Publications by authors named "Felipe Cortés-Ledesma"

31 Publications

Topoisomerase IIα represses transcription by enforcing promoter-proximal pausing.

Cell Rep 2021 Apr;35(2):108977

Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Universidad de Sevilla-CSIC-Universidad Pablo de Olavide, Sevilla 41092, Spain; Topology and DNA Breaks Group, Spanish National Cancer Centre (CNIO), Madrid 28029, Spain. Electronic address:

Accumulation of topological stress in the form of DNA supercoiling is inherent to the advance of RNA polymerase II (Pol II) and needs to be resolved by DNA topoisomerases to sustain productive transcriptional elongation. Topoisomerases are therefore considered positive facilitators of transcription. Here, we show that, in contrast to this general assumption, human topoisomerase IIα (TOP2A) activity at promoters represses transcription of immediate early genes such as c-FOS, maintaining them under basal repressed conditions. Thus, TOP2A inhibition creates a particular topological context that results in rapid release from promoter-proximal pausing and transcriptional upregulation, which mimics the typical bursting behavior of these genes in response to physiological stimulus. We therefore describe the control of promoter-proximal pausing by TOP2A as a layer for the regulation of gene expression, which can act as a molecular switch to rapidly activate transcription, possibly by regulating the accumulation of DNA supercoiling at promoter regions.
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http://dx.doi.org/10.1016/j.celrep.2021.108977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052185PMC
April 2021

Genome-wide prediction of topoisomerase IIβ binding by architectural factors and chromatin accessibility.

PLoS Comput Biol 2021 01 19;17(1):e1007814. Epub 2021 Jan 19.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC-Universidad de Sevilla-Universidad Pablo de Olavide, Seville, Spain.

DNA topoisomerase II-β (TOP2B) is fundamental to remove topological problems linked to DNA metabolism and 3D chromatin architecture, but its cut-and-reseal catalytic mechanism can accidentally cause DNA double-strand breaks (DSBs) that can seriously compromise genome integrity. Understanding the factors that determine the genome-wide distribution of TOP2B is therefore not only essential for a complete knowledge of genome dynamics and organization, but also for the implications of TOP2-induced DSBs in the origin of oncogenic translocations and other types of chromosomal rearrangements. Here, we conduct a machine-learning approach for the prediction of TOP2B binding using publicly available sequencing data. We achieve highly accurate predictions, with accessible chromatin and architectural factors being the most informative features. Strikingly, TOP2B is sufficiently explained by only three features: DNase I hypersensitivity, CTCF and cohesin binding, for which genome-wide data are widely available. Based on this, we develop a predictive model for TOP2B genome-wide binding that can be used across cell lines and species, and generate virtual probability tracks that accurately mirror experimental ChIP-seq data. Our results deepen our knowledge on how the accessibility and 3D organization of chromatin determine TOP2B function, and constitute a proof of principle regarding the in silico prediction of sequence-independent chromatin-binding factors.
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http://dx.doi.org/10.1371/journal.pcbi.1007814DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7845959PMC
January 2021

A Genetic Map of the Response to DNA Damage in Human Cells.

Cell 2020 07 9;182(2):481-496.e21. Epub 2020 Jul 9.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, M5G 1X5, Canada; Department of Molecular Genetics, University of Toronto, 1 King's College Circle, Toronto, ON, M5S 1A8, Canada. Electronic address:

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.
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http://dx.doi.org/10.1016/j.cell.2020.05.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384976PMC
July 2020

Ubiquitin stimulated reversal of topoisomerase 2 DNA-protein crosslinks by TDP2.

Nucleic Acids Res 2020 06;48(11):6310-6325

Structural Cell Biology Group, Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, US Department of Health and Human Services, Research Triangle Park, NC 27709, USA.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) reverses Topoisomerase 2 DNA-protein crosslinks (TOP2-DPCs) in a direct-reversal pathway licensed by ZATTZNF451 SUMO2 E3 ligase and SUMOylation of TOP2. TDP2 also binds ubiquitin (Ub), but how Ub regulates TDP2 functions is unknown. Here, we show that TDP2 co-purifies with K63 and K27 poly-Ubiquitinated cellular proteins independently of, and separately from SUMOylated TOP2 complexes. Poly-ubiquitin chains of ≥ Ub3 stimulate TDP2 catalytic activity in nuclear extracts and enhance TDP2 binding of DNA-protein crosslinks in vitro. X-ray crystal structures and small-angle X-ray scattering analysis of TDP2-Ub complexes reveal that the TDP2 UBA domain binds K63-Ub3 in a 1:1 stoichiometric complex that relieves a UBA-regulated autoinhibitory state of TDP2. Our data indicates that that poly-Ub regulates TDP2-catalyzed TOP2-DPC removal, and TDP2 single nucleotide polymorphisms can disrupt the TDP2-Ubiquitin interface.
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http://dx.doi.org/10.1093/nar/gkaa318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293035PMC
June 2020

TDP2 suppresses genomic instability induced by androgens in the epithelial cells of prostate glands.

