Publications by authors named "Félix Prado"

31 Publications

Physical interactions between MCM and Rad51 facilitate replication fork lesion bypass and ssDNA gap filling by non-recombinogenic functions.

Cell Rep 2021 Jul;36(4):109440

Centro Andaluz de Biología Molecular y Medicina Regenerativa-CABIMER, Consejo Superior de Investigaciones Científicas; Universidad de Sevilla; Universidad Pablo de Olavide; Seville, Spain. Electronic address:

The minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these interactions occur within a nuclease-insoluble scaffold enriched in replication/repair factors. Rad51 accumulates in a MCM- and DNA-binding-independent manner and interacts with MCM helicases located outside of the replication origins and forks. MCM, Rad51, and Rad52 accumulate in this scaffold in G1 and are released during the S phase. In the presence of replication-blocking lesions, Cdc7 prevents their release from the scaffold, thus maintaining the interactions. We identify a rad51 mutant that is impaired in its ability to bind to MCM but not to the scaffold. This mutant is proficient in recombination but partially defective in single-stranded DNA (ssDNA) gap filling and replication fork progression through damaged DNA. Therefore, cells accumulate MCM/Rad51/Rad52 complexes at specific nuclear scaffolds in G1 to assist stressed forks through non-recombinogenic functions.
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http://dx.doi.org/10.1016/j.celrep.2021.109440DOI Listing
July 2021

Chromatin modifiers and recombination factors promote a telomere fold-back structure, that is lost during replicative senescence.

PLoS Genet 2020 12 28;16(12):e1008603. Epub 2020 Dec 28.

Institute of Developmental Biology and Neurobiology (IDN), Johannes Gutenberg-Universität, Mainz, Germany.

Telomeres have the ability to adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomere chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence.
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http://dx.doi.org/10.1371/journal.pgen.1008603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793543PMC
December 2020

Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance.

EMBO Rep 2021 01 2;22(1):e50410. Epub 2020 Dec 2.

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

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.
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http://dx.doi.org/10.15252/embr.202050410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7788459PMC
January 2021

In Vivo Binding of Recombination Proteins to Non-DSB DNA Lesions and to Replication Forks.

Methods Mol Biol 2021 ;2153:447-458

Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), CSIC-University of Seville-UPO, Seville, Spain.

Homologous recombination (HR) has been extensively studied in response to DNA double-strand breaks (DSBs). In contrast, much less is known about how HR deals with DNA lesions other than DSBs (e.g., at single-stranded DNA) and replication forks, despite the fact that these DNA structures are associated with most spontaneous recombination events. A major handicap for studying the role of HR at non-DSB DNA lesions and replication forks is the difficulty of discriminating whether a recombination protein is associated with the non-DSB lesion per se or rather with a DSB generated during their processing. Here, we describe a method to follow the in vivo binding of recombination proteins to non-DSB DNA lesions and replication forks. This approach is based on the cleavage and subsequent electrophoretic analysis of the target DNA by the recombination protein fused to the micrococcal nuclease.
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http://dx.doi.org/10.1007/978-1-0716-0644-5_31DOI Listing
March 2021

Comparative study of the implementation of tin and titanium oxide nanoparticles as electrodes materials in Li-ion batteries.

Sci Rep 2020 Mar 26;10(1):5503. Epub 2020 Mar 26.

Departamento de Física de Materiales, Facultad de CC. Físicas, Universidad Complutense de Madrid, 28040, Madrid, Spain.

Transition metal oxides potentially present higher specific capacities than the current anodes based on carbon, providing an increasing energy density as compared to commercial Li-ion batteries. However, many parameters could influence the performance of the batteries, which depend on the processing of the electrode materials leading to different surface properties, sizes or crystalline phases. In this work a comparative study of tin and titanium oxide nanoparticles synthesized by different methods, undoped or Li doped, used as single components or in mixed ratio, or alternatively forming a composite with graphene oxide have been tested demonstrating an enhancement in capacity with Li doping and better cyclability for mixed phases and composite anodes.
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http://dx.doi.org/10.1038/s41598-020-62505-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7099030PMC
March 2020

Actin and Nuclear Envelope Components Influence Ectopic Recombination in the Absence of Swr1.

Genetics 2019 11 18;213(3):819-834. Epub 2019 Sep 18.

Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), Consejo Superior de Investigaciones Científicas-University of Seville-University Pablo de Olavide, Spain

The accuracy of most DNA processes depends on chromatin integrity and dynamics. Our analyses in the yeast show that an absence of Swr1 (the catalytic and scaffold subunit of the chromatin-remodeling complex SWR) leads to the formation of long-duration Rad52, but not RPA, foci and to an increase in intramolecular recombination. These phenotypes are further increased by MMS, zeocin, and ionizing radiation, but not by double-strand breaks, HU, or transcription/replication collisions, suggesting that they are associated with specific DNA lesions. Importantly, these phenotypes can be specifically suppressed by mutations in: (1) chromatin-anchorage internal nuclear membrane components (∆ and ∆); (2) actin and actin regulators ( , , ∆, and ); or (3) the SWR subunit Swc5 and the SWR substrate Htz1 However, they are not suppressed by global disruption of actin filaments or by the absence of Csm4 (a component of the external nuclear membrane that forms a bridging complex with Mps3, thus connecting the actin cytoskeleton with chromatin). Moreover, ∆-induced Rad52 foci and intramolecular recombination are not associated with tethering recombinogenic DNA lesions to the nuclear periphery. In conclusion, the absence of Swr1 impairs efficient recombinational repair of specific DNA lesions by mechanisms that are influenced by SWR subunits, including actin, and nuclear envelope components. We suggest that these recombinational phenotypes might be associated with a pathological effect on homologous recombination of actin-containing complexes.
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http://dx.doi.org/10.1534/genetics.119.302580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827384PMC
November 2019

Crosstalk between chromatin structure, cohesin activity and transcription.

Epigenetics Chromatin 2019 07 22;12(1):47. Epub 2019 Jul 22.

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

Background: A complex interplay between chromatin and topological machineries is critical for genome architecture and function. However, little is known about these reciprocal interactions, even for cohesin, despite its multiple roles in DNA metabolism.

Results: We have used genome-wide analyses to address how cohesins and chromatin structure impact each other in yeast. Cohesin inactivation in scc1-73 mutants during the S and G2 phases causes specific changes in chromatin structure that preferentially take place at promoters; these changes include a significant increase in the occupancy of the - 1 and + 1 nucleosomes. In addition, cohesins play a major role in transcription regulation that is associated with specific promoter chromatin architecture. In scc1-73 cells, downregulated genes are enriched in promoters with short or no nucleosome-free region (NFR) and a fragile "nucleosome - 1/RSC complex" particle. These results, together with a preferential increase in the occupancy of nucleosome - 1 of these genes, suggest that cohesins promote transcription activation by helping RSC to form the NFR. In sharp contrast, the scc1-73 upregulated genes are enriched in promoters with an "open" chromatin structure and are mostly at cohesin-enriched regions, suggesting that a local accumulation of cohesins might help to inhibit transcription. On the other hand, a dramatic loss of chromatin integrity by histone depletion during DNA replication has a moderate effect on the accumulation and distribution of cohesin peaks along the genome.

Conclusions: Our analyses of the interplay between chromatin integrity and cohesin activity suggest that cohesins play a major role in transcription regulation, which is associated with specific chromatin architecture and cohesin-mediated nucleosome alterations of the regulated promoters. In contrast, chromatin integrity plays only a minor role in the binding and distribution of cohesins.
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http://dx.doi.org/10.1186/s13072-019-0293-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647288PMC
July 2019

Homologous Recombination: To Fork and Beyond.

Authors:
Félix Prado

Genes (Basel) 2018 Dec 4;9(12). Epub 2018 Dec 4.

Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), CSIC-University of Seville-University Pablo de Olavide, 41092 Seville, Spain.

Accurate completion of genome duplication is threatened by multiple factors that hamper the advance and stability of the replication forks. Cells need to tolerate many of these blocking lesions to timely complete DNA replication, postponing their repair for later. This process of lesion bypass during DNA damage tolerance can lead to the accumulation of single-strand DNA (ssDNA) fragments behind the fork, which have to be filled in before chromosome segregation. Homologous recombination plays essential roles both at and behind the fork, through fork protection/lesion bypass and post-replicative ssDNA filling processes, respectively. I review here our current knowledge about the recombination mechanisms that operate at and behind the fork in eukaryotes, and how these mechanisms are controlled to prevent unscheduled and toxic recombination intermediates. A unifying model to integrate these mechanisms in a dynamic, replication fork-associated process is proposed from yeast results.
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http://dx.doi.org/10.3390/genes9120603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316604PMC
December 2018

Histone depletion prevents telomere fusions in pre-senescent cells.

PLoS Genet 2018 06 7;14(6):e1007407. Epub 2018 Jun 7.

