Publications by authors named "Niels Mailand"

77 Publications

Ubiquitylation at Stressed Replication Forks: Mechanisms and Functions.

Trends Cell Biol 2021 Feb 18. Epub 2021 Feb 18.

Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, DK-2200 Copenhagen, Denmark; Center for Chromosome Stability, Department of Cellular and Molecular Medicine, University of Copenhagen, DK-2200 Copenhagen, Denmark. Electronic address:

Accurate duplication of chromosomal DNA is vital for faithful transmission of the genome during cell division. However, DNA replication integrity is frequently challenged by genotoxic insults that compromise the progression and stability of replication forks, posing a threat to genome stability. It is becoming clear that the organization of the replisome displays remarkable flexibility in responding to and overcoming a wide spectrum of fork-stalling insults, and that these transactions are dynamically orchestrated and regulated by protein post-translational modifications (PTMs) including ubiquitylation. In this review, we highlight and discuss important recent advances on how ubiquitin-mediated signaling at the replication fork plays a crucial multifaceted role in regulating replisome composition and remodeling its configuration upon replication stress, thereby ensuring high-fidelity duplication of the genome.
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http://dx.doi.org/10.1016/j.tcb.2021.01.008DOI Listing
February 2021

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination.

Mol Cell 2021 03 14;81(5):1084-1099.e6. Epub 2021 Jan 14.

The Novo Nordisk Center for Protein Research (CPR), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark; Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. Electronic address:

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.
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http://dx.doi.org/10.1016/j.molcel.2020.12.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939521PMC
March 2021

The ubiquitin ligase RFWD3 is required for translesion DNA synthesis.

Mol Cell 2021 02 14;81(3):442-458.e9. Epub 2020 Dec 14.

The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark. Electronic address:

Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.
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http://dx.doi.org/10.1016/j.molcel.2020.11.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864614PMC
February 2021

FAM111 protease activity undermines cellular fitness and is amplified by gain-of-function mutations in human disease.

EMBO Rep 2020 10 9;21(10):e50662. Epub 2020 Aug 9.

Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.

Dominant missense mutations in the human serine protease FAM111A underlie perinatally lethal gracile bone dysplasia and Kenny-Caffey syndrome, yet how FAM111A mutations lead to disease is not known. We show that FAM111A proteolytic activity suppresses DNA replication and transcription by displacing key effectors of these processes from chromatin, triggering rapid programmed cell death by Caspase-dependent apoptosis to potently undermine cell viability. Patient-associated point mutations in FAM111A exacerbate these phenotypes by hyperactivating its intrinsic protease activity. Moreover, FAM111A forms a complex with the uncharacterized homologous serine protease FAM111B, point mutations in which cause a hereditary fibrosing poikiloderma syndrome, and we demonstrate that disease-associated FAM111B mutants display amplified proteolytic activity and phenocopy the cellular impact of deregulated FAM111A catalytic activity. Thus, patient-associated FAM111A and FAM111B mutations may drive multisystem disorders via a common gain-of-function mechanism that relieves inhibitory constraints on their protease activities to powerfully undermine cellular fitness.
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http://dx.doi.org/10.15252/embr.202050662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534640PMC
October 2020

Multisite SUMOylation restrains DNA polymerase η interactions with DNA damage sites.

J Biol Chem 2020 06 29;295(25):8350-8362. Epub 2020 Apr 29.

Ubiquitin Signaling Group, Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen, Denmark

Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.
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http://dx.doi.org/10.1074/jbc.RA120.013780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307195PMC
June 2020

Regulation of ETAA1-mediated ATR activation couples DNA replication fidelity and genome stability.

J Cell Biol 2019 12 15;218(12):3943-3953. Epub 2019 Oct 15.

Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, Copenhagen, Denmark

The ATR kinase is a master regulator of the cellular response to DNA replication stress. Activation of ATR relies on dual pathways involving the TopBP1 and ETAA1 proteins, both of which harbor ATR-activating domains (AADs). However, the exact contribution of the recently discovered ETAA1 pathway to ATR signaling in different contexts remains poorly understood. Here, using an unbiased CRISPR-Cas9-based genome-scale screen, we show that the ATR-stimulating function of ETAA1 becomes indispensable for cell fitness and chromosome stability when the fidelity of DNA replication is compromised. We demonstrate that the ATR-activating potential of ETAA1 is controlled by cell cycle- and replication stress-dependent phosphorylation of highly conserved residues within its AAD, and that the stimulatory impact of these modifications is required for the ability of ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. Our findings suggest an important role of ETAA1 in protecting against genome instability arising from incompletely duplicated DNA via regulatory control of its ATR-stimulating potential.
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http://dx.doi.org/10.1083/jcb.201905064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891097PMC
December 2019

The p97-Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF8.

EMBO J 2019 10 15;38(21):e102361. Epub 2019 Oct 15.

Department of Oncology, Cancer Research UK/Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK.

The E3 ubiquitin ligase RNF8 (RING finger protein 8) is a pivotal enzyme for DNA repair. However, RNF8 hyper-accumulation is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Very little is known about the mechanisms regulating RNF8 homeostasis to preserve genome stability. Here, we identify the cellular machinery, composed of the p97/VCP ubiquitin-dependent unfoldase/segregase and the Ataxin 3 (ATX3) deubiquitinase, which together form a physical and functional complex with RNF8 to regulate its proteasome-dependent homeostasis under physiological conditions. Under genotoxic stress, when RNF8 is rapidly recruited to sites of DNA lesions, the p97-ATX3 machinery stimulates the extraction of RNF8 from chromatin to balance DNA repair pathway choice and promote cell survival after ionising radiation (IR). Inactivation of the p97-ATX3 complex affects the non-homologous end joining DNA repair pathway and hypersensitises human cancer cells to IR. We propose that the p97-ATX3 complex is the essential machinery for regulation of RNF8 homeostasis under both physiological and genotoxic conditions and that targeting ATX3 may be a promising strategy to radio-sensitise BRCA-deficient cancers.
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http://dx.doi.org/10.15252/embj.2019102361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826192PMC
October 2019

TRAIP drives replisome disassembly and mitotic DNA repair synthesis at sites of incomplete DNA replication.

Elife 2019 09 23;8. Epub 2019 Sep 23.

MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom.

The faithful segregation of eukaryotic chromosomes in mitosis requires that the genome be duplicated completely prior to anaphase. However, cells with large genomes sometimes fail to complete replication during interphase and instead enter mitosis with regions of incompletely replicated DNA. These regions are processed in early mitosis via a process known as mitotic DNA repair synthesis (MiDAS), but little is known about how cells switch from conventional DNA replication to MiDAS. Using the early embryo of the nematode as a model system, we show that the TRAIP ubiquitin ligase drives replisome disassembly in response to incomplete DNA replication, thereby providing access to replication forks for other factors. Moreover, TRAIP is essential for MiDAS in human cells, and is important in both systems to prevent mitotic segregation errors. Our data indicate that TRAIP is a master regulator of the processing of incomplete DNA replication during mitosis in metazoa.
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http://dx.doi.org/10.7554/eLife.48686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773462PMC
September 2019

An unorthodox partnership in DNA repair pathway choice.

EMBO Rep 2019 11 23;20(11):e49105. Epub 2019 Sep 23.

Protein Signaling Program, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark.

Complex regulatory circuits determine whether DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways, a carefully balanced equilibrium of which is critical for genome stability. In this issue of EMBO Reports, Deng et al [1] report that a novel p53-suppressed long noncoding RNA (lncRNA), PRLH1, interacts with and stabilizes the E3 ubiquitin ligase RNF169 to stimulate HDR-mediated DSB repair and proliferation of p53-deficient cancer cells. These findings suggest a new regulatory principle in modulating DSB repair pathway selection that may contribute to tumorigenesis.
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http://dx.doi.org/10.15252/embr.201949105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831990PMC
November 2019

Structural Basis of BRCC36 Function in DNA Repair and Immune Regulation.

Mol Cell 2019 08 25;75(3):483-497.e9. Epub 2019 Jun 25.