Genes Cells 2020 Jul 5;25(7):450-465. Epub 2020 May 5.

Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

Androgens stimulate the proliferation of epithelial cells in the prostate by activating topoisomerase 2 (TOP2) and regulating the transcription of target genes. TOP2 resolves the entanglement of genomic DNA by transiently generating double-strand breaks (DSBs), where TOP2 homodimers covalently bind to 5' DSB ends, called TOP2-DNA cleavage complexes (TOP2ccs). When TOP2 fails to rejoin TOP2ccs generating stalled TOP2ccs, tyrosyl DNA phosphodiesterase-2 (TDP2) removes 5' TOP2 adducts from stalled TOP2ccs prior to the ligation of the DSBs by nonhomologous end joining (NHEJ), the dominant DSB repair pathway in G /G phases. We previously showed that estrogens frequently generate stalled TOP2ccs in G /G phases. Here, we show that physiological concentrations of androgens induce several DSBs in individual human prostate cancer cells during G phase, and loss of TDP2 causes a five times higher number of androgen-induced chromosome breaks in mitotic chromosome spreads. Intraperitoneally injected androgens induce several DSBs in individual epithelial cells of the prostate in TDP2-deficient mice, even at 20 hr postinjection. In conclusion, physiological concentrations of androgens have very strong genotoxicity, most likely by generating stalled TOP2ccs.
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http://dx.doi.org/10.1111/gtc.12770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497232PMC
July 2020

Endogenous topoisomerase II-mediated DNA breaks drive thymic cancer predisposition linked to ATM deficiency.

Nat Commun 2020 02 14;11(1):910. Epub 2020 Feb 14.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC-Universidad de Sevilla-Universidad Pablo de Olavide, Sevilla, 41092, Spain.

The ATM kinase is a master regulator of the DNA damage response to double-strand breaks (DSBs) and a well-established tumour suppressor whose loss is the cause of the neurodegenerative and cancer-prone syndrome Ataxia-Telangiectasia (A-T). A-T patients and Atm mouse models are particularly predisposed to develop lymphoid cancers derived from deficient repair of RAG-induced DSBs during V(D)J recombination. Here, we unexpectedly find that specifically disturbing the repair of DSBs produced by DNA topoisomerase II (TOP2) by genetically removing the highly specialised repair enzyme TDP2 increases the incidence of thymic tumours in Atm mice. Furthermore, we find that TOP2 strongly colocalizes with RAG, both genome-wide and at V(D)J recombination sites, resulting in an increased endogenous chromosomal fragility of these regions. Thus, our findings demonstrate a strong causal relationship between endogenous TOP2-induced DSBs and cancer development, confirming these lesions as major drivers of ATM-deficient lymphoid malignancies, and potentially other conditions and cancer types.
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http://dx.doi.org/10.1038/s41467-020-14638-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021672PMC
February 2020

"An End to a Means": How DNA-End Structure Shapes the Double-Strand Break Repair Process.

Front Mol Biosci 2019 10;6:153. Epub 2020 Jan 10.

Andalusian Center of Molecular Biology and Regenerative Medicine (CABIMER-CSIC-University of Seville-Pablo de Olavide University), Seville, Spain.

Endogenously-arising DNA double-strand breaks (DSBs) rarely harbor canonical 5'-phosphate, 3'-hydroxyl moieties at the ends, which are, regardless of the pathway used, ultimately required for their repair. Cells are therefore endowed with a wide variety of enzymes that can deal with these chemical and structural variations and guarantee the formation of ligatable termini. An important distinction is whether the ends are directly "unblocked" by specific enzymatic activities without affecting the integrity of the DNA molecule and its sequence, or whether they are "processed" by unspecific nucleases that remove nucleotides from the termini. DNA end structure and configuration, therefore, shape the repair process, its requirements, and, importantly, its final outcome. Thus, the molecular mechanisms that coordinate and integrate the cellular response to blocked DSBs, although still largely unexplored, can be particularly relevant for maintaining genome integrity and avoiding malignant transformation and cancer.
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http://dx.doi.org/10.3389/fmolb.2019.00153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965357PMC
January 2020

GSE4 peptide suppresses oxidative and telomere deficiencies in ataxia telangiectasia patient cells.

Cell Death Differ 2019 Oct 22;26(10):1998-2014. Epub 2019 Jan 22.

