Éditeur(s) : HAL CCSD BioMed Central
Résumé : International audience
BACKGROUND: Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. RESULTS: We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes -- normally the hottest region in the genome. CONCLUSIONS: Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.
ISSN: 1471-2164

inserm-00849779
http://www.hal.inserm.fr/inserm-00849779
http://www.hal.inserm.fr/inserm-00849779/document
http://www.hal.inserm.fr/inserm-00849779/file/1471-2164-14-493.pdf
DOI : 10.1186/1471-2164-14-493

Éditeur(s) : HAL CCSD Nature Publishing Group
Résumé : International audience
Meiotic recombination predominantly occurs at discrete genomic loci called recombination hotspots, but the features defining these areas are still largely unknown (reviewed in refs 1-5). To allow a comprehensive analysis of hotspot-associated DNA and chromatin characteristics, we developed a direct molecular approach for mapping meiotic DNA double-strand breaks that initiate recombination. Here we present the genome-wide distribution of recombination initiation sites in the mouse genome. Hotspot centres are mapped with approximately 200-nucleotide precision, which allows analysis of the fine structural details of the preferred recombination sites. We determine that hotspots share a centrally distributed consensus motif, possess a nucleotide skew that changes polarity at the centres of hotspots and have an intrinsic preference to be occupied by a nucleosome. Furthermore, we find that the vast majority of recombination initiation sites in mouse males are associated with testis-specific trimethylation of lysine 4 on histone H3 that is distinct from histone H3 lysine 4 trimethylation marks associated with transcription. The recombination map presented here has been derived from a homogeneous mouse population with a defined genetic background and therefore lends itself to extensive future experimental exploration. We note that the mapping technique developed here does not depend on the availability of genetic markers and hence can be easily adapted to other species with complex genomes. Our findings uncover several fundamental features of mammalian recombination hotspots and underline the power of the new recombination map for future studies of genetic recombination, genome stability and evolution.
ISSN: 0028-0836

hal-00877681
https://hal.archives-ouvertes.fr/hal-00877681
DOI : 10.1038/nature09869
PUBMED : 21460839

Éditeur(s) : HAL CCSD Cold Spring Harbor Laboratory Press
Résumé : International audience
Meiotic DNA double-stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population, and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method--single-stranded DNA (ssDNA) sequencing (SSDS)--that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.
ISSN: 1088-9051

hal-00877675
https://hal.archives-ouvertes.fr/hal-00877675
DOI : 10.1101/gr.130583.111
PUBMED : 22367190

Éditeur(s) : Université de Bretagne Occidentale
Résumé : Archaea are micro-organisms present in all ecosystems, but are majoritary present in extremophilic environments. Hyperthermophilic Archaea, as Pyrococcus abyssi, are permanently exposed at temperature which can increase the rate of spontaneous DNA damage. It is probable that hyperthermophilic Archaea possess efficient molecular mechanism to duplicate, maintain and stabilize their genomes. The aim of this project was to investigate the interaction network involved in the process of DNA replication and DNA repair. The methodological approach consists in the coupling of pull-down with mass spectrometry identification of interacting partners. A preliminary interaction network was identified that was composed of new proteins as well as unsuspected interactions between known components of DNA replication machinery. The main results consist in: (1) the nuclease Pab2263, which interacts with PCNA, is a novel player in DNA metabolism. (2) PCNA is likely part of a macrocomplex with the ubiquitary proteins Mre11 and Rad50 suggesting a role of this complex in the repair of double strand break in connection with a replication fork. (3) Fen1 and DNA primase interact physically and can collaborate in vitro to resolve intermediate step of the base excision repair pathway. These results enhance our understanding of DNA repair process in Archaea.
Les Archaea sont des micro-organismes rencontrés dans tous les écosystèmes, mais qui apparaissent comme majoritaires dans les environnements dits extrêmes. Les archaea hyperthermophiles, comme Pyrococcus abyssi sont en permanence exposées à des températures qui peuvent augmenter le taux de dommages de l'ADN, pourtant, le taux de mutations spontanés chez ces micro-organismes est similaire à celui des espèces modèles mésophiles. Il est ainsi probable que les hyperthermophiles possèdent des systèmes particulièrement efficaces pour dupliquer, maintenir et stabiliser leur génome. L'objectif de ce projet était d'explorer le réseau d'interaction impliqué dans les processus de réplication et de réparation de l'ADN. L'approche méthodologique mise en oeuvre a consisté à coupler la capture de partenaires d'interaction par pull-down avec leur identification par spectrométrie de masse. J'ai pu ainsi mettre en évidence, au sein l'extrait cellulaire de P. abyssi, un réseau préliminaire reliant des protéines de la maintenance génomique. Nous avons non seulement mis en évidence de nouvelles protéines impliquées probablement dans des mécanismes de réparation, mais également des nouvelles interactions non suspectées entre des composants déjà caractérisés. Les principaux résultats sont les suivants : (1) La nucléase Pab2263, partenaire du PCNA, est un nouvel acteur du métabolisme de l'ADN. (2) Le PCNA forme un macrocomplexe avec les protéines ubiquitaires Mre11 et Rad50 suggérant un rôle de ce complexe dans la réparation des cassures double brin de l'ADN lors de la réplication. (3) Les protéines Fen1 et l'ADN primase interagissent physiquement et peuvent collaborer in vitro pour résoudre une étape intermédiaire de la voie de réparation par excision de base. Ces résultats enrichissent notre compréhension des processus de réparation de l'ADN chez les archées.