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, 4058 Basel, Switzerland; University of Basel, Petersplatz 10, 4003 Basel, Switzerland. Electronic address:

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.
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http://dx.doi.org/10.1016/j.molcel.2019.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695476PMC
August 2019

GIGYF1/2-Driven Cooperation between ZNF598 and TTP in Posttranscriptional Regulation of Inflammatory Signaling.

Cell Rep 2019 03;26(13):3511-3521.e4

Department of Cellular and Molecular Medicine, Center for Healthy Aging, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

Inflammatory signaling is restricted through degradation and the translational repression of cytokine mRNAs. A key factor in this regulation is tristetraprolin (TTP), an RNA-binding protein (RBP) that recruits RNA-destabilizing factors and the translation inhibitory complex 4EHP-GIGYF1/2 to AU-rich element (ARE)-containing mRNAs. Here, we show that the RBP ZNF598 contributes to the same regulatory module in a TTP-like manner. Similar to TTP, ZNF598 harbors three proline-rich motifs that bind the GYF domain of GIGYF1. RNA sequencing experiments showed that ZNF598 is required for the regulation of known TTP targets, including IL-8 and CSF2 mRNA. Furthermore, we demonstrate that ZNF598 binds to IL-8 mRNA, but not TNF mRNA. Collectively, our findings highlight that ZNF598 functions as an RBP that buffers the level of a range of mRNAs. We propose that ZNF598 is a TTP-like factor that can contribute to the regulation of the inflammatory potential of cytokine-producing cells.
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http://dx.doi.org/10.1016/j.celrep.2019.03.006DOI Listing
March 2019

SUMOylation promotes protective responses to DNA-protein crosslinks.

EMBO J 2019 04 26;38(8). Epub 2019 Mar 26.

Ubiquitin Signaling Group, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark

DNA-protein crosslinks (DPCs) are highly cytotoxic lesions that obstruct essential DNA transactions and whose resolution is critical for cell and organismal fitness. However, the mechanisms by which cells respond to and overcome DPCs remain incompletely understood. Recent studies unveiled a dedicated DPC repair pathway in higher eukaryotes involving the SprT-type metalloprotease SPRTN/DVC1, which proteolytically processes DPCs during DNA replication in a ubiquitin-regulated manner. Here, we show that chemically induced and defined enzymatic DPCs trigger potent chromatin SUMOylation responses targeting the crosslinked proteins and associated factors. Consequently, inhibiting SUMOylation compromises DPC clearance and cellular fitness. We demonstrate that ACRC/GCNA family SprT proteases interact with SUMO and establish important physiological roles of GCNA-1 and SUMOylation in promoting germ cell and embryonic survival upon DPC formation. Our findings provide first global insights into signaling responses to DPCs and reveal an evolutionarily conserved function of SUMOylation in facilitating responses to these lesions in metazoans that may complement replication-coupled DPC resolution processes.
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http://dx.doi.org/10.15252/embj.2019101496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463212PMC
April 2019

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Cell 2018 05 12;173(4):972-988.e23. Epub 2018 Apr 12.

Proteomics Program, the Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark; Center for Chromosome Stability (CCS), Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark. Electronic address:

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.
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http://dx.doi.org/10.1016/j.cell.2018.03.050DOI Listing
May 2018

ZUFSP Deubiquitylates K63-Linked Polyubiquitin Chains to Promote Genome Stability.

Mol Cell 2018 04 22;70(1):165-174.e6. Epub 2018 Mar 22.

Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark; Center for Chromosome Stability, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark. Electronic address:

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.
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http://dx.doi.org/10.1016/j.molcel.2018.02.024DOI Listing
April 2018

p38-MK2 signaling axis regulates RNA metabolism after UV-light-induced DNA damage.

Nat Commun 2018 03 9;9(1):1017. Epub 2018 Mar 9.

Institute of Molecular Biology (IMB), Ackermannweg 4, 55128, Mainz, Germany.

