Publications by authors named "Raimundo Freire"

68 Publications

Ubiquitin-specific protease 7 as a potential therapeutic target in dogs with hematopoietic malignancies.

J Vet Intern Med 2021 Mar 2;35(2):1041-1051. Epub 2021 Mar 2.

Unidad de Investigación, Hospital Universitario de Canarias, FIISC, La Laguna, Tenerife, Spain.

Background: Ubiquitin-specific protease 7 (USP7) belongs to the group of deubiquitinating enzymes (DUBs), which remove ubiquitin which controls various cellular processes such as chromosome segregation, DNA repair, gene expression, protein localization, kinase activity, protein degradation, cell cycle progression, and apoptosis. It is critical for several important functions in the cell, and therefore dysregulation of USP7 can contribute to tumorigenesis.

Objectives: Alterations in the USP7 protein have been identified in various malignancies of humans. Our aim was to examine whether USP7 could be a potential therapeutic target in hematopoietic cancers of dogs.

Methods: The expression level of USP7 in lymphocytes from healthy dogs and canine lymphoma cells was determined, and the effect of USP7 inhibition on the vital functions of canine cancer cells was examined.

Results: We showed that USP7 was overexpressed in lymphomas in dogs. The USP7 inhibitor P5091 has selective cytotoxic activity in canine lymphoma and leukemia cell lines. Our results indicate that inhibition of USP7 leads to a disruption of cell cycle progression, and triggers DNA damage and apoptosis. The observed proapoptotic effect of the USP7 inhibitor most likely is not dependent on the p53 pathway.

Conclusions And Clinical Importance: Our results suggest that USP7 could be explored as a potential therapeutic target in dogs with lymphoma. The effectiveness of USP7 inhibition in malignant cells is predicted to be independent of their p53 status.
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http://dx.doi.org/10.1111/jvim.16082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995420PMC
March 2021

The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients.

Biochem Biophys Res Commun 2021 03 22;543:45-49. Epub 2021 Jan 22.

Unidad de Investigación, Hospital Universitario de Canarias-FIISC, Ofra s/n, 38320, La Laguna, Tenerife, Spain; Instituto de Tecnologías Biomédicas, Universidad de La Laguna, 38200, La Laguna, Tenerife, Spain; Universidad Fernando Pessoa Canarias, 35450, Las Palmas de Gran Canaria, Spain. Electronic address:

In order to control the COVID-19 pandemic caused by SARS-CoV-2 infection, serious progress has been made to identify infected patients and to detect patients with a positive immune response against the virus. Currently, attempts to generate a vaccine against the coronavirus are ongoing. To understand SARS-CoV-2 immunoreactivity, we compared the IgG antibody response against SARS-CoV-2 in infected versus control patients by dot blot using recombinant viral particle proteins: N (Nucleocapsid), M (Membrane) and S (Spike). In addition, we used different protein fragments of the N and S protein to map immune epitopes. Most of the COVID-19 patients presented a specific immune response against the full length and fragments of the N protein and, to lesser extent, against a fragment containing amino acids 300-685 of the S protein. In contrast, immunoreactivity against other S protein fragments or the M protein was low. This response is specific for COVID-19 patients as very few of the control patients displayed immunoreactivity, likely reflecting an immune response against other coronaviruses. Altogether, our results may help develop method(s) for measuring COVID-19 antibody response, selectivity of methods detecting such SARS-CoV-2 antibodies and vaccine development.
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http://dx.doi.org/10.1016/j.bbrc.2021.01.073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825866PMC
March 2021

Vps13 is required for timely removal of nurse cell corpses.

Development 2020 10 29;147(20). Epub 2020 Oct 29.

Department of Biomedical Sciences of Cells and Systems, University Medical Center Groningen, The University of Groningen, 9713 AV, Groningen, The Netherlands

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells.
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http://dx.doi.org/10.1242/dev.191759DOI Listing
October 2020

Implications of Variants in Cellular Function and Susceptibility to Cancer.

Cancers (Basel) 2020 Aug 24;12(9). Epub 2020 Aug 24.

New preventive and therapeutic strategies, Centro de Neurociências e Biologia Celular, Universidade de Coimbra, 3004-504 Coimbra, Portugal.

