated ATM activity18, which may not be achieved in OTUD5-deficient cells. This localized, concentrated ATM

activity has been ascribed to hyper-accumulation of the MRN complex, mediated via the C-terminal BRCT-domain of 53BP118. Although 53BP1 recruitment seemed unperturbed, the reduced RIF1 foci

in one of the OTUD5 KO cell lines suggest incomplete phosphorylation of the 53BP1 N-terminus. It remains possible that incomplete ATM-dependent 53BP1 phosphorylation contributes to the

reduced KAP1 phosphorylation in OTUD5 deficient cells. Furthermore, telomere deprotection via TRF2 loss/inactivation strictly triggers ATM activation as ATR activation remains inhibited via

shelterin factor POT15. This precludes phosphorylation of ATM substrates via the ATR kinase, which has been shown to compensate for loss of ATM in global NHEJ58. This lack of compensatory

KAP1 phosphorylation via ATR adds to the exquisite sensitivity of telomeric NHEJ to disturbances in ATM activation. The need for robust KAP1 phosphorylation at deprotected telomeres is

evident in the impaired end-joining observed in OTUD5-deficient cells, where KAP1 phosphorylation is lacking and end-joining at deprotected telomeres is restored upon ectopic overexpression

of phosphomimic KAP1. The significance of KAP1S824 phosphorylation in telomeric DNA repair shares similarities with its previously reported function in DNA repair at heterochromatin16,17.

Different from euchromatin, a substantial challenge for DNA repair at heterochromatin is its compact, sterically non-permissive conformation. Without chromatin relaxation, it is difficult to

repair DNA lesions at heterochromatin, as access of DNA repair factors is restricted. Among ATM substrates, KAP1 is central to heterochromatic DNA repair as KAP1S824 phosphorylation is

necessary for the eviction of CHD3 and other heterochromatin factors from heterochromatin to achieve a relaxed conformation that is beneficial to DNA repair19. Ablation of KAP1 or CHD3, or

complementation with a phosphomimic KAP1S824D, is sufficient to bypass the requirement of ATM, demonstrating a pivotal function for KAP1S824 phosphorylation in facilitating heterochromatic

DNA repair, by promoting local chromatin decompaction. Our data here reveal that DNA repair at telomeres phenotypically resembles that of heterochromatin _prima facie_, in the sense that

KAP1S824 phosphorylation is required for efficient DNA repair in both genomic contexts. However, intriguingly, it has been reported that deprotected telomeres do not require, nor undergo,

chromatin decompaction for DNA repair to take place59,60, suggesting that KAP1S824 phosphorylation is unlikely to promote telomere end-joining through decompaction of telomeric chromatin. In

line with this notion, contrary to KAP1 depletion, loss of CHD3 did not elevate telomere fusion. This indicates that despite the mutual requirement of ample KAP1 phosphorylation for DNA

repair at heterochromatin and deprotected telomeres, the underlying mechanism is likely different for the two genomic contexts. Thus, the mechanism underlying the requirement of KAP1

phosphorylation at deprotected telomeres, is not immediately clear and requires further exploration in future studies. One interesting possibility to investigate relates to the ability of

both KAP1 and shelterin components TRF1 and TRF2 to interact with nuclear lamina components61,62,63,64. The interaction of KAP1 with nuclear lamina components has been postulated to

contribute to the transcriptional silencing of KAP1-associated heterochromatin, by tethering heterochromatin to the nuclear lamina62,65. In a similar fashion, KAP1 may tether telomeric DNA

to the nuclear lamina, in a way, involving transcriptional control or not, that negatively impacts telomeric DNA repair and is modulated by KAP1S824 phosphorylation. In addition, the role of

KAP1 in transcriptional silencing may also independently from nuclear lamina interactions affect DNA repair, given the increasing implications of RNA in DNA repair66. Alternatively, it is

possible that KAP1 phosphorylation serves to promote mobility of the damaged ends, as both deprotected telomeres and heterochromatic DSBs have been shown to have an increased mobility within

the nucleus. This increased mobility has been proposed to increase the chance of two free DNA ends reconnecting41,67. In addition to the facilitation of telomeric DNA repair by KAP1S824

phosphorylation, our data also indicates an unexpected protective role of KAP1 at dysfunctional telomeres by inhibiting NHEJ, as direct ablation of KAP1 considerably increased the frequency

of telomere fusion in the absence of TRF2 function. KAP1 and HP1 are heterochromatin scaffold proteins that are enriched at heterochromatic regions and serve as a recruitment hub for histone

methyltransferases, deacetylases, and chromatin remodelers that act in orchestration to promote the formation and maintenance of heterochromatic chromatin state. Despite the knowledge that