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

Upon telomerase inactivation, telomeres gradually shorten with each cell division until cells enter replicative senescence. In Saccharomyces cerevisiae, the kinases Mec1/ATR and Tel1/ATM protect the genome during pre-senescence by preventing telomere-telomere fusions (T-TFs) and the subsequent genetic instability associated with fusion-bridge-breakage cycles. Here we report that T-TFs in mec1Δ tel1Δ cells can be suppressed by reducing the pool of available histones. This protection associates neither with changes in bulk telomere length nor with major changes in the structure of subtelomeric chromatin. We show that the absence of Mec1 and Tel1 strongly augments double-strand break (DSB) repair by non-homologous end joining (NHEJ), which might contribute to the high frequency of T-TFs in mec1Δ tel1Δ cells. However, histone depletion does not prevent telomere fusions by inhibiting NHEJ, which is actually increased in histone-depleted cells. Rather, histone depletion protects telomeres from fusions by homologous recombination (HR), even though HR is proficient in maintaining the proliferative state of pre-senescent mec1Δ tel1Δ cells. Therefore, HR during pre-senescence not only helps stalled replication forks but also prevents T-TFs by a mechanism that, in contrast to the previous one, is promoted by a reduction in the histone pool and can occur in the absence of Rad51. Our results further suggest that the Mec1-dependent depletion of histones that occurs during pre-senescence in cells without telomerase (tlc1Δ) prevents T-TFs by favoring the processing of unprotected telomeres by Rad51-independent HR.
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http://dx.doi.org/10.1371/journal.pgen.1007407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991667PMC
June 2018

Functional Impact of the H2A.Z Histone Variant During Meiosis in .

Genetics 2018 08 31;209(4):997-1015. Epub 2018 May 31.

Institute of Functional Biology and Genomics, Consejo Superior de Investigaciones Científicas and University of Salamanca, 37007 Salamanca, Spain

Among the collection of chromatin modifications that influence its function and structure, the substitution of canonical histones by the so-called histone variants is one of the most prominent actions. Since crucial meiotic transactions are modulated by chromatin, here we investigate the functional contribution of the H2A.Z histone variant during both unperturbed meiosis and upon challenging conditions where the meiotic recombination checkpoint is triggered in budding yeast by the absence of the synaptonemal complex component Zip1 We have found that H2A.Z localizes to meiotic chromosomes in an SWR1-dependent manner. Although meiotic recombination is not substantially altered, the mutant (lacking H2A.Z) shows inefficient meiotic progression, impaired sporulation, and reduced spore viability. These phenotypes are likely accounted for by the misregulation of meiotic gene expression landscape observed in In the mutant, the absence of H2A.Z results in a tighter meiotic arrest imposed by the meiotic recombination checkpoint. We have found that Mec1-dependent Hop1-T318 phosphorylation and the ensuing Mek1 activation are not significantly altered in ; however, downstream checkpoint targets, such as the meiosis I-promoting factors Ndt80, Cdc5, and Clb1, are drastically downregulated. The study of the checkpoint response in has also allowed us to reveal the existence of an additional function of the Swe1 kinase, independent of CDK inhibitory phosphorylation, which is relevant to restrain meiotic cell cycle progression. In summary, our study shows that the H2A.Z histone variant impacts various aspects of meiotic development adding further insight into the relevance of chromatin dynamics for accurate gametogenesis.
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http://dx.doi.org/10.1534/genetics.118.301110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6063244PMC
August 2018

Regulation of Replication Fork Advance and Stability by Nucleosome Assembly.

Genes (Basel) 2017 Jan 24;8(2). Epub 2017 Jan 24.

Department of Genome Biology, Andalusian Molecular Biology and Regenerative Medicine Center (CABIMER), Spanish National Research Council (CSIC), Seville 41092, Spain.

The advance of replication forks to duplicate chromosomes in dividing cells requires the disassembly of nucleosomes ahead of the fork and the rapid assembly of parental and de novo histones at the newly synthesized strands behind the fork. Replication-coupled chromatin assembly provides a unique opportunity to regulate fork advance and stability. Through post-translational histone modifications and tightly regulated physical and genetic interactions between chromatin assembly factors and replisome components, chromatin assembly: (1) controls the rate of DNA synthesis and adjusts it to histone availability; (2) provides a mechanism to protect the integrity of the advancing fork; and (3) regulates the mechanisms of DNA damage tolerance in response to replication-blocking lesions. Uncoupling DNA synthesis from nucleosome assembly has deleterious effects on genome integrity and cell cycle progression and is linked to genetic diseases, cancer, and aging.
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http://dx.doi.org/10.3390/genes8020049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333038PMC
January 2017

Histone availability as a strategy to control gene expression.