Claspin is a multifunctional protein that participates in physiological processes essential for cell homeostasis that are often defective in cancer, namely due to genetic changes. It is conceivable that Claspin gene () alterations may contribute to cancer development. Therefore, germline alterations were characterized in sporadic and familial breast cancer and glioma samples, as well as in six cancer cell lines. Their association to cancer susceptibility and functional impact were investigated. Eight variants were identified (c.-68C>T, c.17G>A, c.1574A>G, c.2230T>C, c.2028+16G>A, c.3595-3597del, and c.3839C>T). c.1574A>G (p.Asn525Ser) was significantly associated with breast cancer and was shown to cause partial exon skipping and decreased Claspin expression and Chk1 activation in a minigene splicing assay and in signalling experiments, respectively. c.2028+16G>A was significantly associated with familial breast cancer and glioma, whereas c.2230T>C (p.Ser744Pro), was exclusively detected in breast cancer and glioma patients, but not in healthy controls. The remaining variants lacked a significant association with cancer. Nevertheless, the c.-68C>T promoter variant increased transcriptional activity in a luciferase assay. In conclusion, some of the variants identified in the present study appear to modulate Claspin's function by altering transcription and RNA processing, as well as Chk1 activation.
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http://dx.doi.org/10.3390/cancers12092396DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565888PMC
August 2020

Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling.

NAR Cancer 2020 Jun 1;2(2):zcaa006. Epub 2020 May 1.

Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.

Most cancer cells experience oncogene-induced replication stress and, as a result, exhibit high intrinsic activation of the ATR kinase. Although cancer cells often become more dependent on ATR for survival, the precise mechanism by which ATR signaling ensures cancer cell fitness and viability remains incompletely understood. Here, we find that intrinsic ATR signaling is crucial for the ability of cancer cells to promote DNA end resection, the first step in homology-directed DNA repair. Inhibition of ATR over multiple cell division cycles depletes the pool of pro-resection factors and prevents the engagement of RAD51 as well as RAD52 at nuclear foci, leading to toxic DNA-PKcs signaling and hypersensitivity to PARP inhibitors. The effect is markedly distinct from acute ATR inhibition, which blocks RAD51-mediated repair but not resection and engagement of RAD52. Our findings reveal a key pro-resection function for ATR and define how ATR inhibitors can be used for effective manipulation of DNA end resection capacity and DNA repair outcomes in cancer cells.
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http://dx.doi.org/10.1093/narcan/zcaa006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380482PMC
June 2020

OZF is a Claspin-interacting protein essential to maintain the replication fork progression rate under replication stress.

FASEB J 2020 05 8;34(5):6907-6919. Epub 2020 Apr 8.

Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain.

DNA replication is essential for cell proliferation and is one of the cell cycle stages where DNA is more vulnerable. Replication stress is a prominent property of tumor cells and an emerging target for cancer therapy. Although it is not directly involved in nucleotide incorporation, Claspin is a protein with relevant functions in DNA replication. It harbors a DNA-binding domain that interacts preferentially with branched or forked DNA molecules. It also acts as a platform for the interaction of proteins related to DNA damage checkpoint activation, DNA repair, DNA replication origin firing, and fork progression. In order to find new proteins potentially involved in the regulation of DNA replication, we performed a two-hybrid screen to discover new Claspin-binding proteins. This system allowed us to identify the zinc-finger protein OZF (ZNF146) as a new Claspin-interacting protein. OZF is also present at replication forks and co-immunoprecipitates not only with Claspin but also with other replisome components. Interestingly, OZF depletion does not affect DNA replication in a normal cell cycle, but its depletion induces a reduction in the fork progression rate under replication stress conditions. Our results suggest that OZF is a Claspin-binding protein with a specific function in fork progression under replication stress.
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http://dx.doi.org/10.1096/fj.201901926RDOI Listing
May 2020

PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks.

Nucleic Acids Res 2020 05;48(9):4915-4927

Unidad de Investigación, Hospital Universitario de Canarias, Tenerife, Spain.

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.
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http://dx.doi.org/10.1093/nar/gkaa196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229830PMC
May 2020

TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts.

Nat Commun 2020 03 9;11(1):1274. Epub 2020 Mar 9.

Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, OX3 7DQ, UK.

Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate-a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc)-is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase (TDP1), which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3' phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. Here we find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.
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http://dx.doi.org/10.1038/s41467-020-15000-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062751PMC
March 2020

PHF6 promotes non-homologous end joining and G2 checkpoint recovery.