Instituto de Investigaciones Biomédicas CSIC/UAM, IDiPaz, C/ Arturo Duperier, 4, 28029, Madrid, Spain.

Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H0 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.
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http://dx.doi.org/10.1038/s41418-018-0272-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748109PMC
October 2019

ZATT (ZNF451)-mediated resolution of topoisomerase 2 DNA-protein cross-links.

Science 2017 09 14;357(6358):1412-1416. Epub 2017 Sep 14.

Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences (NIEHS), Research Triangle Park, NC 27709, USA.

Topoisomerase 2 (TOP2) DNA transactions proceed via formation of the TOP2 cleavage complex (TOP2cc), a covalent enzyme-DNA reaction intermediate that is vulnerable to trapping by potent anticancer TOP2 drugs. How genotoxic TOP2 DNA-protein cross-links are resolved is unclear. We found that the SUMO (small ubiquitin-related modifier) ligase ZATT (ZNF451) is a multifunctional DNA repair factor that controls cellular responses to TOP2 damage. ZATT binding to TOP2cc facilitates a proteasome-independent tyrosyl-DNA phosphodiesterase 2 (TDP2) hydrolase activity on stalled TOP2cc. The ZATT SUMO ligase activity further promotes TDP2 interactions with SUMOylated TOP2, regulating efficient TDP2 recruitment through a "split-SIM" SUMO2 engagement platform. These findings uncover a ZATT-TDP2-catalyzed and SUMO2-modulated pathway for direct resolution of TOP2cc.
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http://dx.doi.org/10.1126/science.aam6468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623066PMC
September 2017

Chd7 is indispensable for mammalian brain development through activation of a neuronal differentiation programme.

Nat Commun 2017 03 20;8:14758. Epub 2017 Mar 20.

Division of Molecular Neurogenetics, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 280, Heidelberg 69120, Germany.

Mutations in chromatin modifier genes are frequently associated with neurodevelopmental diseases. We herein demonstrate that the chromodomain helicase DNA-binding protein 7 (Chd7), frequently associated with CHARGE syndrome, is indispensable for normal cerebellar development. Genetic inactivation of Chd7 in cerebellar granule neuron progenitors leads to cerebellar hypoplasia in mice, due to the impairment of granule neuron differentiation, induction of apoptosis and abnormal localization of Purkinje cells, which closely recapitulates known clinical features in the cerebella of CHARGE patients. Combinatory molecular analyses reveal that Chd7 is required for the maintenance of open chromatin and thus activation of genes essential for granule neuron differentiation. We further demonstrate that both Chd7 and Top2b are necessary for the transcription of a set of long neuronal genes in cerebellar granule neurons. Altogether, our comprehensive analyses reveal a mechanism with chromatin remodellers governing brain development via controlling a core transcriptional programme for cell-specific differentiation.
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http://dx.doi.org/10.1038/ncomms14758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364396PMC
March 2017

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining.

DNA Repair (Amst) 2017 03 17;51:31-45. Epub 2017 Jan 17.

Departamento Bioquímica Vegetal y Biología Molecular, Universidad de Sevilla, Sevilla 41092, Spain; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla/CSIC, Sevilla 41092, Spain. Electronic address:

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.
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http://dx.doi.org/10.1016/j.dnarep.2017.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444907PMC
March 2017

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections.

mBio 2015 Dec 29;7(1):e01931-15. Epub 2015 Dec 29.

Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA

Unlabelled: Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5' tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5' end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis) in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections.

Importance: Picornaviruses are one of the most prevalent groups of viruses that infect humans and livestock worldwide. These viruses include the human pathogens belonging to the Enterovirus genus, such as poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus. Diseases caused by enteroviruses pose a major problem for public health and have significant economic impact. Poliovirus can cause paralytic poliomyelitis. CVB3 can cause hand, foot, and mouth disease and myocarditis. Human rhinovirus is the causative agent of the common cold, which has a severe economic impact due to lost productivity and severe health consequences in individuals with respiratory dysfunction, such as asthma. By gaining a better understanding of the enterovirus replication cycle, antiviral drugs against enteroviruses may be developed. Here, we report that the absence of the cellular enzyme TDP2 can significantly decrease viral yields of poliovirus, CVB3, and human rhinovirus, making TDP2 a potential target for an antiviral against enterovirus infections.
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http://dx.doi.org/10.1128/mBio.01931-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725011PMC
December 2015

Non-redundant Functions of ATM and DNA-PKcs in Response to DNA Double-Strand Breaks.

Cell Rep 2015 Nov 12;13(8):1598-609. Epub 2015 Nov 12.

