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ABSTRACT: Novel combinatorial associations of genetic traits arise from homologous recombination between parental chromosomes during meiosis. Histone H3 lysine 4 trimethylation marks meiotic recombination hotspots in yeast and mammals, but how this ubiquitous chromatin modification relates to initiation of Spo11-dependent double-strand breaks (DSB) remains unknown. Here, we show that the tethering of the PHD-containing protein, Spp1, a component of the COMPASS complex to recombinationally cold regions is sufficient to induce DSB formation. Furthermore, we found that Spp1 physically interacts with Mer2, a key protein of the differentiated chromosomal axis required for DSB formation. Thus, Spp1, by interacting with H3K4me3 and Mer2, promotes recruitment of potential meiotic DSB sites to the chromosomal axis, allowing Spo11 cleavage at nearby nucleosome-depleted regions.
Science 11/2012; · 31.20 Impact Factor
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ABSTRACT: Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC-rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC(3) G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites' distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.
PLoS Genetics 11/2012; 8(11):e1003033. · 8.69 Impact Factor
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ABSTRACT: CEB25 is a human minisatellite locus, composed of slightly polymorphic 52-nucleotide (nt) tandem repeats. Genetically, most if not all individuals of the human population are heterozygous, carrying alleles ranging from 0.5 to 20 kb, maintained by mendelian inheritance but also subject to germline instability. To provide insights on the biological role of CEB25, we interrogated its structural features. We report the NMR structure of the G-quadruplex formed by the conserved 26-nt G-rich fragment of the CEB25 motif. In K(+) solution, this sequence forms a propeller-type parallel-stranded G-quadruplex involving a 9-nt central double-chain-reversal loop. This long loop is anchored to the 5'-end of the sequence by an A·T Watson-Crick base pair and a potential G·A noncanonical base pair. These base pairs contribute to the stability of the overall G-quadruplexstructure, as measured by an increase of about 17 kcal/mol in enthalpy or 6 °C in melting temperature. Further, we demonstrate that such a monomorphic structure is formed within longer sequence contexts folding into a pearl-necklace structure.
Journal of the American Chemical Society 02/2012; 134(13):5807-16. · 9.91 Impact Factor
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ABSTRACT: A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.
Human Mutation 09/2011; 32(12):1470-80. · 5.69 Impact Factor
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ABSTRACT: G-quadruplexes are four-stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G-quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G-quadruplex-prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild-type cells but is very frequently destabilized upon treatment with the potent Phen-DC(3) G-quadruplex ligand, or in the absence of the G-quadruplex-unwinding Pif1 helicase, only when the G-rich strand is the template of leading-strand replication. The orientation-dependent instability is associated with the formation of Rad51-Rad52-dependent X-shaped intermediates during replication detected by two-dimensional (2D) gels, and relies on the presence of intact G-quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G-quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G-rich telomeres.
The EMBO Journal 08/2011; 30(19):4033-46. · 9.20 Impact Factor
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ABSTRACT: Srs2 is a 3'-5' DNA helicase that regulates many aspects of DNA metabolism in Saccharomyces cerevisiae. It is best known for its ability to counteract homologous recombination by dismantling Rad51 filaments, but is also involved in checkpoint activation, adaptation and recovery, and in resolution of late recombination intermediates. To further address its biological roles and uncover new genetic interactions, we examined the consequences of overexpressing SRS2 as well as two helicase-dead mutants, srs2-K41A and srs2-K41R, in the collection of 4827 yeast haploid deletion mutants. We identified 274 genes affecting a large variety of cellular functions that are required for cell growth when SRS2 or its mutants are overexpressed. Further analysis of these interactions reveals that Srs2 acts independently of its helicase function at replication forks likely through its recruitment by the sumoylated PCNA replication clamp. This helicase-independent function is responsible for the negative interactions with DNA metabolism genes and for the toxicity of SRS2 overexpression in many of the diverse cellular pathways revealed in our screens.
