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Raimundo Freire
Universidad Fernando Pessoa Canarias, 35450 Las Palmas de Gran Canaria, Spain

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Journal article
Published: 07 July 2021 in COVID
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A novel beta coronavirus that emerged in late December 2019 triggered a global pandemic. Diagnostic methods for rapid identification of infected individuals were established in new biotechnological approaches. Vaccine production and application to individuals and measurement of SARS-CoV-2 antibodies also began. Serum samples from 240 health care workers were collected at three-month intervals over nine months. Indirect SARS-CoV-2 nucleocapsid IgG ELISA tests were used to identify humoral immune responses. All seropositive individuals and those with borderline ELISA values were tested with a specifically generated multipanel nucleocapsid fragment immunoblot. Of the 240 individuals, 24 showed seroconversion in ELISA after experiencing COVID-19. All of them showed a positive reaction against the full-length nucleocapsid protein in the immunoblot. The highest reactivity was seen either against fragment N(100–300) or in a minority against the posterior part N(200–419). In general, the staining pattern of COVID-19 patients showed four phenotypes. In contrast, three individuals classified as borderline by ELISA reacted exclusively with fragments N(1–220) and N(100–300) containing the octamer amino acid sequence FYYLGTGP, which is identical in human coronaviruses sharing this sequence with SARS-CoV-2. These represent a unique and thus fifth phenotype. This work suggests the existence of distinct phenotypic patterns of IgG production towards N-protein subdomains.

ACS Style

Sahra Pajenda; Sebastian Kapps; Thomas Reiter; Raimundo Freire; Veronique Smits; Ludwig Wagner; Daniela Gerges; Wolfgang Winnicki; Gere Sunder-Plassmann; Alice Schmidt. Antibody Response against the SARS-CoV-2 Nucleocapsid Protein and Its Subdomains—Identification of Pre-Immunization Status by Human Coronaviruses with Multipanel Nucleocapsid Fragment Immunoblotting. COVID 2021, 1, 105 -114.

AMA Style

Sahra Pajenda, Sebastian Kapps, Thomas Reiter, Raimundo Freire, Veronique Smits, Ludwig Wagner, Daniela Gerges, Wolfgang Winnicki, Gere Sunder-Plassmann, Alice Schmidt. Antibody Response against the SARS-CoV-2 Nucleocapsid Protein and Its Subdomains—Identification of Pre-Immunization Status by Human Coronaviruses with Multipanel Nucleocapsid Fragment Immunoblotting. COVID. 2021; 1 (1):105-114.

Chicago/Turabian Style

Sahra Pajenda; Sebastian Kapps; Thomas Reiter; Raimundo Freire; Veronique Smits; Ludwig Wagner; Daniela Gerges; Wolfgang Winnicki; Gere Sunder-Plassmann; Alice Schmidt. 2021. "Antibody Response against the SARS-CoV-2 Nucleocapsid Protein and Its Subdomains—Identification of Pre-Immunization Status by Human Coronaviruses with Multipanel Nucleocapsid Fragment Immunoblotting." COVID 1, no. 1: 105-114.

Preprint content
Published: 10 April 2021
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DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here we report that testis-specific RAD1 disruption resulted in impaired DSB repair, germ cell depletion and infertility. UnlikeHus1orRad9adisruption,Rad1loss also caused defects in homolog synapsis, ATR signaling and meiotic sex chromosome inactivation. Comprehensive testis phosphoproteomics revealed that RAD1 and ATR coordinately regulate numerous proteins involved in DSB repair, meiotic silencing, synaptonemal complex formation, and cohesion. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

ACS Style

Catalina Pereira; Gerardo A. Arroyo-Martinez; Matthew Z. Guo; Michael S. Downey; Emma R. Kelly; Kathryn J. Grive; Shantha K. Mahadevaiah; Jennie Sims; Vitor Marcel Faça; Charlton Tsai; Carl J. Schiltz; Niek Wit; Heinz Jacobs; Nathan L. Clark; Raimundo Freire; James M. A. Turner; Amy M. Lyndaker; Miguel A. Brieño-Enríquez; Paula E. Cohen; Marcus B. Smolka; Robert S. Weiss. Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis. 2021, 1 .