RNA Biol 2017 03 21;14(3):281-286. Epub 2016 May 21.

a Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC) , Seville , Spain.

Histone proteins are main structural components of the chromatin and major determinants of gene regulation. Expression of canonical histone genes is strictly controlled during the cell cycle in order to couple DNA replication with histone deposition. Indeed, reductions in the levels of canonical histones or defects in chromatin assembly cause genetic instability. Early data from yeast demonstrated that severe histone depletion also causes strong gene expression changes. We have recently reported that a moderated depletion of canonical histones in human cells leads to an open chromatin configuration, which in turn increases RNA polymerase II elongation rates and causes pre-mRNA splicing defects. Interestingly, some of the observed defects accompany the scheduled histone depletion that is associated with several senescence and aging processes. Thus, our comparison of induced and naturally-occurring histone depletion processes suggests that a programmed reduction of the level of canonical histones might be a strategy to control gene expression during specific physiological processes.
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http://dx.doi.org/10.1080/15476286.2016.1189071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367251PMC
March 2017

Defective histone supply causes condensin-dependent chromatin alterations, SAC activation and chromosome decatenation impairment.

Nucleic Acids Res 2016 Apr 28;44(7):3479-80. Epub 2016 Jan 28.

Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain

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http://dx.doi.org/10.1093/nar/gkw058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838366PMC
April 2016

Defective histone supply causes changes in RNA polymerase II elongation rate and cotranscriptional pre-mRNA splicing.

Proc Natl Acad Sci U S A 2015 Dec 17;112(48):14840-5. Epub 2015 Nov 17.

Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas, E-41092, Seville, Spain;

RNA polymerase II (RNAPII) transcription elongation is a highly regulated process that greatly influences mRNA levels as well as pre-mRNA splicing. Despite many studies in vitro, how chromatin modulates RNAPII elongation in vivo is still unclear. Here, we show that a decrease in the level of available canonical histones leads to more accessible chromatin with decreased levels of canonical histones and variants H2A.X and H2A.Z and increased levels of H3.3. With this altered chromatin structure, the RNAPII elongation rate increases, and the kinetics of pre-mRNA splicing is delayed with respect to RNAPII elongation. Consistent with the kinetic model of cotranscriptional splicing, the rapid RNAPII elongation induced by histone depletion promotes the skipping of variable exons in the CD44 gene. Indeed, a slowly elongating mutant of RNAPII was able to rescue this defect, indicating that the defective splicing induced by histone depletion is a direct consequence of the increased elongation rate. In addition, genome-wide analysis evidenced that histone reduction promotes widespread alterations in pre-mRNA processing, including intron retention and changes in alternative splicing. Our data demonstrate that pre-mRNA splicing may be regulated by chromatin structure through the modulation of the RNAPII elongation rate.
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http://dx.doi.org/10.1073/pnas.1506760112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4672771PMC
December 2015

Defective histone supply causes condensin-dependent chromatin alterations, SAC activation and chromosome decatenation impairment.

Nucleic Acids Res 2014 Nov 9;42(20):12469-82. Epub 2014 Oct 9.

Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain

The structural organization of chromosomes is essential for their correct function and dynamics during the cell cycle. The assembly of DNA into chromatin provides the substrate for topoisomerases and condensins, which introduce the different levels of superhelical torsion required for DNA metabolism. In particular, Top2 and condensin are directly involved in both the resolution of precatenanes that form during replication and the formation of the intramolecular loop that detects tension at the centromeric chromatin during chromosome biorientation. Here we show that histone depletion activates the spindle assembly checkpoint (SAC) and impairs sister chromatid decatenation, leading to chromosome mis-segregation and lethality in the absence of the SAC. We demonstrate that histone depletion impairs chromosome biorientation and activates the Aurora-dependent pathway, which detects tension problems at the kinetochore. Interestingly, SAC activation is suppressed by the absence of Top2 and Smc2, an essential component of condensin. Indeed, smc2-8 suppresses catenanes accumulation, mitotic arrest and growth defects induced by histone depletion at semi-permissive temperature. Remarkably, SAC activation by histone depletion is associated with condensin-mediated alterations of the centromeric chromatin. Therefore, our results reveal the importance of a precise interplay between histone supply and condensin/Top2 for pericentric chromatin structure, precatenanes resolution and centromere biorientation.
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http://dx.doi.org/10.1093/nar/gku927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4227775PMC
November 2014

Homologous recombination maintenance of genome integrity during DNA damage tolerance.