EMBO Rep 2020 01 29;21(1):e48460. Epub 2019 Nov 29.

Unidad de Investigación, Hospital Universitario de Canarias, La Laguna, Tenerife, Spain.

The cellular response to DNA breaks is influenced by chromatin compaction. To identify chromatin regulators involved in the DNA damage response, we screened for genes that affect recovery following DNA damage using an RNAi library of chromatin regulators. We identified genes involved in chromatin remodeling, sister chromatid cohesion, and histone acetylation not previously associated with checkpoint recovery. Among these is the PHD finger protein 6 (PHF6), a gene mutated in Börjeson-Forssman-Lehmann syndrome and leukemic cancers. We find that loss of PHF6 dramatically compromises checkpoint recovery in G2 phase cells. Moreover, PHF6 is rapidly recruited to sites of DNA lesions in a PARP-dependent manner and required for efficient DNA repair through classical non-homologous end joining. These results indicate that PHF6 is a novel DNA damage response regulator that promotes end joining-mediated repair, thereby stimulating timely recovery from the G2 checkpoint.
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http://dx.doi.org/10.15252/embr.201948460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6944915PMC
January 2020

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

GSK3-β Stimulates Claspin Degradation via β-TrCP Ubiquitin Ligase and Alters Cancer Cell Survival.

Cancers (Basel) 2019 Jul 29;11(8). Epub 2019 Jul 29.

Unidad de Investigación, Hospital Universitario de Canarias, Ofra s/n, La Cuesta, 38320 La Laguna, Tenerife, Spain.

Claspin is essential for activating the DNA damage checkpoint effector kinase Chk1, a target in oncotherapy. Claspin functions are tightly correlated to Claspin protein stability, regulated by ubiquitin-dependent proteasomal degradation. Here we identify Glycogen Synthase Kinase 3-β (GSK3-β) as a new regulator of Claspin stability. Interestingly, as Chk1, GSK3-β is a therapeutic target in cancer. GSK3-β inhibition or knockdown stabilizes Claspin, whereas a GSK3-β constitutively active form reduces Claspin protein levels by ubiquitination and proteasome-mediated degradation. Our results also suggest that GSK3-β modulates the interaction of Claspin with β-TrCP, a critical E3 ubiquitin ligase that regulates Claspin stability. Importantly, GSK3-β knock down increases Chk1 activation in response to DNA damage in a Claspin-dependent manner. Therefore, Chk1 activation could be a pro-survival mechanism that becomes activated upon GSK3-β inhibition. Importantly, treating triple negative breast cancer cell lines with Chk1 or GSK3-β inhibitors alone or in combination, demonstrates that Chk1/GSK3-β double inhibition restrains cell growth and triggers more apoptosis compared to individual treatments, thereby revealing novel possibilities for a combination therapy for cancer.
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http://dx.doi.org/10.3390/cancers11081073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721324PMC
July 2019

Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint.

Chromosoma 2019 09 12;128(3):297-316. Epub 2019 Mar 12.

Instituto de Biología Funcional y Genómica (IBFG), Consejo Superior de Investigaciones Científicas (CSIC) and University of Salamanca, 37007, Salamanca, Spain.

The meiotic recombination checkpoint blocks meiotic cell cycle progression in response to synapsis and/or recombination defects to prevent aberrant chromosome segregation. The evolutionarily conserved budding yeast Pch2 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1 adaptor at T318. In the wild type, Pch2 localizes to synapsed chromosomes and to the unsynapsed rDNA region (nucleolus), excluding Hop1. In contrast, in synaptonemal complex (SC)-defective zip1Δ mutants, which undergo checkpoint activation, Pch2 is detected only on the nucleolus. Alterations in some epigenetic marks that lead to Pch2 dispersion from the nucleolus suppress zip1Δ-induced checkpoint arrest. These observations have led to the notion that Pch2 nucleolar localization could be important for the meiotic recombination checkpoint. Here we investigate how Pch2 chromosomal distribution impacts checkpoint function. We have generated and characterized several mutations that alter Pch2 localization pattern resulting in aberrant Hop1 distribution and compromised meiotic checkpoint response. Besides the AAA+ signature, we have identified a basic motif in the extended N-terminal domain critical for Pch2's checkpoint function and localization. We have also examined the functional relevance of the described Orc1-Pch2 interaction. Both proteins colocalize in the rDNA, and Orc1 depletion during meiotic prophase prevents Pch2 targeting to the rDNA allowing unwanted Hop1 accumulation on this region. However, Pch2 association with SC components remains intact in the absence of Orc1. We finally show that checkpoint activation is not affected by the lack of Orc1 demonstrating that, in contrast to previous hypotheses, nucleolar localization of Pch2 is actually dispensable for the meiotic checkpoint.
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http://dx.doi.org/10.1007/s00412-019-00696-7DOI Listing
September 2019

Bio-inspired Printed Monopole Antenna Applied to Partial Discharge Detection.