AMA Style

Catalina Pereira, Gerardo A. Arroyo-Martinez, Matthew Z. Guo, Michael S. Downey, Emma R. Kelly, Kathryn J. Grive, Shantha K. Mahadevaiah, Jennie Sims, Vitor Marcel Faça, Charlton Tsai, Carl J. Schiltz, Niek Wit, Heinz Jacobs, Nathan L. Clark, Raimundo Freire, James M. A. Turner, Amy M. Lyndaker, Miguel A. Brieño-Enríquez, Paula E. Cohen, Marcus B. Smolka, Robert S. Weiss. Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis. . 2021; ():1.

Chicago/Turabian Style

Catalina Pereira; Gerardo A. Arroyo-Martinez; Matthew Z. Guo; Michael S. Downey; Emma R. Kelly; Kathryn J. Grive; Shantha K. Mahadevaiah; Jennie Sims; Vitor Marcel Faça; Charlton Tsai; Carl J. Schiltz; Niek Wit; Heinz Jacobs; Nathan L. Clark; Raimundo Freire; James M. A. Turner; Amy M. Lyndaker; Miguel A. Brieño-Enríquez; Paula E. Cohen; Marcus B. Smolka; Robert S. Weiss. 2021. "Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis." , no. : 1.

Standard article
Published: 02 March 2021 in Journal of Veterinary Internal Medicine
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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.

ACS Style

Aleksandra Pawlak; Joanna Bajzert; Katarzyna Bugiel; Beatriz Hernández Suárez; Justyna Kutkowska; Andrzej Rapak; Wojciech Hildebrand; Bożena Obmińska‐Mrukowicz; Raimundo Freire; Veronique A. J. Smits. Ubiquitin‐specific protease 7 as a potential therapeutic target in dogs with hematopoietic malignancies. Journal of Veterinary Internal Medicine 2021, 35, 1041 -1051.

AMA Style

Aleksandra Pawlak, Joanna Bajzert, Katarzyna Bugiel, Beatriz Hernández Suárez, Justyna Kutkowska, Andrzej Rapak, Wojciech Hildebrand, Bożena Obmińska‐Mrukowicz, Raimundo Freire, Veronique A. J. Smits. Ubiquitin‐specific protease 7 as a potential therapeutic target in dogs with hematopoietic malignancies. Journal of Veterinary Internal Medicine. 2021; 35 (2):1041-1051.

Chicago/Turabian Style

Aleksandra Pawlak; Joanna Bajzert; Katarzyna Bugiel; Beatriz Hernández Suárez; Justyna Kutkowska; Andrzej Rapak; Wojciech Hildebrand; Bożena Obmińska‐Mrukowicz; Raimundo Freire; Veronique A. J. Smits. 2021. "Ubiquitin‐specific protease 7 as a potential therapeutic target in dogs with hematopoietic malignancies." Journal of Veterinary Internal Medicine 35, no. 2: 1041-1051.

Short communication
Published: 22 January 2021 in Biochemical and Biophysical Research Communications
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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.

ACS Style

Veronique A.J. Smits; Esperanza Hernández-Carralero; María Cristina Paz-Cabrera; Elisa Cabrera; Yeray Hernández-Reyes; Juan Ramón Hernández-Fernaud; David A. Gillespie; Eduardo Salido; Miriam Hernández-Porto; Raimundo Freire. The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients. Biochemical and Biophysical Research Communications 2021, 543, 45 -49.

AMA Style

Veronique A.J. Smits, Esperanza Hernández-Carralero, María Cristina Paz-Cabrera, Elisa Cabrera, Yeray Hernández-Reyes, Juan Ramón Hernández-Fernaud, David A. Gillespie, Eduardo Salido, Miriam Hernández-Porto, Raimundo Freire. The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients. Biochemical and Biophysical Research Communications. 2021; 543 ():45-49.