DNA repair 03/2011; 10(5):506-17. · 4.20 Impact Factor
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ABSTRACT: SUMMARY: We present SVDetect, a program designed to identify genomic structural variations from paired-end and mate-pair next-generation sequencing data produced by the Illumina GA and ABI SOLiD platforms. Applying both sliding-window and clustering strategies, we use anomalously mapped read pairs provided by current short read aligners to localize genomic rearrangements and classify them according to their type, e.g. large insertions-deletions, inversions, duplications and balanced or unbalanced inter-chromosomal translocations. SVDetect outputs predicted structural variants in various file formats for appropriate graphical visualization. AVAILABILITY: Source code and sample data are available at http://svdetect.sourceforge.net/
Bioinformatics 08/2010; 26(15):1895-6. · 5.47 Impact Factor
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ABSTRACT: G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC(3) and Phen-DC(6), two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.
Nucleic Acids Research 03/2010; 38(13):4337-48. · 8.03 Impact Factor
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ABSTRACT: Sexual reproduction depends on the success of faithful chromosome transmission during meiosis to yield viable gametes. Central to meiosis is the process of recombination between paternal and maternal chromosomes, which boosts the genetic diversity of progeny and ensures normal homologous chromosome segregation. Imperfections in meiotic recombination are the source of de novo germline mutations, abnormal gametes, and infertility. Thus, not surprisingly, cells have developed a variety of mechanisms and tight controls to ensure sufficient and well-distributed recombination events within their genomes, the details of which remain to be fully elucidated. Local and genome-wide studies of normal and genetically engineered cells have uncovered a remarkable stochasticity in the number and positioning of recombination events per chromosome and per cell, which reveals an impressive level of flexibility. In this minireview, we summarize our contemporary understanding of meiotic recombination and its control mechanisms, and address the seemingly paradoxical and poorly understood diversity of recombination sites. Flexibility in the distribution of meiotic recombination events within genomes may reside in regulation at the chromatin level, with histone modifications playing a recently recognized role.
FEBS Journal 12/2009; 277(3):571-89. · 3.79 Impact Factor
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ABSTRACT: In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.
PLoS Genetics 06/2009; 5(5):e1000475. · 8.69 Impact Factor
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ABSTRACT: Meiotic recombination is initiated by DNA double-strand breaks (DSBs) that are catalyzed by the type II topoisomerase-like Spo11 protein. Locally, at recombination hot spots, Spo11 introduces DSBs at multiple positions within approximately 75 to 250 bp, corresponding to accessible regions of the chromatin. The molecular basis of this multiplicity of cleavage positions, observed in a population of meiotic cells, remains elusive. To address this issue, we have examined the properties of the Gal4BD-Spo11 fusion protein, which targets meiotic DSBs to regions with Gal4 binding sites (UAS). By single-nucleotide resolution mapping of targeted DSBs, we found that DSB formation was restricted to discrete sites approximately 20 nucleotides from the UAS, defining a "DSB targeting window." Thus, the multiplicity of cleavage positions at natural Spo11 hot spots likely represents binding of Spo11 to different distinct sites within the accessible DNA region in each different meiotic cell. Further, we showed that mutations in the Spo11 moiety affected the DSB distribution in the DSB targeting window and that mutations in the DNA at the Spo11 cleavage site affected DSB position. These results demonstrate that Spo11 itself has sequence preference and contributes to the choice of DSB positions.
Molecular and cellular biology 05/2009; 29(13):3500-16. · 6.06 Impact Factor
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Alain Nicolas
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ABSTRACT: Meiotic recombination is initiated by DNA double-strand breaks (DSBs) formed by the evolutionary conserved Spo11 protein. Along the S. cerevisiae chromosomes, the DSB sites are not evenly distributed and the cleavage frequencies vary 10-100-fold from site to site. Herein are reviewed the methods used in budding yeast to modulate locally and globally the native DSB frequencies, including a powerful method to target Spo11-dependent meiotic DSB in novel chromosomal regions. These methods serve to investigate the control and the mechanism of recombination initiation and modify the natural distribution of meiotic recombination.