Ultraviolet (UV) light radiation induces the formation of bulky photoproducts in the DNA that globally affect transcription and splicing. However, the signaling pathways and mechanisms that link UV-light-induced DNA damage to changes in RNA metabolism remain poorly understood. Here we employ quantitative phosphoproteomics and protein kinase inhibition to provide a systems view on protein phosphorylation patterns induced by UV light and uncover the dependencies of phosphorylation events on the canonical DNA damage signaling by ATM/ATR and the p38 MAP kinase pathway. We identify RNA-binding proteins as primary substrates and 14-3-3 as direct readers of p38-MK2-dependent phosphorylation induced by UV light. Mechanistically, we show that MK2 phosphorylates the RNA-binding subunit of the NELF complex NELFE on Serine 115. NELFE phosphorylation promotes the recruitment of 14-3-3 and rapid dissociation of the NELF complex from chromatin, which is accompanied by RNA polymerase II elongation.
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http://dx.doi.org/10.1038/s41467-018-03417-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845016PMC
March 2018

RADX interacts with single-stranded DNA to promote replication fork stability.

EMBO Rep 2017 11 11;18(11):1991-2003. Epub 2017 Oct 11.

Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark

Single-stranded DNA (ssDNA) regions form as an intermediate in many DNA-associated transactions. Multiple cellular proteins interact with ssDNA via the oligonucleotide/oligosaccharide-binding (OB) fold domain. The heterotrimeric, multi-OB fold domain-containing Replication Protein A (RPA) complex has an essential genome maintenance role, protecting ssDNA regions from nucleolytic degradation and providing a recruitment platform for proteins involved in responses to replication stress and DNA damage. Here, we identify the uncharacterized protein RADX (CXorf57) as an ssDNA-binding factor in human cells. RADX binds ssDNA via an N-terminal OB fold cluster, which mediates its recruitment to sites of replication stress. Deregulation of RADX expression and ssDNA binding leads to enhanced replication fork stalling and degradation, and we provide evidence that a balanced interplay between RADX and RPA ssDNA-binding activities is critical for avoiding these defects. Our findings establish RADX as an important component of cellular pathways that promote DNA replication integrity under basal and stressful conditions by means of multiple ssDNA-binding proteins.
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http://dx.doi.org/10.15252/embr.201744877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666600PMC
November 2017

SCAI promotes DNA double-strand break repair in distinct chromosomal contexts.

Nat Cell Biol 2016 Dec 7;18(12):1357-1366. Epub 2016 Nov 7.

Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose accurate repair by non-homologous end-joining (NHEJ) or homologous recombination (HR) is crucial for genome integrity and is strongly influenced by the local chromatin environment. Here, we identify SCAI (suppressor of cancer cell invasion) as a 53BP1-interacting chromatin-associated protein that promotes the functionality of several DSB repair pathways in mammalian cells. SCAI undergoes prominent enrichment at DSB sites through dual mechanisms involving 53BP1-dependent recruitment to DSB-surrounding chromatin and 53BP1-independent accumulation at resected DSBs. Cells lacking SCAI display reduced DSB repair capacity, hypersensitivity to DSB-inflicting agents and genome instability. We demonstrate that SCAI is a mediator of 53BP1-dependent repair of heterochromatin-associated DSBs, facilitating ATM kinase signalling at DSBs in repressive chromatin environments. Moreover, we establish an important role of SCAI in meiotic recombination, as SCAI deficiency in mice leads to germ cell loss and subfertility associated with impaired retention of the DMC1 recombinase on meiotic chromosomes. Collectively, our findings uncover SCAI as a physiologically important component of both NHEJ- and HR-mediated pathways that potentiates DSB repair efficiency in specific chromatin contexts.
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http://dx.doi.org/10.1038/ncb3436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278951PMC
December 2016

Activation of the ATR kinase by the RPA-binding protein ETAA1.

Nat Cell Biol 2016 Nov 10;18(11):1196-1207. Epub 2016 Oct 10.