Sensors (Basel) 2019 Feb 1;19(3). Epub 2019 Feb 1.

Department of Electrical Engineering (DEE), Federal University of Campina Grande (UFCG), Aprigio Veloso 882, Campina Grande 58429-900, Brazil.

A new, bio-inspired printed monopole antenna (PMA) model is applied to monitor partial discharge (PD) activity in high voltage insulating systems. An optimized sensor was obtained by designing a PMA in accordance with the characteristics of the electromagnetic signal produced by PD. An ultra-wideband (UWB) antenna was obtained by applying the truncated ground plane technique. The patch geometry was bio-inspired by that of the leaf, resulting in a significant reduction in size. To verify the operating frequency and gain of the PMA, measurements were carried out in an anechoic chamber. The results show that the antenna operating bandwidth covers most of the frequency range of PD occurrence. Moreover, the antenna presented a good sensitivity (mean gain of 3.63 dBi). The antenna performance was evaluated through comparative results with the standard IEC 60270 method. For this purpose, simultaneous tests were carried out in a PD generator arrangement, composed by an oil cell with point-to-plane electrode configurations. The developed PMA can be classified as an optimized sensor for PD detection and suitable for substation application, since it is able to measure PD radiated signals with half the voltage levels obtained from the IEC method and is immune to corona discharges.
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http://dx.doi.org/10.3390/s19030628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386966PMC
February 2019

Chk1 KA1 domain auto-phosphorylation stimulates biological activity and is linked to rapid proteasomal degradation.

Sci Rep 2018 12 3;8(1):17536. Epub 2018 Dec 3.

Instituto de Tecnologías Biomédicas, Centro de Investigaciones Biomédicas de Canarias, Facultad de Medicina, Campus Ciencias de la Salud, Universidad de La Laguna, La Laguna, 38071, Tenerife, Spain.

The DNA damage-activated protein kinase Chk1 is known to undergo auto-phosphorylation, however the sites and functional significance of this modification remain poorly understood. We have identified two novel Chk1 auto-phosphorylation sites, threonines 378 and 382 (T378/382), located in a highly conserved motif within the C-terminal Kinase Associated 1 (KA1) domain. T378/382 occur within optimal consensus Chk1 phosphorylation motifs and substitution with phospho-mimetic aspartic acid residues results in a constitutively active mutant Chk1 kinase (Chk1-DD) that arrests cell cycle progression in G2 phase of the cell cycle in the absence of DNA damage. Remarkably, the mutant Chk1-DD protein is also subject to very rapid proteasomal degradation, with a half-life approximately one tenth that of wild-type Chk1. Consistent with this, T378/T382 auto-phosphorylation also accelerates the proteasomal degradation of constitutively active Chk1 KA1 domain structural mutants. T378/382 auto-phosphorylation and accelerated degradation of wild-type Chk1 occurs at low levels during unperturbed growth, but surprisingly, is not augmented in response to genotoxic stress. Taken together, these observations demonstrate that Chk1 T378/T382 auto-phosphorylation within the KA1 domain is linked to kinase activation and rapid proteasomal degradation, and suggest a non-canonical mechanism of regulation.
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http://dx.doi.org/10.1038/s41598-018-35616-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277497PMC
December 2018

Control of DNA Replication Initiation by Ubiquitin.

Cells 2018 Sep 20;7(10). Epub 2018 Sep 20.

Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n, 38320 La Laguna, Tenerife, Spain.