Université de Toulouse, UPS, LBCMCP, 118 route de Narbonne, 31062 Toulouse, France; CNRS, LBCMCP, 31062 Toulouse, France. Electronic address:

DNA double-strand breaks (DSBs) elicit the so-called DNA damage response (DDR), largely relying on ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs), two members of the PI3K-like kinase family, whose respective functions during the sequential steps of the DDR remains controversial. Using the DIvA system (DSB inducible via AsiSI) combined with high-resolution mapping and advanced microscopy, we uncovered that both ATM and DNA-PKcs spread in cis on a confined region surrounding DSBs, independently of the pathway used for repair. However, once recruited, these kinases exhibit non-overlapping functions on end joining and γH2AX domain establishment. More specifically, we found that ATM is required to ensure the association of multiple DSBs within "repair foci." Our results suggest that ATM acts not only on chromatin marks but also on higher-order chromatin organization to ensure repair accuracy and survival.
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http://dx.doi.org/10.1016/j.celrep.2015.10.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670905PMC
November 2015

Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

PLoS One 2015 16;10(6):e0128401. Epub 2015 Jun 16.

Department of Microbiology and Immunology, Hershey, The Pennsylvania State University, College of Medicine, Hershey, Pennsylvania, United States of America.

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5' end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5' DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5' DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5' end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0128401PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469307PMC
April 2016

TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function.

Nat Genet 2014 May 23;46(5):516-21. Epub 2014 Mar 23.

Genome Damage and Stability Centre, School of Biological Sciences, University of Sussex, Sussex, UK.

Topoisomerase II (TOP2) removes torsional stress from DNA and facilitates gene transcription by introducing transient DNA double-strand breaks (DSBs). Such DSBs are normally rejoined by TOP2 but on occasion can become abortive and remain unsealed. Here we identify homozygous mutations in the TDP2 gene encoding tyrosyl DNA phosphodiesterase-2, an enzyme that repairs 'abortive' TOP2-induced DSBs, in individuals with intellectual disability, seizures and ataxia. We show that cells from affected individuals are hypersensitive to TOP2-induced DSBs and that loss of TDP2 inhibits TOP2-dependent gene transcription in cultured human cells and in mouse post-mitotic neurons following abortive TOP2 activity. Notably, TDP2 is also required for normal levels of many gene transcripts in developing mouse brain, including numerous gene transcripts associated with neurological function and/or disease, and for normal interneuron density in mouse cerebellum. Collectively, these data implicate chromosome breakage by TOP2 as an endogenous threat to gene transcription and to normal neuronal development and maintenance.
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http://dx.doi.org/10.1038/ng.2929DOI Listing
May 2014

ATM specifically mediates repair of double-strand breaks with blocked DNA ends.

Nat Commun 2014 Feb 27;5:3347. Epub 2014 Feb 27.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), CSIC-Universidad de Sevilla (Departamento de Genética), Sevilla 41092, Spain.

Ataxia telangiectasia is caused by mutations in ATM and represents a paradigm for cancer predisposition and neurodegenerative syndromes linked to deficiencies in the DNA-damage response. The role of ATM as a key regulator of signalling following DNA double-strand breaks (DSBs) has been dissected in extraordinary detail, but the impact of this process on DSB repair still remains controversial. Here we develop novel genetic and molecular tools to modify the structure of DSB ends and demonstrate that ATM is indeed required for efficient and accurate DSB repair, preventing cell death and genome instability, but exclusively when the ends are irreversibly blocked. We therefore identify the nature of ATM involvement in DSB repair, presenting blocked DNA ends as a possible pathogenic trigger of ataxia telangiectasia and related disorders.
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http://dx.doi.org/10.1038/ncomms4347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948078PMC
February 2014

TDP2-dependent non-homologous end-joining protects against topoisomerase II-induced DNA breaks and genome instability in cells and in vivo.

PLoS Genet 2013 7;9(3):e1003226. Epub 2013 Mar 7.

Genome Damage and Stability Centre, University of Sussex, Falmer, United Kingdom.