Droits : info:eu-repo/semantics/openAccess
http://archimer.ifremer.fr/doc/2008/these-3722.pdf
http://archimer.ifremer.fr/doc/00000/3722/

Éditeur(s) : HAL CCSD American Association for the Advancement of Science
Résumé : International audience
DNA double-strand breaks (DSBs) are introduced in meiosis to initiate recombination and generate crossovers, the reciprocal exchanges of genetic material between parental chromosomes. Here, we present high-resolution maps of meiotic DSBs in individual human genomes. Comparing DSB maps between individuals shows that along with DNA binding by PRDM9, additional factors may dictate the efficiency of DSB formation. We find evidence for both GC-biased gene conversion and mutagenesis around meiotic DSB hotspots, while frequent colocalization of DSB hotspots with chromosome rearrangement breakpoints implicates the aberrant repair of meiotic DSBs in genomic disorders. Furthermore, our data indicate that DSB frequency is a major determinant of crossover rate. These maps provide new insights into the regulation of meiotic recombination and the impact of meiotic recombination on genome function.
ISSN: 0036-8075

hal-01123717
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01123717
PUBMED : 25395542
DOI : 10.1126/science.1256442

Éditeur(s) : HAL CCSD Elsevier
Résumé : International audience
Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (B[a]P), are widely distributed environmental contaminants exerting toxic effects such as genotoxicity and carcinogenicity, mainly associated with aryl hydrocarbon receptor (AhR) activation and the subsequent induction of cytochromes P-450 (CYP) 1-metabolizing enzymes. We previously reported an up-regulation of AhR expression and activity in primary cultures of human T lymphocyte by a physiological activation. Despite the suggested link between exposure to PAHs and the risk of lymphoma, the potential of activated human T lymphocytes to metabolize AhR exogenous ligands such as B[a]P, and produce DNA damage has not been investigated. In the present study, we characterized the genotoxic response of primary activated T lymphocytes to B[a] P. We demonstrated that, following T lymphocyte activation, B[a]P treatment triggers a marked increase in CYP1 expression and activity generating, upon metabolic activation, DNA adducts and double-strand breaks (DSBs) after a 48-h treatment. At this time point, B[a]P also induces a DNA damage response with ataxia telangiectasia mutated kinase activation, thus producing a p53-dependent response and T lymphocyte survival. B[a]P activates DSB repair by mobilizing homologous recombination machinery but also induces gene mutations in activated human T lymphocytes which could consequently drive a cancer process. In conclusion, primary cultures of activated human T lymphocytes represent a good model for studying genotoxic effects of environmental contaminants such as PAHs, and predicting human health issues.
ISSN: 0006-2952

hal-01560145
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560145
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560145/document
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01560145/file/Liamin%20et%20al.%20-%20Benzo%5Ba%5Dpyrene-induced%20DNA%20damage%20associated%20with%20.pdf
DOI : 10.1016/j.bcp.2017.04.025

Éditeur(s) : HAL CCSD Cold Spring Harbor Laboratory Press
Résumé : International audience
Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation
ISSN: 0890-9369

hal-01281480
https://hal-univ-rennes1.archives-ouvertes.fr/hal-01281480
DOI : 10.1101/gad.270009.115
PUBMED : 26833728