Differing Requirements for RAD51 and DMC1 in Meiotic Pairing of Centromeres and Chromosome Arms in Arabidopsis thaliana

During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains

SINE Retroposons Can Be Used In Vivo as Nucleation Centers for De Novo Methylation

SINEs (short interspersed elements) are an abundant class of transposable elements found in a wide variety of eukaryotes. Using the genomic sequencing technique, we observed that plant S1 SINE retroposons mainly integrate in hypomethylated DNA regions and are targeted by methylases. Methylation can then spread from the SINE into flanking genomic sequences, creating distal epigenetic modifications. This methylation spreading is vectorially directed upstream or downstream of the S1 element, suggesting that it could be facilitated when a potentially good methylatable sequence is single stranded during DNA replication, particularly when located on the lagging strand. Replication of a short methylated DNA region could thus lead to the de novo methylation of upstream or downstream adjacent sequences

Identification of Arvicola terrestris scherman Sperm Antigens for Immune Contraceptive Purposes

International audienceThe cyclical proliferation of the wild fossorial rodent Arvicola terrestris scherman (ATS) is critical in mid-mountain ecosystems of several European countries. Our goal is to develop an immunocontraceptive vaccine to control their fertility, as a sustainable alternative to chemical poisons currently used. Indeed, these chemicals cause the death of ATS predators and animals sharing their ecosystem, and current laws progressively limit their use, making the development of a targeted vaccination strategy an interesting and efficient alternative. In order to identify species-specific sperm antigens, male and female ATS received subcutaneous injections of whole ATS spermatozoa to elicit an immune response. The analysis of the immune sera led to the identification of 120 immunogenic proteins of sperm cells. Of these, 15 were strictly sperm-specific and located in different regions of the male gamete. Some of these antigens are proteins involved in molecular events essential to the reproductive process, such as sperm-egg interaction, acrosomal reaction, or sperm motility. This approach not only identified a panel of immunogenic proteins from ATS sperm cells, but also demonstrated that some of these proteins trigger an immune response in both male and female ATS. These spermatic antigens are good candidates for the development of a contraceptive vaccine

Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana

International audienceHomologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51-DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three 'non-meiotic' paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells

The Structure-Specific Endonucleases MUS81 and SEND1 are essential for telomere stability in Arabidopsis.

International audienceStructure-specific endonucleases act to repair potentially toxic structures produced by recombination and DNA replication, ensuring proper segregation of the genetic material to daughter cells during mitosis and meiosis. Arabidopsis thaliana has two putative homologs of the resolvase (structure-specific endonuclease): GEN1/Yen1. Knockout of resolvase genes GEN1 and SEND1, individually or together, has no detectable effect on growth, fertility, or sensitivity to DNA damage. However, combined absence of the endonucleases MUS81 and SEND1 results in severe developmental defects, spontaneous cell death, and genome instability. A similar effect is not seen in mus81 gen1 plants, which develop normally and are fertile. Absence of RAD51 does not rescue mus81 send1, pointing to roles of these proteins in DNA replication rather than DNA break repair. The enrichment of S-phase histone γ-H2AX foci and a striking loss of telomeric DNA in mus81 send1 further support this interpretation. SEND1 has at most a minor role in resolution of the Holliday junction but acts as an essential backup to MUS81 for resolution of toxic replication structures to ensure genome stability and to maintain telomere integrity

Chromosome arm pairing during meiotic prophase I in WT, <i>xrcc3</i>, and <i>rad51C</i> plants.

<p>Percentages of WT, <i>xrcc3</i>, or <i>rad51C</i> meiocytes showing paired or unpaired signals in FISH using mid-arm probes on chromosomes 2 or 1. Numbers of nuclei are given in brackets.</p

Pairing of centromeres and 5S rDNA loci in <i>rad51</i>, <i>dmc1</i>, <i>rad51 dmc1</i>, and <i>spo11-1</i> mutants.

<p>(A) Zygo-pachytene of <i>rad51</i>, <i>dmc1</i>, <i>rad51 dmc1</i> and <i>spo11-1</i> showing centromeres (green) and three pericentromeric 5S rDNA signals (red) in <i>rad51</i> or more in <i>dmc1</i>, <i>rad51 dmc1</i> and <i>spo11-1</i> mutants. (Scale bar: 10 µm.). (B) shows progression in centromere coupling through prophase I for each mutant with 8 to 10 (fully unpaired), 1 to 3 (clustering), 6 to 7 (partially paired) and 4 or 5 centromeres (complete pairing). (C) shows percentages of pairing of 5S rDNA loci in zygo-pachytene nuclei of each mutant. Homologous centromere pairing is observed in <i>rad51</i> whereas non-homologous centromere coupling is predominant in <i>dmc1</i>, <i>rad51 dmc1</i> and <i>spo11-1</i> mutants.</p

Mean numbers of centromere FISH signals though prophase I in recombination defective lines.

<p>In all mutants, zygotene and pachytene figures were grouped into the zygo-pachytene stage. Numbers of meiocytes analysed are given in brackets.</p