Anticancer topoisomerase "poisons" exploit the break-and-rejoining mechanism of topoisomerase II (TOP2) to generate TOP2-linked DNA double-strand breaks (DSBs). This characteristic underlies the clinical efficacy of TOP2 poisons, but is also implicated in chromosomal translocations and genome instability associated with secondary, treatment-related, haematological malignancy. Despite this relevance for cancer therapy, the mechanistic aspects governing repair of TOP2-induced DSBs and the physiological consequences that absent or aberrant repair can have are still poorly understood. To address these deficits, we employed cells and mice lacking tyrosyl DNA phosphodiesterase 2 (TDP2), an enzyme that hydrolyses 5'-phosphotyrosyl bonds at TOP2-associated DSBs, and studied their response to TOP2 poisons. Our results demonstrate that TDP2 functions in non-homologous end-joining (NHEJ) and liberates DSB termini that are competent for ligation. Moreover, we show that the absence of TDP2 in cells impairs not only the capacity to repair TOP2-induced DSBs but also the accuracy of the process, thus compromising genome integrity. Most importantly, we find this TDP2-dependent NHEJ mechanism to be physiologically relevant, as Tdp2-deleted mice are sensitive to TOP2-induced damage, displaying marked lymphoid toxicity, severe intestinal damage, and increased genome instability in the bone marrow. Collectively, our data reveal TDP2-mediated error-free NHEJ as an efficient and accurate mechanism to repair TOP2-induced DSBs. Given the widespread use of TOP2 poisons in cancer chemotherapy, this raises the possibility of TDP2 being an important etiological factor in the response of tumours to this type of agent and in the development of treatment-related malignancy.
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http://dx.doi.org/10.1371/journal.pgen.1003226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3592926PMC
June 2013

Competing roles of DNA end resection and non-homologous end joining functions in the repair of replication-born double-strand breaks by sister-chromatid recombination.

Nucleic Acids Res 2013 Feb 18;41(3):1669-83. Epub 2012 Dec 18.

Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av. Américo Vespucio s/n, 41092 Seville, Spain.

While regulating the choice between homologous recombination and non-homologous end joining (NHEJ) as mechanisms of double-strand break (DSB) repair is exerted at several steps, the key step is DNA end resection, which in Saccharomyces cerevisiae is controlled by the MRX complex and the Sgs1 DNA helicase or the Sae2 and Exo1 nucleases. To assay the role of DNA resection in sister-chromatid recombination (SCR) as the major repair mechanism of spontaneous DSBs, we used a circular minichromosome system for the repair of replication-born DSBs by SCR in yeast. We provide evidence that MRX, particularly its Mre11 nuclease activity, and Sae2 are required for SCR-mediated repair of DSBs. The phenotype of nuclease-deficient MRX mutants is suppressed by ablation of Yku70 or overexpression of Exo1, suggesting a competition between NHEJ and resection factors for DNA ends arising during replication. In addition, we observe partially redundant roles for Sgs1 and Exo1 in SCR, with a more prominent role for Sgs1. Using human U2OS cells, we also show that the competitive nature of these reactions is likely evolutionarily conserved. These results further our understanding of the role of DNA resection in repair of replication-born DSBs revealing unanticipated differences between these events and repair of enzymatically induced DSBs.
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http://dx.doi.org/10.1093/nar/gks1274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561951PMC
February 2013

TDP2/TTRAP is the major 5'-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular resistance to topoisomerase II-induced DNA damage.

J Biol Chem 2011 Jan 28;286(1):403-9. Epub 2010 Oct 28.

Genome Damage and Stability Centre, Science Park Road, University of Sussex, Falmer, Brighton, BN1 9RQ, United Kingdom.

Topoisomerase II (Top2) activity involves an intermediate in which the topoisomerase is covalently bound to a DNA double-strand break via a 5'-phosphotyrosyl bond. Although these intermediates are normally transient, they can be stabilized by antitumor agents that act as Top2 "poisons," resulting in the induction of cytotoxic double-strand breaks, and they are implicated in the formation of site-specific translocations that are commonly associated with cancer. Recently, we revealed that TRAF and TNF receptor-associated protein (TTRAP) is a 5'-tyrosyl DNA phosphodiesterase (5'-TDP) that can cleave 5'-phosphotyrosyl bonds, and we denoted this protein tyrosyl DNA phosphodiesterase-2 (TDP2). Here, we have generated TDP2-deleted DT40 cells, and we show that TDP2 is the major if not the only 5'-TDP activity present in vertebrate cells. We also show that TDP2-deleted DT40 cells are highly sensitive to the anticancer Top2 poison, etoposide, but are not hypersensitive to the Top1 poison camptothecin or the DNA-alkyating agent methyl methanesulfonate. These data identify an important mechanism for resistance to Top2-induced chromosome breakage and raise the possibility that TDP2 is a significant factor in cancer development and treatment.
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http://dx.doi.org/10.1074/jbc.M110.181016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012998PMC
January 2011

A human 5'-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage.

Nature 2009 Oct;461(7264):674-8

Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton, Sussex BN1 9RQ, UK.