Methods in molecular biology (Clifton, N.J.) 01/2009; 557:27-33.
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ABSTRACT: Meiotic recombination is triggered by programmed DNA double-strand breaks (DSBs), which are catalyzed by Spo11 protein in a type II topoisomerase-like manner. Meiotic DSBs can be detected directly using physical assays (gel electrophoresis, Southern blotting, and indirect end-labeling) applied to samples of genomic DNA from sporulating cultures of budding and fission yeast. Such assays are extremely useful for quantifying and characterizing many aspects of the initiation of meiotic recombination, including the timing of DSB formation relative to other events, the distribution of DSBs across the genome, and the influence on DSB formation of mutations in recombination factors and other gene products. By varying the type of gel electrophoresis and other parameters, the spatial resolution of DSB analysis can range from single nucleotides up to whole yeast chromosomes.
Methods in molecular biology (Clifton, N.J.) 01/2009; 557:117-42.
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ABSTRACT: The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome-wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double-strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild-type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB-prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.
The EMBO Journal 01/2009; 28(2):99-111. · 9.20 Impact Factor
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ABSTRACT: Cadmium (Cd(2+)) is a ubiquitous environmental pollutant and human carcinogen. The molecular basis of its toxicity remains unclear. Here, to identify the landscape of genes and cell functions involved in cadmium resistance, we have screened the Saccharomyces cerevisiae deletion collection for mutants sensitive to cadmium exposure. Among the 4866 ORFs tested, we identified 73 genes whose inactivation confers increased sensitivity to Cd(2+). Most were previously unknown to play a role in cadmium tolerance and we observed little correlation between the cadmium sensitivity of a gene deletant and the variation in the transcriptional activity of that gene in response to cadmium. These genes encode proteins involved in various functions: intracellular transport, stress response and gene expression. Analysis of the sensitive phenotype of our "Cd(2+)-sensitive mutant collection" to arsenite, cobalt, mercury and H(2)O(2) revealed 17 genes specifically involved in cadmium-induced response. Among them we found RAD27 and subsequently DNA2 which encode for proteins involved in DNA repair and replication. Analysis of the Cd(2+)-sensitivity of RAD27 (rad27-G67S) and DNA2 (dna2-1) separation of function alleles revealed that their activities necessary for Okazaki fragment processing are essential in conditions of cadmium exposure. Consistently, we observed that wild-type cells exposed to cadmium display an enhanced frequency of forward mutations to canavanine resistance and minisatellite destabilisation. Taken together these results provide a global picture of the genetic requirement for cadmium tolerance in yeast and strongly suggest that DNA replication, through the step of Okazaki fragment processing, is a target of cadmium toxicity.
DNA Repair 09/2008; 7(8):1262-75. · 4.14 Impact Factor
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Juri Hikiba,
Kouji Hirota,
Wataru Kagawa,
Shukuko Ikawa,
Takashi Kinebuchi,
Isao Sakane,
Yoshimasa Takizawa,
Shigeyuki Yokoyama,
Béatrice Mandon-Pépin, Alain Nicolas,
Takehiko Shibata,
Kunihiro Ohta,
Hitoshi Kurumizaka
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ABSTRACT: The M200V polymorphism of the human DMC1 protein, which is an essential, meiosis-specific DNA recombinase, was found in an infertile patient, raising the question of whether this homozygous human DMC1-M200V polymorphism may cause infertility by affecting the function of the human DMC1 protein. In the present study, we determined the crystal structure of the human DMC1-M200V variant in the octameric-ring form. Biochemical analyses revealed that the human DMC1-M200V variant had reduced stability, and was moderately defective in catalyzing in vitro recombination reactions. The corresponding M194V mutation introduced in the Schizosaccharomyces pombe dmc1 gene caused a significant decrease in the meiotic homologous recombination frequency. Together, these structural, biochemical and genetic results provide extensive evidence that the human DMC1-M200V mutation impairs its function, supporting the previous interpretation that this single-nucleotide polymorphism is a source of human infertility.