The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically, this requires a conserved domain in ETAA1 that potently and directly stimulates ATR kinase activity independently of TopBP1. Simultaneous loss of ETAA1 and TopBP1 gives rise to synthetic lethality characterized by massive genome instability and abrogation of ATR-dependent signalling. Our findings demonstrate that parallel TopBP1- and ETAA1-mediated pathways underlie ATR activation and that their combined action is essential for coping with replication stress.
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http://dx.doi.org/10.1038/ncb3422DOI Listing
November 2016

Writers, Readers, and Erasers of Histone Ubiquitylation in DNA Double-Strand Break Repair.

Front Genet 2016 28;7:122. Epub 2016 Jun 28.

Ubiquitin Signaling Group, Protein Signaling Program, Faculty of Health and Medical Sciences, The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen Copenhagen, Denmark.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions, whose faulty repair may alter the content and organization of cellular genomes. To counteract this threat, numerous signaling and repair proteins are recruited hierarchically to the chromatin areas surrounding DSBs to facilitate accurate lesion repair and restoration of genome integrity. In vertebrate cells, ubiquitin-dependent modifications of histones adjacent to DSBs by RNF8, RNF168, and other ubiquitin ligases have a key role in promoting the assembly of repair protein complexes, serving as direct recruitment platforms for a range of genome caretaker proteins and their associated factors. These DNA damage-induced chromatin ubiquitylation marks provide an essential component of a histone code for DSB repair that is controlled by multifaceted regulatory circuits, underscoring its importance for genome stability maintenance. In this review, we provide a comprehensive account of how DSB-induced histone ubiquitylation is sensed, decoded and modulated by an elaborate array of repair factors and regulators. We discuss how these mechanisms impact DSB repair pathway choice and functionality for optimal protection of genome integrity, as well as cell and organismal fitness.
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http://dx.doi.org/10.3389/fgene.2016.00122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923129PMC
July 2016

H4K20me0 marks post-replicative chromatin and recruits the TONSL–MMS22L DNA repair complex.

Nature 2016 06 22;534(7609):714-718. Epub 2016 Jun 22.

Biotech Research and Innovation Centre (BRIC), Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.

After DNA replication, chromosomal processes including DNA repair and transcription take place in the context of sister chromatids. While cell cycle regulation can guide these processes globally, mechanisms to distinguish pre- and post-replicative states locally remain unknown. Here we reveal that new histones incorporated during DNA replication provide a signature of post-replicative chromatin, read by the human TONSL–MMS22L homologous recombination complex. We identify the TONSL ankyrin repeat domain (ARD) as a reader of histone H4 tails unmethylated at K20 (H4K20me0), which are specific to new histones incorporated during DNA replication and mark post-replicative chromatin until the G2/M phase of the cell cycle. Accordingly, TONSL–MMS22L binds new histones H3–H4 both before and after incorporation into nucleosomes, remaining on replicated chromatin until late G2/M. H4K20me0 recognition is required for TONSL–MMS22L binding to chromatin and accumulation at challenged replication forks and DNA lesions. Consequently, TONSL ARD mutants are toxic, compromising genome stability, cell viability and resistance to replication stress. Together, these data reveal a histone-reader-based mechanism for recognizing the post-replicative state, offering a new angle to understand DNA repair with the potential for targeted cancer therapy.
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http://dx.doi.org/10.1038/nature18312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939875PMC
June 2016

Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers.

Nat Rev Mol Cell Biol 2016 05;17(6):379-94

Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs) orchestrates and regulates cellular responses to DSBs at multiple levels, often involving extensive crosstalk between these modifications. Recent findings have revealed compelling insights into the complex mechanisms by which ubiquitin and UBLs regulate protein interactions with DSB sites to promote accurate lesion repair and protection of genome integrity in mammalian cells. These advances offer new therapeutic opportunities for diseases linked to genetic instability.
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http://dx.doi.org/10.1038/nrm.2016.58DOI Listing
May 2016

Structural Analysis of a Complex between Small Ubiquitin-like Modifier 1 (SUMO1) and the ZZ Domain of CREB-binding Protein (CBP/p300) Reveals a New Interaction Surface on SUMO.

J Biol Chem 2016 Jun 25;291(24):12658-12672. Epub 2016 Apr 25.