Chicago/Turabian Style

Veronique A.J. Smits; Esperanza Hernández-Carralero; María Cristina Paz-Cabrera; Elisa Cabrera; Yeray Hernández-Reyes; Juan Ramón Hernández-Fernaud; David A. Gillespie; Eduardo Salido; Miriam Hernández-Porto; Raimundo Freire. 2021. "The Nucleocapsid protein triggers the main humoral immune response in COVID-19 patients." Biochemical and Biophysical Research Communications 543, no. : 45-49.

Journal article
Published: 24 August 2020 in Cancers
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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 (CLSPN) alterations may contribute to cancer development. Therefore, CLSPN 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). CLSPN 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. CLSPN 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 CLSPN variants identified in the present study appear to modulate Claspin’s function by altering CLSPN transcription and RNA processing, as well as Chk1 activation.

ACS Style

Diana Azenha; Santiago Hernandez-Perez; Yuse Martin; Marta S. Viegas; Alexandra Martins; Maria C. Lopes; Eric W. -F. Lam; Raimundo Freire; Teresa C. Martins. Implications of CLSPN Variants in Cellular Function and Susceptibility to Cancer. Cancers 2020, 12, 2396 .

AMA Style

Diana Azenha, Santiago Hernandez-Perez, Yuse Martin, Marta S. Viegas, Alexandra Martins, Maria C. Lopes, Eric W. -F. Lam, Raimundo Freire, Teresa C. Martins. Implications of CLSPN Variants in Cellular Function and Susceptibility to Cancer. Cancers. 2020; 12 (9):2396.

Chicago/Turabian Style

Diana Azenha; Santiago Hernandez-Perez; Yuse Martin; Marta S. Viegas; Alexandra Martins; Maria C. Lopes; Eric W. -F. Lam; Raimundo Freire; Teresa C. Martins. 2020. "Implications of CLSPN Variants in Cellular Function and Susceptibility to Cancer." Cancers 12, no. 9: 2396.

Journal article
Published: 01 May 2020 in NAR Cancer
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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.

ACS Style

Diego Dibitetto; Jennie R Sims; Carolline F R Ascenção; Kevin Feng; Dongsung Kim; Susannah Oberly; Raimundo Freire; Marcus B Smolka. Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling. NAR Cancer 2020, 2, zcaa006 .

AMA Style

Diego Dibitetto, Jennie R Sims, Carolline F R Ascenção, Kevin Feng, Dongsung Kim, Susannah Oberly, Raimundo Freire, Marcus B Smolka. Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling. NAR Cancer. 2020; 2 (2):zcaa006.

Chicago/Turabian Style

Diego Dibitetto; Jennie R Sims; Carolline F R Ascenção; Kevin Feng; Dongsung Kim; Susannah Oberly; Raimundo Freire; Marcus B Smolka. 2020. "Intrinsic ATR signaling shapes DNA end resection and suppresses toxic DNA-PKcs signaling." NAR Cancer 2, no. 2: zcaa006.

Research article
Published: 08 April 2020 in The FASEB Journal
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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.

ACS Style

Sonia Feu; Fernando Unzueta; Alba Llopis; Jennifer I. Semple; Amaia Ercilla; Sandra Guaita‐Esteruelas; Montserrat Jaumot; Raimundo Freire; Neus Agell. OZF is a Claspin‐interacting protein essential to maintain the replication fork progression rate under replication stress. The FASEB Journal 2020, 34, 6907 -6919.

AMA Style

Sonia Feu, Fernando Unzueta, Alba Llopis, Jennifer I. Semple, Amaia Ercilla, Sandra Guaita‐Esteruelas, Montserrat Jaumot, Raimundo Freire, Neus Agell. OZF is a Claspin‐interacting protein essential to maintain the replication fork progression rate under replication stress. The FASEB Journal. 2020; 34 (5):6907-6919.

Chicago/Turabian Style

Sonia Feu; Fernando Unzueta; Alba Llopis; Jennifer I. Semple; Amaia Ercilla; Sandra Guaita‐Esteruelas; Montserrat Jaumot; Raimundo Freire; Neus Agell. 2020. "OZF is a Claspin‐interacting protein essential to maintain the replication fork progression rate under replication stress." The FASEB Journal 34, no. 5: 6907-6919.