Topoisomerases regulate DNA topology and are fundamental to many aspects of chromosome metabolism. Their activity involves the transient cleavage of DNA, which, if it occurs near sites of endogenous DNA damage or in the presence of topoisomerase poisons, can result in abortive topoisomerase-induced DNA strand breaks. These breaks feature covalent linkage of the enzyme to the DNA termini by a 3'- or 5'-phosphotyrosyl bond and are implicated in hereditary human disease, chromosomal instability and cancer, and underlie the clinical efficacy of an important class of anti-tumour poisons. The importance of liberating DNA termini from trapped topoisomerase is illustrated by the progressive neurodegenerative disease observed in individuals containing a mutation in tyrosyl-DNA phosphodiesterase 1 (TDP1), an enzyme that cleaves 3'-phosphotyrosyl bonds. However, a complementary human enzyme that cleaves 5'-phosphotyrosyl bonds has not been reported, despite the effect of DNA double-strand breaks containing such termini on chromosome instability and cancer. Here we identify such an enzyme in human cells and show that this activity efficiently restores 5'-phosphate termini at DNA double-strand breaks in preparation for DNA ligation. This enzyme, TTRAP, is a member of the Mg(2+)/Mn(2+)-dependent family of phosphodiesterases. Cellular depletion of TTRAP results in increased susceptibility and sensitivity to topoisomerase-II-induced DNA double-strand breaks. TTRAP is, to our knowledge, the first human 5'-tyrosyl DNA phosphodiesterase to be identified, and we suggest that this enzyme is denoted tyrosyl DNA phosphodiesterase-2 (TDP2).
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http://dx.doi.org/10.1038/nature08444DOI Listing
October 2009

The Dot1 histone methyltransferase and the Rad9 checkpoint adaptor contribute to cohesin-dependent double-strand break repair by sister chromatid recombination in Saccharomyces cerevisiae.

Genetics 2009 Jun 30;182(2):437-46. Epub 2009 Mar 30.

Universidad de Salamanca, Salamanca, Spain.

Genomic integrity is threatened by multiple sources of DNA damage. DNA double-strand breaks (DSBs) are among the most dangerous types of DNA lesions and can be generated by endogenous or exogenous agents, but they can arise also during DNA replication. Sister chromatid recombination (SCR) is a key mechanism for the repair of DSBs generated during replication and it is fundamental for maintaining genomic stability. Proper repair relies on several factors, among which histone modifications play important roles in the response to DSBs. Here, we study the role of the histone H3K79 methyltransferase Dot1 in the repair by SCR of replication-dependent HO-induced DSBs, as a way to assess its function in homologous recombination. We show that Dot1, the Rad9 DNA damage checkpoint adaptor, and phosphorylation of histone H2A (gammaH2A) are required for efficient SCR. Moreover, we show that Dot1 and Rad9 promote DSB-induced loading of cohesin onto chromatin. We propose that recruitment of Rad9 to DSB sites mediated by gammaH2A and H3K79 methylation contributes to DSB repair via SCR by regulating cohesin binding to damage sites. Therefore, our results contribute to an understanding of how different chromatin modifications impinge on DNA repair mechanisms, which are fundamental for maintaining genomic stability.
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http://dx.doi.org/10.1534/genetics.109.101899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691753PMC
June 2009

CDK targets Sae2 to control DNA-end resection and homologous recombination.

Nature 2008 Oct 20;455(7213):689-92. Epub 2008 Aug 20.

The Wellcome Trust and Cancer Research UK Gurdon Institute, and Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

DNA double-strand breaks (DSBs) are repaired by two principal mechanisms: non-homologous end-joining (NHEJ) and homologous recombination (HR). HR is the most accurate DSB repair mechanism but is generally restricted to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as a repair template. By contrast, NHEJ operates throughout the cell cycle but assumes most importance in G1 (refs 4, 6). The choice between repair pathways is governed by cyclin-dependent protein kinases (CDKs), with a major site of control being at the level of DSB resection, an event that is necessary for HR but not NHEJ, and which takes place most effectively in S and G2 (refs 2, 5). Here we establish that cell-cycle control of DSB resection in Saccharomyces cerevisiae results from the phosphorylation by CDK of an evolutionarily conserved motif in the Sae2 protein. We show that mutating Ser 267 of Sae2 to a non-phosphorylatable residue causes phenotypes comparable to those of a sae2Delta null mutant, including hypersensitivity to camptothecin, defective sporulation, reduced hairpin-induced recombination, severely impaired DNA-end processing and faulty assembly and disassembly of HR factors. Furthermore, a Sae2 mutation that mimics constitutive Ser 267 phosphorylation complements these phenotypes and overcomes the necessity of CDK activity for DSB resection. The Sae2 mutations also cause cell-cycle-stage specific hypersensitivity to DNA damage and affect the balance between HR and NHEJ. These findings therefore provide a mechanistic basis for cell-cycle control of DSB repair and highlight the importance of regulating DSB resection.
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http://dx.doi.org/10.1038/nature07215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2635538PMC
October 2008

APLF (C2orf13) is a novel component of poly(ADP-ribose) signaling in mammalian cells.