Authors:
Félix Prado

Mol Cell Oncol 2014 Apr-Jun;1(2):e957039. Epub 2014 Oct 29.

Departamento de Biología Molecular; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER) ; Consejo Superior de Investigaciones Científicas (CSIC) ; Seville, Spain.

The DNA strand exchange protein Rad51 provides a safe mechanism for the repair of DNA breaks using the information of a homologous DNA template. Homologous recombination (HR) also plays a key role in the response to DNA damage that impairs the advance of the replication forks by providing mechanisms to circumvent the lesion and fill in the tracks of single-stranded DNA that are generated during the process of lesion bypass. These activities postpone repair of the blocking lesion to ensure that DNA replication is completed in a timely manner. Experimental evidence generated over the last few years indicates that HR participates in this DNA damage tolerance response together with additional error-free (template switch) and error-prone (translesion synthesis) mechanisms through intricate connections, which are presented here. The choice between repair and tolerance, and the mechanism of tolerance, is critical to avoid increased mutagenesis and/or genome rearrangements, which are both hallmarks of cancer.
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http://dx.doi.org/10.4161/23723548.2014.957039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4905194PMC
June 2016

Genetic instability is prevented by Mrc1-dependent spatio-temporal separation of replicative and repair activities of homologous recombination: homologous recombination tolerates replicative stress by Mrc1-regulated replication and repair activities operating at S and G2 in distinct subnuclear compartments.

Authors:
Félix Prado

Bioessays 2014 May 26;36(5):451-62. Epub 2014 Feb 26.

Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Consejo Superior de Investigaciones Científicas (CSIC), Seville, Spain.

Homologous recombination (HR) is required to protect and restart stressed replication forks. Paradoxically, the Mrc1 branch of the S phase checkpoints, which is activated by replicative stress, prevents HR repair at breaks and arrested forks. Indeed, the mechanisms underlying HR can threaten genome integrity if not properly regulated. Thus, understanding how cells avoid genetic instability associated with replicative stress, a hallmark of cancer, is still a challenge. Here I discuss recent results that support a model by which HR responds to replication stress through replicative and repair activities that operate at different stages of the cell cycle (S and G2, respectively) and in distinct subnuclear structures. Remarkably, the replication checkpoint appears to control this scenario by inhibiting the assembly of HR repair centers at stressed forks during S phase, thereby avoiding genetic instability.
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http://dx.doi.org/10.1002/bies.201300161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4312893PMC
May 2014

Rad51 replication fork recruitment is required for DNA damage tolerance.

EMBO J 2013 May 5;32(9):1307-21. Epub 2013 Apr 5.

Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa, Consejo Superior de Investigaciones Científicas, Seville, Spain.

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non-repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non-DSB DNA lesions, presumably single-stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle-regulated replicative and repair functions.
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http://dx.doi.org/10.1038/emboj.2013.73DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642682PMC
May 2013

Nucleosome assembly and genome integrity: The fork is the link.

Bioarchitecture 2012 Jan;2(1):6-10

Departamento de Biología Molecular; Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER); Consejo Superior de Investigaciones Científicas (CSIC); Seville, Spain.

Maintaining the stability of the replication forks is one of the main tasks of the DNA damage response. Specifically, checkpoint mechanisms detect stressed forks and prevent their collapse. In the published report reviewed here we have shown that defective chromatin assembly in cells lacking either H3K56 acetylation or the chromatin assembly factors CAF1 and Rtt106 affects the integrity of advancing replication forks, despite the presence of functional checkpoints. This loss of replication intermediates is exacerbated in the absence of Rad52, suggesting that collapsed forks are rescued by homologous recombination and providing an explanation for the accumulation of recombinogenic DNA damage displayed by these mutants. These phenotypes mimic those obtained by a partial reduction in the pool of available histones and are consistent with a model in which defective histone deposition uncouples DNA synthesis and nucleosome assembly, thus making the fork more susceptible to collapse. Here, we review these findings and discuss the possibility that defects in the lagging strand represent a major source of fork instability in chromatin assembly mutants.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383716PMC
http://dx.doi.org/10.4161/bioa.19737DOI Listing
January 2012

Histone H3K56 acetylation, CAF1, and Rtt106 coordinate nucleosome assembly and stability of advancing replication forks.

PLoS Genet 2011 Nov 10;7(11):e1002376. Epub 2011 Nov 10.