Eukaryotic cells divide by accomplishing a program of events in which the replication of the genome is a fundamental part. To ensure all cells have an accurate copy of the genome, DNA replication occurs only once per cell cycle and is controlled by numerous pathways. A key step in this process is the initiation of DNA replication in which certain regions of DNA are marked as competent to replicate. Moreover, initiation of DNA replication needs to be coordinated with other cell cycle processes. At the molecular level, initiation of DNA replication relies, among other mechanisms, upon post-translational modifications, including the conjugation and hydrolysis of ubiquitin. An example is the precise control of the levels of the DNA replication initiation protein Cdt1 and its inhibitor Geminin by ubiquitin-mediated proteasomal degradation. This control ensures that DNA replication occurs with the right timing during the cell cycle, thereby avoiding re-replication events. Here, we review the events that involve ubiquitin signalling during DNA replication initiation, and how they are linked to human disease.
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http://dx.doi.org/10.3390/cells7100146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211026PMC
September 2018

A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression.

Nucleic Acids Res 2018 11;46(19):10132-10144

Oncode Institute, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here, we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which gives rise to aneuploid progeny.
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http://dx.doi.org/10.1093/nar/gky786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6212793PMC
November 2018

Histone Ubiquitination by the DNA Damage Response Is Required for Efficient DNA Replication in Unperturbed S Phase.

Mol Cell 2018 09 16;71(6):897-910.e8. Epub 2018 Aug 16.

Institute of Molecular Cancer Research, University of Zurich, Zurich 8057, Switzerland. Electronic address:

Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.
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http://dx.doi.org/10.1016/j.molcel.2018.07.011DOI Listing
September 2018

ATR-mediated proteome remodeling is a major determinant of homologous recombination capacity in cancer cells.

Nucleic Acids Res 2018 09;46(16):8311-8325

Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853, USA.

The ATR kinase is crucial for genome maintenance, but the mechanisms by which ATR controls the DNA repair machinery are not fully understood. Here, we find that long-term chronic inhibition of ATR signaling severely impairs the ability of cells to utilize homologous recombination (HR)-mediated DNA repair. Proteomic analysis shows that chronic ATR inhibition depletes the abundance of key HR factors, suggesting that spontaneous ATR signaling enhances the capacity of cells to use HR-mediated repair by controlling the abundance of the HR machinery. Notably, ATR controls the abundance of HR factors largely via CHK1-dependent transcription, and can also promote stabilization of specific HR proteins. Cancer cells exhibit a strong dependency on ATR signaling for maintaining elevated levels of HR factors, and we propose that increased constitutive ATR signaling caused by augmented replication stress in cancer cells drives the enhanced HR capacity observed in certain tumor types. Overall, these findings define a major pro-HR function for ATR and have important implications for therapy by providing rationale for sensitizing HR-proficient cancer cells to PARP inhibitors.
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http://dx.doi.org/10.1093/nar/gky625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6144784PMC
September 2018

Claspin - checkpoint adaptor and DNA replication factor.

FEBS J 2019 02 29;286(3):441-455. Epub 2018 Jun 29.

Facultad de Medicina, Instituto de Tecnologías Biomédicas, Centro de Investigaciones Biomédicas de Canarias, Universidad de La Laguna, Tenerife, Spain.

Claspin was discovered as a Chk1-interacting protein necessary for Chk1 phosphorylation and activation by the upstream kinase, ATR, in response to DNA synthesis inhibition in Xenopus oocyte extracts. Subsequent investigations have defined a molecular model in which Claspin acts as an adaptor or scaffold protein to facilitate activation of Chk1 by ATR within a multiprotein complex that forms on single-stranded DNA at stalled replication forks and sites of DNA damage. Interestingly, Claspin is an unstable protein whose degradation via the proteasome is tightly regulated via ubiquitination and controlled by multiple ubiquitin ligases and deubiquitinases. As a result, Claspin levels fluctuate during the cell cycle, contributing to the regulation of checkpoint proficiency and playing a key role in terminating checkpoint-mediated cell cycle arrest. In addition to its role in signalling genotoxic stress, Claspin is required to maintain normal rates of replication fork progression during unperturbed DNA replication and may contribute to the regulation of replication origin firing. Consistent with this, Claspin can bind directly to DNA, with particular affinity for branched or forked molecules, and it interacts with multiple protein components of the replisome. As expected for a protein with key roles in checkpoint signalling and genome duplication, aberrations of Claspin expression and structure have been observed in cancer. Claspin is furthermore targeted to facilitate viral replication and plays a role in suppressing cellular DNA synthesis in response to nongenotoxic endoplasmic reticulum stress. Here, we review the functions and regulation of Claspin with a focus on areas of active research.
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http://dx.doi.org/10.1111/febs.14594DOI Listing
February 2019

Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.