KAP1 and HP1 are enriched in the telomeric proteome in both human and mouse cells51,53, little is known about their functional roles at telomeres. Intriguingly, human telomeres were reported

to not be enriched in heterochromatic histone signatures like H3K9me3 or H3K27me349. Thus, it is enigmatic why these heterochromatin factors are present despite that there is no

heterochromatin to maintain. Our data on KAP1 implies the involvement of heterochromatin factors in the maintenance and protection of telomeres against unwarranted DNA repair. This provides

a plausible functional explanation for the presence of heterochromatin factors at telomeres. It is worth exploring through what mechanisms KAP1, and potentially HP1, inhibit telomeric DNA

repair, especially since KAP1 is frequently upregulated in cancers, with correlation to enhanced invasiveness and poor survival in patients68,69,70. Further understanding on the link between

KAP1 and telomeres may potentiate advancement of our understanding on telomere biology in cancers. Altogether, our work identifies an important role for OTUD5 in promoting DDR and

particularly KAP1S824 phosphorylation through an OTUD5-UBR5-ATMIN-ATM axis. Loss of OTUD5 confers a DNA repair defect most prevalent in regions enriched in KAP1, both at deprotected

telomeres and heterochromatic DSBs. In addition, our work points at an unanticipated role of KAP1 in suppressing toxic end-joining activity at dysfunctional telomeres, the mechanistic basis

of which is interesting to explore in future studies. METHODS CELL CULTURE HEK293T, U2OS and NIH3T3 cells were obtained from ATCC. _Trf2__−/−__;p53__−/−_;TRF2ts MEFs (TRF2ts MEFs) were

generated from _Trf2__flox/-_;_p53__−/−_ MEFs as previously described71. _Trf2__flox/-__;p53__−/−_ MEFs and _Trf1__F/F__;Trf2__F/F__;Ku70__−/−__;p53__−/−__;Cre-ER__T2_ MEFs were obtained

from T. de Lange10. HEK293T cells harboring the DSB-Spectrum_V1 reporter system were a gift from H. van Attikum38. HeLa cells with doxycycline-inducible shTRF2 were a gift from J. Lingner36.

All cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS, Sigma), 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, and 2 mM

l-Glutamine (Gibco, Life Technologies), at 37 °C in a humidified atmosphere with 5% CO2, except for TRF2ts MEFs, which were cultured at 32 °C. All cell lines were routinely tested for

mycoplasma contamination and scored negatively. FACS-BASED DETECTION OF PKAP1S824 TRF2ts MEFs were trypsinized and resuspended in ice cold PBS/10% FCS, centrifuged at 268 × _g_ for 5 min at

4 °C. Cells pellets were resuspended in 0.5 ml ice-cold PBS and fixed while vortexing, by adding dropwise 4.5 ml of ice-cold 100% methanol. Cells were then incubated overnight at −20 °C

before starting the staining. After adding 5 ml of PBS, fixed cells were centrifuged for 5 min at 420 × _g_. Cell pellets were resuspended in 500 µl PBS/0.5% BSA (PBA) and cells were

permeabilized with 0.1% Triton X-100/PBA on ice for 15 min, followed by 3 washes with PBA and incubation with anti-pKAP1S824 antibody (1:250; Bethyl A300-767A) for 3 h at room temperature.

After 3 washes with PBA cells were incubated with anti-rabbit Alexa 647 (1:5000, Invitrogen A21246) for 1 h at room temperature. Cells were washed 3 times in PBA and finally resuspended in

PBA. FACS was performed with the LSRFortessa Cell Analyzer (BD). FACS data were analyzed by FlowJo 10.4.2 software. ATD SCREEN For the ATD screen, a tailored lentiviral shRNA library

targeting 123 ubiquitin system factors was assembled from the Mission shRNA library (Sigma). A full list of shRNAs is included in Supplementary Data 2. Infected cells were selected by

puromycin, split into triplicates that were each cultured at 39 °C for 3 h and stained for pKAP1S824 as described above. The total G1 cell population was FACS-sorted for the 5% cells with

the lowest pKAP1 signal, from which the shRNA enrichment was determined relative to the untreated control cell population. Genomic DNA (gDNA) was extracted from the sorted cells using the