Mol Cell Biol 2008 Jul 12;28(14):4620-8. Epub 2008 May 12.

Genome Damage and Stability Centre, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, United Kingdom.

APLF is a novel protein of unknown function that accumulates at sites of chromosomal DNA strand breakage via forkhead-associated (FHA) domain-mediated interactions with XRCC1 and XRCC4. APLF can also accumulate at sites of chromosomal DNA strand breaks independently of the FHA domain via an unidentified mechanism that requires a highly conserved C-terminal tandem zinc finger domain. Here, we show that the zinc finger domain binds tightly to poly(ADP-ribose), a polymeric posttranslational modification synthesized transiently at sites of chromosomal damage to accelerate DNA strand break repair reactions. Protein poly(ADP-ribosyl)ation is tightly regulated and defects in either its synthesis or degradation slow global rates of chromosomal single-strand break repair. Interestingly, APLF negatively affects poly(ADP-ribosyl)ation in vitro, and this activity is dependent on its capacity to bind the polymer. In addition, transient overexpression in human A549 cells of full-length APLF or a C-terminal fragment encoding the tandem zinc finger domain greatly suppresses the appearance of poly(ADP-ribose), in a zinc finger-dependent manner. We conclude that APLF can accumulate at sites of chromosomal damage via zinc finger-mediated binding to poly(ADP-ribose) and is a novel component of poly(ADP-ribose) signaling in mammalian cells.
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http://dx.doi.org/10.1128/MCB.02243-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447129PMC
July 2008

Different genetic requirements for repair of replication-born double-strand breaks by sister-chromatid recombination and break-induced replication.

Nucleic Acids Res 2007 28;35(19):6560-70. Epub 2007 Sep 28.

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, and Departamento de Biología Molecular, CABIMER, CSIC-Universidad de Sevilla, Av. Américo Vespucio s/n, 41092 SEVILLA, Spain.

Homologous recombination (HR) is the major mechanism used to repair double-strand breaks (DSBs) that result from replication, but a study of repair of DSBs specifically induced during S-phase is lacking. Using an inverted-repeat assay in which a DSB is generated by the encountering of the replication fork with nicks, we can physically detect repair by sister-chromatid recombination (SCR) and intra-chromatid break-induced replication (IC-BIR). As expected, both events depend on Rad52, but, in contrast to previous data, both require Rad59, suggesting a prominent role of Rad59 in repair of replication-born DSBs. In the absence of Rad51, SCR is severely affected while IC-BIR increases, a phenotype that is also observed in the absence of Rad54 but not of its paralog Rdh54/Tid1. These data are consistent with SCR occurring by Rad51-dependent mechanisms assisted by Rad54, and indicate that in the absence of strand exchange-dependent SCR, breaks can be channeled to IC-BIR, which works efficiently in the absence of Rad51. Our study provides molecular evidence for inversions between repeats occurring by BIR followed by single-strand annealing (SSA) in the absence of strand exchange.
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http://dx.doi.org/10.1093/nar/gkm488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2095809PMC
December 2007

SMC proteins, new players in the maintenance of genomic stability.

Cell Cycle 2007 Apr 2;6(8):914-8. Epub 2007 Apr 2.

Department of Molecular Biology, CABIMER, CSIC-Universidad de Sevilla, Sevilla, Spain.

Homologous recombination (HR) is one of the key mechanisms responsible for the repair of DNA double-strand breaks (DSBs), including those that occur during DNA replication. Recent studies in yeast and mammals have uncovered that the SMC complexes cohesins and Smc5-Smc6 are recruited to induced DSBs, and play a role in the maintenance of genome stability by favouring SCR as the main recombinational DSB repair mechanism. These new results raise intriguing questions such as whether SMC proteins might play a functional role at collapsed replication forks, which may represent the main source of spontaneous recombinogenic damage. A deeper knowledge of the role of SMC proteins in DSB repair should contribute to a better understanding of chromosome dynamics and stability.
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http://dx.doi.org/10.4161/cc.6.8.4107DOI Listing
April 2007

Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination.

Nat Cell Biol 2006 Sep 6;8(9):1032-4. Epub 2006 Aug 6.

Cell Cycle Group, MRC Clinical Sciences Centre, Imperial College London, Du Cane Road, London W12 0NN, UK.