Nucleic Acids Research 08/2008; 36(12):4181-90. · 8.03 Impact Factor
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ABSTRACT: The autonomously replicating sequence binding factor 1 (Abf1) was initially identified as an essential DNA replication factor and later shown to be a component of the regulatory network controlling mitotic and meiotic cell cycle progression in budding yeast. The protein is thought to exert its functions via specific interaction with its target site as part of distinct protein complexes, but its roles during mitotic growth and meiotic development are only partially understood. Here, we report a comprehensive approach aiming at the identification of direct Abf1-target genes expressed during fermentation, respiration, and sporulation. Computational prediction of the protein's target sites was integrated with a genome-wide DNA binding assay in growing and sporulating cells. The resulting data were combined with the output of expression profiling studies using wild-type versus temperature-sensitive alleles. This work identified 434 protein-coding loci as being transcriptionally dependent on Abf1. More than 60% of their putative promoter regions contained a computationally predicted Abf1 binding site and/or were bound by Abf1 in vivo, identifying them as direct targets. The present study revealed numerous loci previously unknown to be under Abf1 control, and it yielded evidence for the protein's variable DNA binding pattern during mitotic growth and meiotic development.
Molecular biology of the cell 06/2008; 19(5):2193-207. · 5.98 Impact Factor
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ABSTRACT: The goal of this study was to determine whether mutations of meiotic genes, such as disrupted meiotic cDNA (DMC1), MutS homolog (MSH4), MSH5, and S. cerevisiae homolog (SPO11), were associated with premature ovarian failure (POF).
Case-control study.
Blood sampling, karyotype, hormonal dosage, ultrasound, and ovarian biopsy were carried out on most patients. However, the main outcome measure was the sequencing of genomic DNA from peripheral blood samples of 41 women with POF and 36 fertile women (controls).
A single heterozygous missense mutation, substitution of a cytosine residue with thymidine in exon 2 of MSH5, was found in two Caucasian women in whom POF developed at 18 and 36 years of age. This mutation resulted in replacement of a non-polar amino acid (proline) with a polar amino acid (serine) at position 29 (P29S). Neither 36 control women nor 39 other patients with POF possessed this genetic perturbation. Another POF patient of African origin showed a homozygous nucleotide change in the tenth of DMC1 gene that led to an alteration of the amino acid composition of the protein (M200V).
The symptoms of infertility observed in the DMC1 homozygote mutation carrier and in both patients with a heterozygous substitution in exon 2 of the MSH5 gene provide indirect evidence of the role of genes involved in meiotic recombination in the regulation of ovarian function. MSH5 and DMC1 mutations may be one explanation for POF, albeit uncommon.
European Journal of Endocrinology 02/2008; 158(1):107-15. · 3.42 Impact Factor
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ABSTRACT: During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1. We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Delta cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Delta cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.
PLoS Genetics 12/2007; 3(11):e223. · 8.69 Impact Factor
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ABSTRACT: Treatment of eukaryotic cells with 8-methoxypsoralen plus UVA irradiation (8-MOP/UVA) induces pyrimidine monoadducts and interstrand crosslinks and initiates a cascade of events leading to cytotoxic, mutagenic and carcinogenic responses. Transcriptional activation plays an important part in these responses. Our previous study in Saccharomyces cerevisiae showed that the repair of these lesions involves the transient formation of DNA double-strand breaks and the enhanced expression of landmark DNA damage response genes such as RAD51, RNR2 and DUN1, as well as the Mec1/Rad53 kinase signaling cascade. We have now used DNA microarrays to examine genome-wide transcriptional changes produced after induction of 8-MOP/UVA photolesions. We found that 128 genes were strongly induced and 29 genes strongly repressed. Modifications in gene expression concern numerous biological processes. Compared to other genotoxic treatments, c. 42% of the response genes were specific to 8-MOP/UVA treatment. In addition to common DNA damage response genes and genes induced by environmental stresses, a large fraction of 8-MOP/UVA response genes correspond to membrane-related functions.
FEMS Yeast Research 10/2007; 7(6):866-78. · 2.40 Impact Factor