FASEB J 2018 09 17;32(9):4984-4999. Epub 2018 Apr 17.

Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute, Herston, Queensland, Australia.

Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein 55 kDa (CEP55) is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 (TEX14) protein, has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic ( Cep55) mice aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55 male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men.-Sinha, D., Kalimutho, M., Bowles, J., Chan, A.-L., Merriner, D. J., Bain, A. L., Simmons, J. L., Freire, R., Lopez, J. A., Hobbs, R. M., O'Bryan, M. K., Khanna, K. K. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.
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http://dx.doi.org/10.1096/fj.201701096RRDOI Listing
September 2018

SUMO regulates p21Cip1 intracellular distribution and with p21Cip1 facilitates multiprotein complex formation in the nucleolus upon DNA damage.

PLoS One 2017 5;12(6):e0178925. Epub 2017 Jun 5.

Departament Biomedicina, Universitat de Barcelona, IDIBAPS, Barcelona, Spain.

We previously showed that p21Cip1 transits through the nucleolus on its way from the nucleus to the cytoplasm and that DNA damage inhibits this transit and induces the formation of p21Cip1-containing intranucleolar bodies (INoBs). Here, we demonstrate that these INoBs also contain SUMO-1 and UBC9, the E2 SUMO-conjugating enzyme. Furthermore, whereas wild type SUMO-1 localized in INoBs, a SUMO-1 mutant, which is unable to conjugate with proteins, does not, suggesting the presence of SUMOylated proteins at INoBs. Moreover, depletion of the SUMO-conjugating enzyme UBC9 or the sumo hydrolase SENP2 changed p21Cip1 intracellular distribution. In addition to SUMO-1 and p21Cip1, cell cycle regulators and DNA damage checkpoint proteins, including Cdk2, Cyclin E, PCNA, p53 and Mdm2, and PML were also detected in INoBs. Importantly, depletion of UBC9 or p21Cip1 impacted INoB biogenesis and the nucleolar accumulation of the cell cycle regulators and DNA damage checkpoint proteins following DNA damage. The impact of p21Cip1 and SUMO-1 on the accumulation of proteins in INoBs extends also to CRM1, a nuclear exportin that is also important for protein translocation from the cytoplasm to the nucleolus. Thus, SUMO and p21Cip1 regulate the transit of proteins through the nucleolus, and that disruption of nucleolar export by DNA damage induces SUMO and p21Cip1 to act as hub proteins to form a multiprotein complex in the nucleolus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178925PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459497PMC
September 2017

TOPBP1 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1.

J Cell Biol 2017 03 22;216(3):623-639. Epub 2017 Feb 22.

Department of Molecular Biology and Genetics, Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY 14853

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1-TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair.
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http://dx.doi.org/10.1083/jcb.201607031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5350513PMC
March 2017

Drosophila Vps13 Is Required for Protein Homeostasis in the Brain.

PLoS One 2017 20;12(1):e0170106. Epub 2017 Jan 20.

Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands.

Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170106PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5249141PMC
August 2017

Metalloprotease SPRTN/DVC1 Orchestrates Replication-Coupled DNA-Protein Crosslink Repair.

Mol Cell 2016 11 27;64(4):704-719. Epub 2016 Oct 27.

Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK. Electronic address:

The cytotoxicity of DNA-protein crosslinks (DPCs) is largely ascribed to their ability to block the progression of DNA replication. DPCs frequently occur in cells, either as a consequence of metabolism or exogenous agents, but the mechanism of DPC repair is not completely understood. Here, we characterize SPRTN as a specialized DNA-dependent and DNA replication-coupled metalloprotease for DPC repair. SPRTN cleaves various DNA binding substrates during S-phase progression and thus protects proliferative cells from DPC toxicity. Ruijs-Aalfs syndrome (RJALS) patient cells with monogenic and biallelic mutations in SPRTN are hypersensitive to DPC-inducing agents due to a defect in DNA replication fork progression and the inability to eliminate DPCs. We propose that SPRTN protease represents a specialized DNA replication-coupled DPC repair pathway essential for DNA replication progression and genome stability. Defective SPRTN-dependent clearance of DPCs is the molecular mechanism underlying RJALS, and DPCs are contributing to accelerated aging and cancer.
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http://dx.doi.org/10.1016/j.molcel.2016.09.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5128727PMC
November 2016

Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection.