DNA double-strand breaks (DSB) can arise during DNA replication, or after exposure to DNA-damaging agents, and their correct repair is fundamental for cell survival and genomic stability. Here, we show that the Smc5-Smc6 complex is recruited to DSBs de novo to support their repair by homologous recombination between sister chromatids. In addition, we demonstrate that Smc5-Smc6 is necessary to suppress gross chromosomal rearrangements. Our findings show that the Smc5-Smc6 complex is essential for genome stability as it promotes repair of DSBs by error-free sister-chromatid recombination (SCR), thereby suppressing inappropriate non-sister recombination events.
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http://dx.doi.org/10.1038/ncb1466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493748PMC
September 2006

Double-strand breaks arising by replication through a nick are repaired by cohesin-dependent sister-chromatid exchange.

EMBO Rep 2006 Sep 4;7(9):919-26. Epub 2006 Aug 4.

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avd. Reina Mercedes 6, 41012 Sevilla, Spain.

Molecular studies on double-strand break (DSB) repair in mitosis are usually performed with enzymatically induced DSBs, but spontaneous DSBs might arise because of replication failures, for example when replication encounters nicks. To study repair of replication-born DSBs, we defined a system in Saccharomyces cerevisiae for the induction of a site-specific single-strand break. We show that a 21-base pair (bp) HO site is cleaved at only one strand by the HO endonuclease, with the resulting nick being converted into a DSB by replication during the S phase. Repair of such replication-born DSBs occurs by sister-chromatid exchange (SCE). We provide molecular evidence that cohesins are required for repair of replication-born DSBs by SCE, as determined in smc3, scc1 and scc2 mutants, but not for other recombinational repair events. This work opens new perspectives to understand the importance of single-strand breaks as a source of recombination and the relevance of cohesion in the repair of replication-born DSBs.
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http://dx.doi.org/10.1038/sj.embor.7400774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1559660PMC
September 2006

A novel yeast mutation, rad52-L89F, causes a specific defect in Rad51-independent recombination that correlates with a reduced ability of Rad52-L89F to interact with Rad59.

Genetics 2004 Sep;168(1):553-7

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41012, Spain.

We isolated a novel rad52 mutation, rad52-L89F, which specifically impairs recombination in rad51Delta cells. rad52-L89F displays phenotypes similar to rad59Delta and encodes a mutant protein impaired in its ability to interact with Rad59. These results support the idea that Rad59 acts in homologous recombination via physical interaction with Rad52.
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http://dx.doi.org/10.1534/genetics.104.030551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448092PMC
September 2004

The absence of the yeast chromatin assembly factor Asf1 increases genomic instability and sister chromatid exchange.

EMBO Rep 2004 May 8;5(5):497-502. Epub 2004 Apr 8.

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avda. Reina Mercedes 6, 41012 Sevilla, Spain.

Histone chaperone Asf1 participates in heterochromatin silencing, DNA repair and regulation of gene expression, and promotes the assembly of DNA into chromatin in vitro. To determine the influence of Asf1 on genetic stability, we have analysed the effect of asf1Delta on homologous recombination. In accordance with a defect in nucleosome assembly, asf1Delta leads to a loss of negative supercoiling in plasmids. Importantly, asf1Delta increases spontaneous recombination between inverted DNA sequences. This increase correlates with an accumulation of double-strand breaks (DSBs) as determined by immunodetection of phosphorylated histone H2A and fluorescent detection of Rad52-YFP foci during S and G2/M phases. In addition, asf1Delta shows high levels of sister chromatid exchange (SCE) and is proficient in DSB-induced SCE as determined by physical analysis. Our results suggest that defective chromatin assembly caused by asf1Delta leads to DSBs that can be repaired by SCE, affecting genetic stability.
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http://dx.doi.org/10.1038/sj.embor.7400128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1299049PMC
May 2004

Equal sister chromatid exchange is a major mechanism of double-strand break repair in yeast.

Mol Cell 2003 Jun;11(6):1661-71

Departamento de Genética, Facultad de Biología, Universidad de Sevilla, 41012 Seville, Spain.

Equal sister chromatid exchange (SCE) has been thought to be an important mechanism of double-strand break (DSB) repair in eukaryotes, but this has never been proven due to the difficulty of distinguishing SCE products from parental molecules. To evaluate the biological relevance of equal SCE in DSB repair and to understand the underlying molecular mechanism, we developed recombination substrates for the analysis of DSB repair by SCE in yeast. In these substrates, most breaks are limited to one chromatid, allowing the intact sister chromatid to serve as the repair template; both equal and unequal SCE can be detected. We show that equal SCE is a major mechanism of DSB repair, is Rad51 dependent, and is stimulated by Rad59 and Mre11. Our work provides a physical analysis of mitotically occurring SCE in vivo and opens new perspectives for the study and understanding of DSB repair in eukaryotes.
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http://dx.doi.org/10.1016/s1097-2765(03)00183-7DOI Listing
June 2003