Journal article
Published: 30 March 2020 in Nucleic Acids Research
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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.

ACS Style

Ignacio Alonso-De Vega; Maria Cristina Paz-Cabrera; Magdalena B Rother; Wouter W Wiegant; Cintia Checa Rodríguez; Juan Ramon Hernandez Fernaud; Pablo Huertas; Raimundo Freire; Haico Van Attikum; Veronique A J Smits. PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks. Nucleic Acids Research 2020, 48, 4915 -4927.

AMA Style

Ignacio Alonso-De Vega, Maria Cristina Paz-Cabrera, Magdalena B Rother, Wouter W Wiegant, Cintia Checa Rodríguez, Juan Ramon Hernandez Fernaud, Pablo Huertas, Raimundo Freire, Haico Van Attikum, Veronique A J Smits. PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks. Nucleic Acids Research. 2020; 48 (9):4915-4927.

Chicago/Turabian Style

Ignacio Alonso-De Vega; Maria Cristina Paz-Cabrera; Magdalena B Rother; Wouter W Wiegant; Cintia Checa Rodríguez; Juan Ramon Hernandez Fernaud; Pablo Huertas; Raimundo Freire; Haico Van Attikum; Veronique A J Smits. 2020. "PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks." Nucleic Acids Research 48, no. 9: 4915-4927.

Journal article
Published: 09 March 2020 in Nature Communications
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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.

ACS Style

John Fielden; Katherine Wiseman; Ignacio Torrecilla; Shudong Li; Samuel Hume; Shih-Chieh Chiang; Annamaria Ruggiano; Abhay Narayan Singh; Raimundo Freire; Sylvana Hassanieh; Enric Domingo; Iolanda Vendrell; Roman Fischer; Benedikt M. Kessler; Timothy S. Maughan; Sherif F. El-Khamisy; Kristijan Ramadan. TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. Nature Communications 2020, 11, 1 -16.

AMA Style

John Fielden, Katherine Wiseman, Ignacio Torrecilla, Shudong Li, Samuel Hume, Shih-Chieh Chiang, Annamaria Ruggiano, Abhay Narayan Singh, Raimundo Freire, Sylvana Hassanieh, Enric Domingo, Iolanda Vendrell, Roman Fischer, Benedikt M. Kessler, Timothy S. Maughan, Sherif F. El-Khamisy, Kristijan Ramadan. TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts. Nature Communications. 2020; 11 (1):1-16.

Chicago/Turabian Style

John Fielden; Katherine Wiseman; Ignacio Torrecilla; Shudong Li; Samuel Hume; Shih-Chieh Chiang; Annamaria Ruggiano; Abhay Narayan Singh; Raimundo Freire; Sylvana Hassanieh; Enric Domingo; Iolanda Vendrell; Roman Fischer; Benedikt M. Kessler; Timothy S. Maughan; Sherif F. El-Khamisy; Kristijan Ramadan. 2020. "TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts." Nature Communications 11, no. 1: 1-16.

Preprint content
Published: 14 January 2020
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DNA end resection is a key step in homologous recombination-mediated DNA repair. The ability to manipulate resection capacity is expected to be a powerful strategy to rationally modulate DNA repair outcomes in cancer cells and induce selective cell lethality. However, clinically compatible strategies to manipulate resection are not yet well established. Here we find that long-term inhibition of the ATR kinase has a drastic effect on DNA end resection. Inhibition of ATR over multiple cell division cycles depletes the pool of pro-resection factors and prevents RAD51 as well as RAD52-mediated DNA repair, leading to toxic end-joining and hypersensitivity to PARP inhibitors. The effect is markedly distinct from acute ATR inhibition, which blocks RAD51-mediated repair but not resection and RAD52-mediated repair. 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.

ACS Style

Diego DiBitetto; Jennie R. Sims; Carolline F. R. Ascencao; Kevin Feng; Raimundo Freire; Marcus B. Smolka. ATR Inhibitors as Potent Modulators of DNA End Resection Capacity. 2020, 1 .