Departamento de Biología Molecular, Centro Andaluz de Biología Molecular y Medicina Regenerativa, Consejo Superior de Investigaciones Científicas, Seville, Spain.

Chromatin assembly mutants accumulate recombinogenic DNA damage and are sensitive to genotoxic agents. Here we have analyzed why impairment of the H3K56 acetylation-dependent CAF1 and Rtt106 chromatin assembly pathways, which have redundant roles in H3/H4 deposition during DNA replication, leads to genetic instability. We show that the absence of H3K56 acetylation or the simultaneous knock out of CAF1 and Rtt106 increases homologous recombination by affecting the integrity of advancing replication forks, while they have a minor effect on stalled replication fork stability in response to the replication inhibitor hydroxyurea. This defect in replication fork integrity is not due to defective checkpoints. In contrast, H3K56 acetylation protects against replicative DNA damaging agents by DNA repair/tolerance mechanisms that do not require CAF1/Rtt106 and are likely subsequent to the process of replication-coupled nucleosome deposition. We propose that the tight connection between DNA synthesis and histone deposition during DNA replication mediated by H3K56ac/CAF1/Rtt106 provides a mechanism for the stabilization of advancing replication forks and the maintenance of genome integrity, while H3K56 acetylation has an additional, CAF1/Rtt106-independent function in the response to replicative DNA damage.
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http://dx.doi.org/10.1371/journal.pgen.1002376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3213180PMC
November 2011

The SWR1 histone replacement complex causes genetic instability and genome-wide transcription misregulation in the absence of H2A.Z.

PLoS One 2010 Aug 12;5(8):e12143. Epub 2010 Aug 12.

Department of Molecular Biology, CABIMER-CSIC, Seville, Spain.

The SWR1 complex replaces the canonical histone H2A with the variant H2A.Z (Htz1 in yeast) at specific chromatin regions. This dynamic alteration in nucleosome structure provides a molecular mechanism to regulate transcription, gene silencing, chromosome segregation and DNA repair. Here we show that genetic instability, sensitivity to drugs impairing different cellular processes and genome-wide transcriptional misregulation in htz1Delta can be partially or totally suppressed if SWR1 is not formed (swr1Delta), if it forms but cannot bind to chromatin (swc2Delta) or if it binds to chromatin but lacks histone replacement activity (swc5Delta and the ATPase-dead swr1-K727G). These results suggest that in htz1Delta the nucleosome remodelling activity of SWR1 affects chromatin integrity because of an attempt to replace H2A with Htz1 in the absence of the latter. This would impair transcription and, either directly or indirectly, other cellular processes. Specifically, we show that in htz1Delta, the SWR1 complex causes an accumulation of recombinogenic DNA damage by a mechanism dependent on phosphorylation of H2A at Ser129, a modification that occurs in response to DNA damage, suggesting that the SWR1 complex impairs the repair of spontaneous DNA damage in htz1Delta. In addition, SWR1 causes DSBs sensitivity in htz1Delta; consistently, in the absence of Htz1 the SWR1 complex bound near an endonuclease HO-induced DSB at the mating-type (MAT) locus impairs DSB-induced checkpoint activation. Our results support a stepwise mechanism for the replacement of H2A with Htz1 and demonstrate that a tight control of this mechanism is essential to regulate chromatin dynamics but also to prevent the deleterious consequences of an incomplete nucleosome remodelling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0012143PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2920830PMC
August 2010

Chromatin assembly controls replication fork stability.

EMBO Rep 2009 Jul 22;10(7):790-6. Epub 2009 May 22.

Departamento de Biología Molecular, CABIMER-CSIC, Seville, Spain.

During DNA replication, the advance of replication forks is tightly connected with chromatin assembly, a process that can be impaired by the partial depletion of histone H4 leading to recombinogenic DNA damage. Here, we show that the partial depletion of H4 is rapidly followed by the collapse of unperturbed and stalled replication forks, even though the S-phase checkpoints remain functional. This collapse is characterized by a reduction in the amount of replication intermediates, but an increase in single Ys relative to bubbles, defects in the integrity of the replisome and an accumulation of DNA double-strand breaks. This collapse is also associated with an accumulation of Rad52-dependent X-shaped molecules. Consistently, a Rad52-dependent--although Rad51-independent--mechanism is able to rescue these broken replication forks. Our findings reveal that correct nucleosome deposition is required for replication fork stability, and provide molecular evidence for homologous recombination as an efficient mechanism of replication fork restart.
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http://dx.doi.org/10.1038/embor.2009.67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2727422PMC
July 2009

Replication fork progression is impaired by transcription in hyperrecombinant yeast cells lacking a functional THO complex.