From the Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark,; Amgen Inc., Thousand Oaks, California 91320. Electronic address:

We have recently discovered that the ZZ zinc finger domain represents a novel small ubiquitin-like modifier (SUMO) binding motif. In this study we identify the binding epitopes in the ZZ domain of CBP (CREB-binding protein) and SUMO1 using NMR spectroscopy. The binding site on SUMO1 represents a unique epitope for SUMO interaction spatially opposite to that observed for canonical SUMO interaction motifs (SIMs). HADDOCK docking simulations using chemical shift perturbations and residual dipolar couplings was employed to obtain a structural model for the ZZ domain-SUMO1 complex. Isothermal titration calorimetry experiments support this model by showing that the mutation of key residues in the binding site abolishes binding and that SUMO1 can simultaneously and non-cooperatively bind both the ZZ domain and a canonical SIM motif. The binding dynamics of SUMO1 was further characterized using (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersions, which define the off rates for the ZZ domain and SIM motif and show that the dynamic binding process has different characteristics for the two cases. Furthermore, in the absence of bound ligands SUMO1 transiently samples a high energy conformation, which might be involved in ligand binding.
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http://dx.doi.org/10.1074/jbc.M115.711325DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4933466PMC
June 2016

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress.

J Cell Biol 2016 Jan 28;212(1):63-75. Epub 2015 Dec 28.

Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, 2200 Copenhagen, Denmark

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.
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http://dx.doi.org/10.1083/jcb.201506071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4700480PMC
January 2016

p38- and MK2-dependent signalling promotes stress-induced centriolar satellite remodelling via 14-3-3-dependent sequestration of CEP131/AZI1.

Nat Commun 2015 Nov 30;6:10075. Epub 2015 Nov 30.

Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, Copenhagen DK-2200, Denmark.

Centriolar satellites (CS) are small granular structures that cluster in the vicinity of centrosomes. CS are highly susceptible to stress stimuli, triggering abrupt displacement of key CS factors. Here we discover a linear p38-MK2-14-3-3 signalling pathway that specifically targets CEP131 to trigger CS remodelling after cell stress. We identify CEP131 as a substrate of the p38 effector kinase MK2 and pinpoint S47 and S78 as critical MK2 phosphorylation sites in CEP131. Ultraviolet-induced phosphorylation of these residues generates direct binding sites for 14-3-3 proteins, which sequester CEP131 in the cytoplasm to block formation of new CS, thereby leading to rapid depletion of these structures. Mutating S47 and S78 in CEP131 is sufficient to abolish stress-induced CS reorganization, demonstrating that CEP131 is the key regulatory target of MK2 and 14-3-3 in these structures. Our findings reveal the molecular mechanism underlying dynamic CS remodelling to modulate centrosome functions on cell stress.
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http://dx.doi.org/10.1038/ncomms10075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674683PMC
November 2015

RNF138 joins the HR team.

Nat Cell Biol 2015 Nov;17(11):1375-7

Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark.

Two studies show that the E3 ubiquitin ligase RNF138 is recruited to DNA double-strand break sites, where it ubiquitylates key repair factors to promote DNA-end resection and homologous recombination. These findings add insights into the multilayered regulatory mechanisms underlying DNA double-strand break repair pathway choice in mammalian cells.
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http://dx.doi.org/10.1038/ncb3262DOI Listing
November 2015

Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage.

Nature 2015 Nov 21;527(7578):389-93. Epub 2015 Oct 21.

Ubiquitin Signaling Group, Protein Signaling Program, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, DK-2200 Copenhagen, Denmark.