AMA Style

Diego DiBitetto, Jennie R. Sims, Carolline F. R. Ascencao, Kevin Feng, Raimundo Freire, Marcus B. Smolka. ATR Inhibitors as Potent Modulators of DNA End Resection Capacity. . 2020; ():1.

Chicago/Turabian Style

Diego DiBitetto; Jennie R. Sims; Carolline F. R. Ascencao; Kevin Feng; Raimundo Freire; Marcus B. Smolka. 2020. "ATR Inhibitors as Potent Modulators of DNA End Resection Capacity." , no. : 1.

Journal article
Published: 29 November 2019 in EMBO reports
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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.

ACS Style

Daniël O Warmerdam; Ignacio Alonso‐De Vega; Wouter W Wiegant; Bram Van Den Broek; Magdalena B Rother; Rob Wolthuis; Raimundo Freire; Haico Van Attikum; René H Medema; Veronique Aj Smits. PHF6 promotes non‐homologous end joining and G2 checkpoint recovery. EMBO reports 2019, 21, e48460 .

AMA Style

Daniël O Warmerdam, Ignacio Alonso‐De Vega, Wouter W Wiegant, Bram Van Den Broek, Magdalena B Rother, Rob Wolthuis, Raimundo Freire, Haico Van Attikum, René H Medema, Veronique Aj Smits. PHF6 promotes non‐homologous end joining and G2 checkpoint recovery. EMBO reports. 2019; 21 (1):e48460.

Chicago/Turabian Style

Daniël O Warmerdam; Ignacio Alonso‐De Vega; Wouter W Wiegant; Bram Van Den Broek; Magdalena B Rother; Rob Wolthuis; Raimundo Freire; Haico Van Attikum; René H Medema; Veronique Aj Smits. 2019. "PHF6 promotes non‐homologous end joining and G2 checkpoint recovery." EMBO reports 21, no. 1: e48460.

Journal article
Published: 15 October 2019 in The EMBO Journal
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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.

ACS Style

Abhay Narayan Singh; Judith Oehler; Ignacio Torrecilla; Susan Kilgas; Shudong Li; Bruno Vaz; Claire Guérillon; John Fielden; Esperanza Hernández-Carralero; Elisa Cabrera; Iain Dc Tullis; Mayura Meerang; Paul R Barber; Raimundo Freire; Jason Parsons; Borivoj Vojnovic; Anne E Kiltie; Niels Mailand; Kristijan Ramadan. The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8. The EMBO Journal 2019, 38, e102361 .

AMA Style

Abhay Narayan Singh, Judith Oehler, Ignacio Torrecilla, Susan Kilgas, Shudong Li, Bruno Vaz, Claire Guérillon, John Fielden, Esperanza Hernández-Carralero, Elisa Cabrera, Iain Dc Tullis, Mayura Meerang, Paul R Barber, Raimundo Freire, Jason Parsons, Borivoj Vojnovic, Anne E Kiltie, Niels Mailand, Kristijan Ramadan. The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8. The EMBO Journal. 2019; 38 (21):e102361.

Chicago/Turabian Style

Abhay Narayan Singh; Judith Oehler; Ignacio Torrecilla; Susan Kilgas; Shudong Li; Bruno Vaz; Claire Guérillon; John Fielden; Esperanza Hernández-Carralero; Elisa Cabrera; Iain Dc Tullis; Mayura Meerang; Paul R Barber; Raimundo Freire; Jason Parsons; Borivoj Vojnovic; Anne E Kiltie; Niels Mailand; Kristijan Ramadan. 2019. "The p97–Ataxin 3 complex regulates homeostasis of the DNA damage response E3 ubiquitin ligase RNF 8." The EMBO Journal 38, no. 21: e102361.

Preprint content
Published: 25 September 2019
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Post-translational histone modifications and chromatin remodelling play a critical role in the mechanisms controlling the integrity of the genome. Here we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating the balance between DNA damage-induced focus formation by 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knock down 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 knock down decreases CtIP and BRCA1 protein and mRNA levels and cells depleted of PHF2 display genome instability and are 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.