Mol Cell Biol 2006 Apr;26(8):3327-34

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

THO/TREX is a conserved, eukaryotic protein complex operating at the interface between transcription and messenger ribonucleoprotein (mRNP) metabolism. THO mutations impair transcription and lead to increased transcription-associated recombination (TAR). These phenotypes are dependent on the nascent mRNA; however, the molecular mechanism by which impaired mRNP biogenesis triggers recombination in THO/TREX mutants is unknown. In this study, we provide evidence that deficient mRNP biogenesis causes slowdown or pausing of the replication fork in hpr1Delta mutants. Impaired replication appears to depend on sequence-specific features since it was observed upon activation of lacZ but not leu2 transcription. Replication fork progression could be partially restored by hammerhead ribozyme-guided self-cleavage of the nascent mRNA. Additionally, hpr1Delta increased the number of S-phase but not G(2)-dependent TAR events as well as the number of budded cells containing Rad52 repair foci. Our results link transcription-dependent genomic instability in THO mutants with impaired replication fork progression, suggesting a molecular basis for a connection between inefficient mRNP biogenesis and genetic instability.
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http://dx.doi.org/10.1128/MCB.26.8.3327-3334.2006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1446968PMC
April 2006

Impairment of replication fork progression mediates RNA polII transcription-associated recombination.

EMBO J 2005 Mar 3;24(6):1267-76. Epub 2005 Mar 3.

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

Homologous recombination safeguards genome integrity, but it can also cause genome instability of important consequences for cell proliferation and organism development. Transcription induces recombination, as shown in prokaryotes and eukaryotes for both spontaneous and developmentally regulated events such as those responsible for immunoglobulin class switching. Deciphering the molecular basis of transcription-associated recombination (TAR) is important in understanding genome instability. Using novel plasmid-borne recombination constructs in Saccharomyces cerevisiae, we show that RNA polymerase II (RNAPII) transcription induces recombination by impairing replication fork progression. RNAPII transcription concomitant to head-on oncoming replication causes a replication fork pause (RFP) that is linked to a significant increase in recombination. However, transcription that is codirectional with replication has little effect on replication fork progression and recombination. Transcription occurring in the absence of replication does not affect either recombination or replication fork progression. The Rrm3 helicase, which is required for replication fork progression through nucleoprotein complexes, facilitates replication through the transcription-dependent RFP site and reduces recombination. Therefore, our work provides evidence that one mechanism responsible for TAR is RNAP-mediated replication impairment.
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http://dx.doi.org/10.1038/sj.emboj.7600602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC556405PMC
March 2005

Partial depletion of histone H4 increases homologous recombination-mediated genetic instability.

Mol Cell Biol 2005 Feb;25(4):1526-36

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

DNA replication can be a source of genetic instability. Given the tight connection between DNA replication and nucleosome assembly, we analyzed the effect of a partial depletion of histone H4 on genetic instability mediated by homologous recombination. A Saccharomyces cerevisiae strain was constructed in which the expression of histone H4 was driven by the regulated tet promoter. In agreement with defective nucleosome assembly, partial depletion of histone H4 led to subtle changes in plasmid superhelical density and chromatin sensitivity to micrococcal nuclease. Under these conditions, homologous recombination between ectopic DNA sequences was increased 20-fold above the wild-type levels. This hyperrecombination was not associated with either defective repair or transcription but with an accumulation of recombinogenic DNA lesions during the S and G(2)/M phases, as determined by an increase in the proportion of budded cells containing Rad52-yellow fluorescent protein foci. Consistently, partial depletion of histone H4 caused a delay during the S and G(2)/M phases. Our results suggest that histone deposition defects lead to the formation of recombinogenic DNA structures during replication that increase genomic instability.
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http://dx.doi.org/10.1128/MCB.25.4.1526-1536.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC548009PMC
February 2005

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

Control of cross-over by single-strand DNA resection.

Trends Genet 2003 Aug;19(8):428-31

Departamento de Genética, Facultad de Biologi;a, Universidad de Sevilla, 41012, Sevilla, Spain.