DNeasy Blood & Tissue Kit (Qiagen). A 2-step PCR recovery of shRNA sequences from gDNA was performed using the Phusion polymerase kit (NEB): PCR 1: Thermocycling conditions: (1) 98 °C

for 30 s; (2) 20 cycles of 98 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; (3) 72 °C for 5 min; (4) hold at 4 °C. Using PCR products from PCR 1 as template for PCR 2: (1) 98 °C for 30 s;

(2) 15 cycles of 98 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min; (3) 72 °C for 5 min; (4) hold at 4 °C. For primers used in PCR 1 and 2, see Supplementary Data 3. PCR products from PCR 2

were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega). Purified PCR products were subsequently sequenced using Illumina Hiseq 2500. shRNA enrichment and gene ranking were

done using MAGeCK-RRA analysis30. LENTIVIRAL EXPRESSION CONSTRUCTS AND LENTIVIRAL PACKAGING Expression constructs of OTUD5 and KAP1 were used for complementation/rescue experiments. For

OTUD5, full-length cDNA of human OTUD5, codon-optimized, was cloned into pCDH-Puro lentiviral expression vector by NheI-EcoRI restriction cloning. Sequence of the codon-optimized cDNA of

OTUD5 is included in Supplementary Fig. 5. For KAP1, the expression construct in pLX304-BLAST vector was obtained from the CCSB-Broad Lentiviral Expression Library72. Lentiviral particles

were packaged as described previously73, by co-transfecting HEK293T cell with the lentiviral transfer constructs and lentivirus packaging vectors pRRE, pRSV-Rev, pMD2.G. Medium was refreshed

once at 16 h post transfection. Lentivirus-containing supernatant was collected at 24–36 h after medium was refreshed. SITE-DIRECTED MUTAGENESIS Mutants of OTUD5 (S177A and C224S) and KAP1

(S824D) were generated by site-directed mutagenesis reactions on pCDH-OTUD5 and pLX304-KAP1 respectively, using the QuikChange II XL directed mutagenesis kit (200521, Agilent) according to

manufacturer’s instructions. Primers containing the desired mutations were designed using the web based QuikChange Primer Design Program (www.agilent.com/genomics/qcpd). PCR’s cycling

parameters were as follows: (1) 95 °C for 1 min; (2) 18 cycles of 95 °C for 50 s, 62 °C for 50 s, 68 °C for 12 min; (3) 68 °C for 7 min; (4) hold at 4 °C. The PCR product was treated with 20

U of DpnI enzyme for 2 h at 37 °C to digest the parental/template DNA. The DpnI-treated PCR mix was transformed into Stbl3 _Escherichia coli_ (C737303, Thermo Fisher Scientific) following

manufacturer’s instructions. Transformed cultures were incubated overnight at 37 °C. Mutations were confirmed by Sanger sequencing. CRISPR/CAS9 GENE EDITING For CRISPR/Cas9-mediated gene

knockout, sgRNA sequences against OTUD5, UBR5, and KAP1 were designed using the Broad Institute’s GPP sgRNA Designer

(https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design). sgRNA sequences were cloned into a LentiCRISPRv2 plasmid according to standard protocols33. Lentiviral particles

are packaged as described above73. The sgRNA sequences used are included in Supplementary Data 4. LentiCRISPRv2 was a gift from Feng Zhang (Addgene plasmid #52961)72. RNA INTERFERENCE For

RNA interference by shRNAs, TRF2ts MEFs were transduced with pLKO.1-puro shRNA lentiviruses from Mission library clones (Sigma). shRNA sequences used for the knockdown of UBE2D3, UBE2M and

OTUD5, and their respective Mission library numbers, are included in Supplementary Data 4. METAPHASE CHROMOSOME ANALYSIS FOR TELOMERE FUSIONS Cell collection, preparation of metaphase

spreads and telomere FISH with a FITC-OO-(CCCTAA)3 peptide nucleic acid custom probe (Biosynthesis) or Alexa488-labeled C-rich Telomere Probe (Eurogentec) for metaphase chromosome analysis

was done as described27. Digital images of metaphases were captured using the Metafer4/MSearch automated metaphase finder system (MetaSystems) equipped with an AxioImager Z2 microscope (Carl

Zeiss). After scanning metaphase preparations at x10 magnification, high-resolution images of metaphases were acquired using a ‘Plan-Apochromat’ ×63/1.40 oil objective. Chromosome fusions

were quantified from >1500 chromosomes per experimental condition. CO-IMMUNOPRECIPITATION HEK293T cells were transfected with 10 µg of pLVU-GFP-OTUD5 using polyethylenimine. At 16 h post

transfection, medium was refreshed. At 48 h post transfection HEK293T cells were lysed in 500 µL of Co-IP Lysis Buffer: 20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% Nonidet P-40, 5% glycerol, 1 mM