ACS Style

Ignacio Alonso-De Vega; M. Cristina Paz-Cabrera; Wouter W. Wiegant; Cintia Checa-Rodríguez; Pablo Huertas; Raimundo Freire; Haico Van Attikum; Veronique A.J. Smits. PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks. 2019, 782490 .

AMA Style

Ignacio Alonso-De Vega, M. Cristina Paz-Cabrera, Wouter W. Wiegant, Cintia Checa-Rodríguez, Pablo Huertas, Raimundo Freire, Haico Van Attikum, Veronique A.J. Smits. PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks. . 2019; ():782490.

Chicago/Turabian Style

Ignacio Alonso-De Vega; M. Cristina Paz-Cabrera; Wouter W. Wiegant; Cintia Checa-Rodríguez; Pablo Huertas; Raimundo Freire; Haico Van Attikum; Veronique A.J. Smits. 2019. "PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks." , no. : 782490.

Journal article
Published: 29 July 2019 in Cancers
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: 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.

ACS Style

Elisa Cabrera; Prahlad Raninga; Kum Kum Khanna; Raimundo Freire. GSK3-β Stimulates Claspin Degradation via β-TrCP Ubiquitin Ligase and Alters Cancer Cell Survival. Cancers 2019, 11, 1073 .

AMA Style

Elisa Cabrera, Prahlad Raninga, Kum Kum Khanna, Raimundo Freire. GSK3-β Stimulates Claspin Degradation via β-TrCP Ubiquitin Ligase and Alters Cancer Cell Survival. Cancers. 2019; 11 (8):1073.

Chicago/Turabian Style

Elisa Cabrera; Prahlad Raninga; Kum Kum Khanna; Raimundo Freire. 2019. "GSK3-β Stimulates Claspin Degradation via β-TrCP Ubiquitin Ligase and Alters Cancer Cell Survival." Cancers 11, no. 8: 1073.

Correction
Published: 08 May 2019 in Oncogene
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The final sentence of the Acknowledgements should be as follows: This work was supported by grants from Instituto de Salud Carlos III (BA15/00092), Spanish Ministry of Economy and Competitiveness/EU-ERDF (SAF2016-80626-R, SAF2013-49149-R, BFU2014-51672-REDC), Fundación CajaCanarias (AP2015/008) to RF, and the Australian National Health and Medical Research (NHMRC program grant to SRL and KKK (APP1017028).

ACS Style

S. Hernández-Pérez; E. Cabrera; Eduardo Salido; Malcolm Lim; L. Reid; S. R Lakhani; K. K. Khanna; J. M. Saunus; R. Freire. Correction: DUB3 and USP7 de-ubiquitinating enzymes control replication inhibitor Geminin: molecular characterization and associations with breast cancer. Oncogene 2019, 38, 4886 -4886.

AMA Style

S. Hernández-Pérez, E. Cabrera, Eduardo Salido, Malcolm Lim, L. Reid, S. R Lakhani, K. K. Khanna, J. M. Saunus, R. Freire. Correction: DUB3 and USP7 de-ubiquitinating enzymes control replication inhibitor Geminin: molecular characterization and associations with breast cancer. Oncogene. 2019; 38 (24):4886-4886.

Chicago/Turabian Style

S. Hernández-Pérez; E. Cabrera; Eduardo Salido; Malcolm Lim; L. Reid; S. R Lakhani; K. K. Khanna; J. M. Saunus; R. Freire. 2019. "Correction: DUB3 and USP7 de-ubiquitinating enzymes control replication inhibitor Geminin: molecular characterization and associations with breast cancer." Oncogene 38, no. 24: 4886-4886.

Original article
Published: 12 March 2019 in Chromosoma
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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 Pch2TRIP13 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1HORMAD 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.

ACS Style

Esther Herruzo; Beatriz Santos; Raimundo Freire; Jesús A. Carballo; Pedro A. San-Segundo. Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint. Chromosoma 2019, 128, 297 -316.

AMA Style

Esther Herruzo, Beatriz Santos, Raimundo Freire, Jesús A. Carballo, Pedro A. San-Segundo. Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint. Chromosoma. 2019; 128 (3):297-316.