Control of DNA cross-overs is necessary for meiotic recombination and genome integrity. The frequency of cross-overs is dependent on homology length and the conversion tract, but the mechanisms underlying the regulation of cross-overs remain unknown. We propose that 5'-end resection, a key intermediate in double-strand break repair, could determine the formation of cross-overs. Extensive DNA resection might favor gene conversion without cross-over by channeling recombination events through synthesis-dependent strand-annealing. In reactions with short regions of homology, resection beyond the homologous sequence would impede Holliday junction formation and, consequently, cross-over. Extensive DNA resection could be an effective mechanism to prevent reciprocal exchanges between dispersed DNA sequences, and thus contribute to the genome stability.
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http://dx.doi.org/10.1016/S0168-9525(03)00173-2DOI Listing
August 2003

Mitotic recombination in Saccharomyces cerevisiae.

Curr Genet 2003 Jan 29;42(4):185-98. Epub 2002 Nov 29.

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

Mitotic homologous recombination (HR) is an important mechanism for the repair of double-strand breakS and errors occurring during DNA replication. It is likely that the recombinational repair of DNA lesions occurs preferentially by sister chromatid exchanges that have no genetic consequences. However, most genetically detectable HR events occur between homologous DNA sequences located at allelic positions in homologous chromosomes, or between DNA repeats located at ectopic positions in either the same, homologous or heterologous chromosomes. Mitotic recombination may occur by multiple mechanisms, including double-strand break repair, synthesis-dependent strand annealing, break-induced replication and single-strand annealing. The occurrence of one recombination mechanism versus another depends on different elements, including the position of the homologous partner, the initiation event, the length of homology of the recombinant molecules and the genotype. The genetics and molecular biology of the yeast Saccharomyces cerevisiae have proved essential for the understanding of mitotic recombination mechanisms in eukaryotes. Here, we review recent genetic yeast data that contribute to our understanding of the different mechanisms of mitotic recombination and the in vivo role of the recombination proteins.
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http://dx.doi.org/10.1007/s00294-002-0346-3DOI Listing
January 2003

Defective nucleotide excision repair in yeast hpr1 and tho2 mutants.

Nucleic Acids Res 2002 May;30(10):2193-201

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

Nucleotide excision repair (NER) and transcription are intimately related. First, TFIIH has a dual role in transcription initiation and NER and, secondly, transcription leads to more efficient repair of damage present in transcribed sequences. It is thought that elongating RNAPII, stalled at a DNA lesion, is used for the loading of the NER machinery in a process termed transcription-coupled repair (TCR). Non-transcribed regions are repaired by the so-called global genome repair (GGR). We have previously defined a number of yeast genes, whose deletions confer transcription-dependent hyper-recombination phenotypes. As these mutations cause impairment of transcription elongation we have assayed whether they also affect DNA repair. We show that null mutations of the HPR1 and THO2 genes, encoding two prominent proteins of the THO complex, increase UV sensitivity of yeast cells lacking GGR. Consistent with this result, molecular analyses of DNA repair of the RPB2 transcribed strand using T4 endo V show that hpr1 and tho2 do indeed impair TCR. However, this effect is not confined to TCR alone because the mutants are slightly affected in GGR. These results indicate that THO affects both transcription and NER. We discuss different alternatives to explain the effect of the THO complex on DNA repair.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC115280PMC
http://dx.doi.org/10.1093/nar/30.10.2193DOI Listing
May 2002

Differential role of the proline-rich domain of nuclear factor 1-C splice variants in DNA binding and transactivation.

J Biol Chem 2002 May 22;277(19):16383-90. Epub 2002 Feb 22.

Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität, E.-Mannkopff-Str. 2, D-35033 Marburg, Germany.

We have addressed the functional significance of the existence of several natural splice variants of NF1-C* differing in their COOH-terminal proline-rich transactivation domain (PRD) by studying their specific DNA binding and transactivation in the yeast Saccharomyces cerevisiae. These parameters yielded the intrinsic transactivation potential (ITP), defined as the activation observed with equal amounts of DNA bound protein. Exchange of 83 amino acids at the COOH-terminal end of the PRD by 16 unrelated amino acids, as found in NF1-C2, and splicing out the central region of the PRD, as found in NF1-C7, enhanced DNA binding in vivo and in vitro. However, the ITP of the splice variants NF1-C2 and NF1-C7 was found to be similar to that of the intact NF1-C1. Additional mutations showed that the ITP of NF1-C requires the synergistic action of the PRD and a novel domain encoded in exons 5 and 6. Intriguingly the carboxyl-terminal domain-like motif encoded in exons 9/10 is not essential for transactivation of a reporter with a single NF1 site but is required for activation of a reporter with six NF1 sites in tandem. Our results imply that differential splicing is used to regulate transcription by generating variants with different DNA binding affinities but similar ITPs.
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http://dx.doi.org/10.1074/jbc.M200418200DOI Listing
May 2002
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