EDTA, protease inhibitors (Roche). To complete lysis, cells were incubated for 30 min at 4 °C in a rotating wheel. Lysates were centrifuged for 10 min at 13,523 × _g_ and supernatants were

collected. Protein concentration was quantified using Pierce BCA assay (Thermo Fisher Scientific) and lysate containing 2 mg of protein was incubated with 15 µL of washed GFP-Trap® magnetic

agarose beads (ChromoTek) overnight at 4 °C in a rotating wheel. Next, beads were washed 3× for 5 min with lysis buffer. For western blot, protein was eluted from beads with 40 µL of 2× SDS

sample buffer and incubating at 95 °C for 5 min. IMMUNOBLOTTING Immunoblotting was done according to standard protocols and as described before29. Briefly, cells were washed with PBS and

collected by scraping in 2× SDS sample buffer followed by boiling at 95 °C. The lysates obtained were sonicated at 30% amplitude for 10 s. Protein concentrations were measured using Pierce

BCA Protein Assay (Thermo Fisher Scientific) and equal amounts of protein were loaded onto precast 4–12% Bis-Tris gels (Invitrogen). After protein transfer at 100 V for 60 min onto 0.45 μm

nitrocellulose membranes (Amersham), membranes were incubated in blocking buffer and subsequently primary antibodies O/N at 4 °C. Primary antibodies and respective working concentrations are

included in Supplementary Data 5. Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Invitrogen), for detection using enhanced chemiluminescence

(SuperSignal West Pico PLUS, Thermo Fisher Scientific) on a Syngene G:BOX, or with IRDye800CW- and IRDye680-labeled secondary antibodies (Li-COR) for detection on the Odyssey CLx Infrared

imager (LI-COR). Blots were analyzed using either the GeneSys software (version 1.6.9.0) or the Image Studio Lite LI-COR software (version 5.2.5). IMMUNOFLUORESCENCE For immunofluorescence,

cells were seeded on 15 mm ø coverslips in 12-well plates and treated as indicated in the individual experiments. Cells were washed with PBS, fixed for 10 min with 2% paraformaldehyde

followed by 10 min permeabilization in 0.5% Triton/PBS. After 1 h of blocking (0.02% Triton, 5% NGS, 5% FCS in PBS), cells were incubated with primary antibody diluted with blocking solution

overnight at 4 °C in a humid chamber. Primary antibodies and respective concentrations used are included in Supplementary Data 5. Next, cells were washed three times with 0.02% Triton/PBS

and incubated with Alexa Fluor 488, 568 or 647 goat anti-mouse or anti-rabbit IgG secondary antibodies (Invitrogen) in blocking solution for 1 h at room temperature in the dark. After

washing three times with 0.02% Triton/PBS, slides were mounted using ProLong Gold Antifade Mountant with DAPI (Invitrogen). Slides were imaged using a LSM 980 confocal with Airyscan2 system

with ×63 oil objective and ZEN software. Image analysis for foci co-localization was performed with the publicly available Foci Analyzer 1.3 macro

(https://github.com/BioImaging-NKI/Foci-analyzer) on ImageJ software. Briefly, maximum intensity projections were first produced from Z-stack images. Nuclei were automatically defined using

the “Stardist nuclei segmentation”. The two channels where the foci were captured were selected for co-localization analysis. Specifically, for the γH2AX-chromocenter co-localization

experiment (Fig. 6), the DAPI channel was selected as one of the two channels for co-localization analysis, “Foci size” was set to “large”, to enable the detection of chromocenters as foci.

RANDOM PLASMID INTEGRATION ASSAY For random plasmid integration assays, 500,000 U2OS cells were seeded in 6 cm dishes 24 h before transfection. With the use of Lipofectamine 2000 (Thermo