Chicago/Turabian Style

Esther Herruzo; Beatriz Santos; Raimundo Freire; Jesús A. Carballo; Pedro A. San-Segundo. 2019. "Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint." Chromosoma 128, no. 3: 297-316.

Preprint content
Published: 05 February 2019
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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 Pch2TRIP13 AAA+ ATPase participates in this pathway by supporting phosphorylation of the Hop1HORMAD 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 on 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.

ACS Style

Esther Herruzo; Beatriz Santos; Raimundo Freire; Jesús A. Carballo; Pedro A. San-Segundo. Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint. 2019, 541367 .

AMA Style

Esther Herruzo, Beatriz Santos, Raimundo Freire, Jesús A. Carballo, Pedro A. San-Segundo. Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint. . 2019; ():541367.

Chicago/Turabian Style

Esther Herruzo; Beatriz Santos; Raimundo Freire; Jesús A. Carballo; Pedro A. San-Segundo. 2019. "Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint." , no. : 541367.

Journal article
Published: 03 December 2018 in Scientific Reports
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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.

ACS Style

Eun-Yeung Gong; Beatriz Hernández; Jessica Hernández Nielsen; Veronique A. J. Smits; Raimundo Freire; David A. Gillespie. Chk1 KA1 domain auto-phosphorylation stimulates biological activity and is linked to rapid proteasomal degradation. Scientific Reports 2018, 8, 17536 .

AMA Style

Eun-Yeung Gong, Beatriz Hernández, Jessica Hernández Nielsen, Veronique A. J. Smits, Raimundo Freire, David A. Gillespie. Chk1 KA1 domain auto-phosphorylation stimulates biological activity and is linked to rapid proteasomal degradation. Scientific Reports. 2018; 8 (1):17536.

Chicago/Turabian Style

Eun-Yeung Gong; Beatriz Hernández; Jessica Hernández Nielsen; Veronique A. J. Smits; Raimundo Freire; David A. Gillespie. 2018. "Chk1 KA1 domain auto-phosphorylation stimulates biological activity and is linked to rapid proteasomal degradation." Scientific Reports 8, no. 1: 17536.

Review
Published: 20 September 2018 in Cells
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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.

ACS Style

Esperanza Hernández-Carralero; Elisa Cabrera; Ignacio Alonso-De Vega; Santiago Hernández-Pérez; Veronique A. J. Smits; Raimundo Freire. Control of DNA Replication Initiation by Ubiquitin. Cells 2018, 7, 146 .

AMA Style

Esperanza Hernández-Carralero, Elisa Cabrera, Ignacio Alonso-De Vega, Santiago Hernández-Pérez, Veronique A. J. Smits, Raimundo Freire. Control of DNA Replication Initiation by Ubiquitin. Cells. 2018; 7 (10):146.

Chicago/Turabian Style

Esperanza Hernández-Carralero; Elisa Cabrera; Ignacio Alonso-De Vega; Santiago Hernández-Pérez; Veronique A. J. Smits; Raimundo Freire. 2018. "Control of DNA Replication Initiation by Ubiquitin." Cells 7, no. 10: 146.

Journal article
Published: 03 September 2018 in Nucleic Acids Research
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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.

ACS Style

Jeroen Van Den Berg; Anna G. Manjón; Karoline Kielbassa; Femke Feringa; Raimundo Freire; René H Medema. A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression. Nucleic Acids Research 2018, 46, 10132 -10144.

AMA Style

Jeroen Van Den Berg, Anna G. Manjón, Karoline Kielbassa, Femke Feringa, Raimundo Freire, René H Medema. A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression. Nucleic Acids Research. 2018; 46 (19):10132-10144.

Chicago/Turabian Style

Jeroen Van Den Berg; Anna G. Manjón; Karoline Kielbassa; Femke Feringa; Raimundo Freire; René H Medema. 2018. "A limited number of double-strand DNA breaks is sufficient to delay cell cycle progression." Nucleic Acids Research 46, no. 19: 10132-10144.