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked ubiquitylation remain elusive. Here we elucidate how RNF8 and UBC13 promote recruitment of RNF168 and downstream factors to DSB sites in human cells. We establish that UBC13-dependent K63-linked ubiquitylation at DSB sites is predominantly mediated by RNF8 but not RNF168, and that H1-type linker histones, but not core histones, represent major chromatin-associated targets of this modification. The RNF168 module (UDM1) recognizing RNF8-generated ubiquitylations is a high-affinity reader of K63-ubiquitylated H1, mechanistically explaining the essential roles of RNF8 and UBC13 in recruiting RNF168 to DSBs. Consistently, reduced expression or chromatin association of linker histones impair accumulation of K63-linked ubiquitin conjugates and repair factors at DSB-flanking chromatin. These results identify histone H1 as a key target of RNF8-UBC13 in DSB signalling and expand the concept of the histone code by showing that posttranslational modifications of linker histones can serve as important marks for recognition by factors involved in genome stability maintenance, and possibly beyond.
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http://dx.doi.org/10.1038/nature15401DOI Listing
November 2015

SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair.

Nat Commun 2015 Jul 7;6:7499. Epub 2015 Jul 7.

Department of Genetics, Cancer Genomics Netherlands Erasmus MC, Wytemaweg 80, Rotterdam 3015 CN, The Netherlands.

XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4(DDB2) ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial for efficient progression of the NER reaction, thereby providing an extra layer of quality control of NER.
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http://dx.doi.org/10.1038/ncomms8499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501428PMC
July 2015

DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links.

Science 2015 May 30;348(6234):1253671. Epub 2015 Apr 30.

Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany. Novo Nordisk Foundation Center for Protein Research, Proteomics Program, University of Copenhagen, DK-2200 Copenhagen, Denmark.

DNA interstrand cross-links (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. We devised a technique called chromatin mass spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly on ICL-containing chromatin. Among numerous prospective DNA repair factors, we identified SLF1 and SLF2, which form a complex with RAD18 and together define a pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides a global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.
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http://dx.doi.org/10.1126/science.1253671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331883PMC
May 2015

Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway.

EMBO J 2015 May 9;34(10):1385-98. Epub 2015 Apr 9.

Ubiquitin Signaling Group, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark

Ubiquitin and ubiquitin-like proteins (UBLs) function in a wide array of cellular processes. UBL5 is an atypical UBL that does not form covalent conjugates with cellular proteins and which has a known role in modulating pre-mRNA splicing. Here, we report an unexpected involvement of human UBL5 in promoting the function of the Fanconi anemia (FA) pathway for repair of DNA interstrand crosslinks (ICLs), mediated by a specific interaction with the central FA pathway component FANCI. UBL5-deficient cells display spliceosome-independent reduction of FANCI protein stability, defective FANCI function in response to DNA damage and hypersensitivity to ICLs. By mapping the sequence determinants underlying UBL5-FANCI binding, we generated separation-of-function mutants to demonstrate that key aspects of FA pathway function, including FANCI-FANCD2 heterodimerization, FANCD2 and FANCI monoubiquitylation and maintenance of chromosome stability after ICLs, are compromised when the UBL5-FANCI interaction is selectively inhibited by mutations in either protein. Together, our findings establish UBL5 as a factor that promotes the functionality of the FA DNA repair pathway.
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http://dx.doi.org/10.15252/embj.201490376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491998PMC
May 2015

Lamin A/C-dependent interaction with 53BP1 promotes cellular responses to DNA damage.

Aging Cell 2015 Apr 23;14(2):162-9. Epub 2015 Jan 23.

School of Biological and Biomedical Sciences, Durham University, Mountjoy Science Park, Durham, DH1 3LE, UK; Ubiquitin Signaling Group, Department of Disease Biology, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, DK-2200, Denmark.

Lamins A/C have been implicated in DNA damage response pathways. We show that the DNA repair protein 53BP1 is a lamin A/C binding protein. In undamaged human dermal fibroblasts (HDF), 53BP1 is a nucleoskeleton protein. 53BP1 binds to lamins A/C via its Tudor domain, and this is abrogated by DNA damage. Lamins A/C regulate 53BP1 levels and consequently lamin A/C-null HDF display a 53BP1 null-like phenotype. Our data favour a model in which lamins A/C maintain a nucleoplasmic pool of 53BP1 in order to facilitate its rapid recruitment to sites of DNA damage and could explain why an absence of lamin A/C accelerates aging.
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http://dx.doi.org/10.1111/acel.12258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4364828PMC
April 2015