Fisher Scientific) cells were transfected with 3 μg of EcoRI/BamHI-linearized peGFP-C1 plasmid that confers G418 resistance upon successful integration. Transfected cells were trypsinized

the following day and seeded in 10 cm dishes at 1000 cells per dish for –G418 controls and 20,000 cells per dish for treatment with G418. Simultaneously, 20,000 cells per sample were seeded

separately for assessment of transfection efficiency by the % GFP-positive cells using FACS. Selection was initiated the following day with G418/Geneticin at 500 µg/ml. At 10–14 days after

seeding, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Dishes were scanned and colony counting was performed using ImageJ (1.52p). NHEJ scores were

calculated by normalizing the number of colonies in +G418 dishes to the plating efficiency (-G418) and transfection efficiency (% GFP). DSB-SPECTRUM_V1 REPORTER The DSB-Spectrum_V1 reporter

system was used to assess the NHEJ-HR balance as described38. Briefly, DSB-Spectrum_V1 reporter HEK293T cells were seeded at 300,000 cells/well in 12-well plates. The next day, cells were

transfected wit pX459-Cas9-sgRNA-mCherry using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. At 48 h post transfection, cells were trypsinized and analyzed by

FACS for the % BFP (NHEJ) and % GFP (HR) of transfected (mCherry+) cells using an LSRFortessa Cell Analyzer (BD). STATISTICAL ANALYSIS AND REPRODUCIBILITY Statistical analyses were performed

using GraphPad Prism (9.2.0) and Microsoft Excel 2016 (16.0.5356.1000). Details on data representation, statistical tests and number of replicate experiments are indicated in the respective

figure legends. Exact p-values are indicated in the figures. REPORTING SUMMARY Further information on research design is available in the Nature Portfolio Reporting Summary linked to this

article. DATA AVAILABILITY All data generated or analyzed during this study are included in this article and its supplementary information files and are available from the corresponding

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repair. _Nat. Commun._ 12, 5421 (2021). Article  ADS  PubMed Central  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Titia de Lange for TRF2I468A expression vector and

_Trf2_flox/-;_p53_−/− MEFs, used to generate TRF2ts MEFs, and for _Trf1__F/F__;Trf2__F/F__;Ku70__−/−__;p53__−/−__;Cre-ER__T2_ MEFs. We thank Haico van Attikum and Bert van der Kooij for

HEK293T cells with the DSB-Spectrum_V1 reporter, Joachim Lingner for the HeLa shTRF2 cells, Thomas Kuilman for MAGeCK-RRA analysis of the screen results, Daniëlle Koot for assistance with

IF-FISH and Brigitte Wevers for contributing to initial optimization of the pKAP1 screen. This work was supported by the European Union’s Horizon 2020 research and innovation program

(Marie-Skłodowska-Curie grant agreement 812829) to J.J.L.J, Dutch Cancer Society project grant KWF 2019-2/12826 to J.J.L.J., and an institutional grant of the Dutch Cancer Society and the

Dutch Ministry of Health, Welfare and Sport to the Netherlands Cancer Institute. AUTHOR INFORMATION Author notes * These authors contributed equally: Shiu Yeung Lam, Ruben van der Lugt.

AUTHORS AND AFFILIATIONS * Division of Oncogenomics, The Netherlands Cancer Institute, Amsterdam, The Netherlands Shiu Yeung Lam, Ruben van der Lugt, Aurora Cerutti, Zeliha Yalçin, Alexander

M. Thouin, Marco Simonetta & Jacqueline J. L. Jacobs Authors * Shiu Yeung Lam View author publications You can also search for this author inPubMed Google Scholar * Ruben van der Lugt

View author publications You can also search for this author inPubMed Google Scholar * Aurora Cerutti View author publications You can also search for this author inPubMed Google Scholar *

Zeliha Yalçin View author publications You can also search for this author inPubMed Google Scholar * Alexander M. Thouin View author publications You can also search for this author inPubMed

 Google Scholar * Marco Simonetta View author publications You can also search for this author inPubMed Google Scholar * Jacqueline J. L. Jacobs View author publications You can also search

for this author inPubMed Google Scholar CONTRIBUTIONS J.J.L.J. conceived the original idea for the screen and together with S.Y.L. and R.v.d.L. conceived the ideas for follow-up work. M.S.

constructed the shRNA library and A.C. conducted the screen. S.Y.L. and R.v.d.L. performed most experiments, Z.Y. assessed telomere fusion upon UBR5 knockdown and A.T. conducted

DSB-spectrum_V1 reporter assays. J.J.L.J. and S.Y.L. co-wrote the manuscript, to which R.v.d.L. contributed with comments, composing part of the figures and responding to peer reviewers.

CORRESPONDING AUTHOR Correspondence to Jacqueline J. L. Jacobs. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION

_Nature Communications_ thanks Younghoon Kee, Tej Pandita, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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OTUD5 promotes end-joining of deprotected telomeres by promoting ATM-dependent phosphorylation of KAP1S824. _Nat Commun_ 15, 8960 (2024). https://doi.org/10.1038/s41467-024-53404-0 Download

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