Nat Commun 2016 08 26;7:12628. Epub 2016 Aug 26.

University of Zurich, Institute of Molecular Cancer Research, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity.
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http://dx.doi.org/10.1038/ncomms12628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007465PMC
August 2016

USP7/HAUSP: A SUMO deubiquitinase at the heart of DNA replication.

Bioessays 2016 09 4;38(9):863-8. Epub 2016 Jul 4.

Unidad de Investigación, Hosptial Universitario de Canarias, Instituto de Tecnologías Biomédicas, La Laguna, Tenerife, Spain.

DNA replication is both highly conserved and controlled. Problematic DNA replication can lead to genomic instability and therefore carcinogenesis. Numerous mechanisms work together to achieve this tight control and increasing evidence suggests that post-translational modifications (phosphorylation, ubiquitination, SUMOylation) of DNA replication proteins play a pivotal role in this process. Here we discuss such modifications in the light of a recent article that describes a novel role for the deubiquitinase (DUB) USP7/HAUSP in the control of DNA replication. USP7 achieves this function by an unusual and novel mechanism, namely deubiquitination of SUMOylated proteins at the replication fork, making USP7 also a SUMO DUB (SDUB). This work extends previous observations of increased levels of SUMO and low levels of ubiquitin at the on-going replication fork. Here, we discuss this novel study, its contribution to the DNA replication and genomic stability field and what questions arise from this work.
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http://dx.doi.org/10.1002/bies.201600096DOI Listing
September 2016

USP37 deubiquitinates Cdt1 and contributes to regulate DNA replication.

Mol Oncol 2016 10 3;10(8):1196-206. Epub 2016 Jun 3.

Unidad de Investigación, Hospital Universitario de Canarias, Instituto de Tecnologías Biomédicas, Ofra s/n, La Laguna, Tenerife, Spain. Electronic address:

DNA replication control is a key process in maintaining genomic integrity. Monitoring DNA replication initiation is particularly important as it needs to be coordinated with other cellular events and should occur only once per cell cycle. Crucial players in the initiation of DNA replication are the ORC protein complex, marking the origin of replication, and the Cdt1 and Cdc6 proteins, that license these origins to replicate by recruiting the MCM2-7 helicase. To accurately achieve its functions, Cdt1 is tightly regulated. Cdt1 levels are high from metaphase and during G1 and low in S/G2 phases of the cell cycle. This control is achieved, among other processes, by ubiquitination and proteasomal degradation. In an overexpression screen for Cdt1 deubiquitinating enzymes, we isolated USP37, to date the first ubiquitin hydrolase controlling Cdt1. USP37 overexpression stabilizes Cdt1, most likely a phosphorylated form of the protein. In contrast, USP37 knock down destabilizes Cdt1, predominantly during G1 and G1/S phases of the cell cycle. USP37 interacts with Cdt1 and is able to de-ubiquitinate Cdt1 in vivo and, USP37 is able to regulate the loading of MCM complexes onto the chromatin. In addition, downregulation of USP37 reduces DNA replication fork speed. Taken together, here we show that the deubiquitinase USP37 plays an important role in the regulation of DNA replication. Whether this is achieved via Cdt1, a central protein in this process, which we have shown to be stabilized by USP37, or via additional factors, remains to be tested.
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http://dx.doi.org/10.1016/j.molonc.2016.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423201PMC
October 2016

HUWE1 interacts with PCNA to alleviate replication stress.

EMBO Rep 2016 06 4;17(6):874-86. Epub 2016 May 4.

Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, USA

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S-phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT-type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA We show that HUWE1-knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono-ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.
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http://dx.doi.org/10.15252/embr.201541685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278616PMC
June 2016

New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress.

Nucleic Acids Res 2016 06 2;44(10):4745-62. Epub 2016 Mar 2.

Departament de Biologia Cellular, Immunologia i Neurociències, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Facultat de Medicina, Universitat de Barcelona, C/ Casanova 143, 08036 Barcelona, Spain

Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C(Cdh1) is activated upon severe replication stress. Activation of APC/C(Cdh1) prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C(Cdh1) activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C(Cdh1) activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C(Cdh1) function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.
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http://dx.doi.org/10.1093/nar/gkw132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889930PMC
June 2016