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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 chromosomes preceding recombination and synapsis. Recent attention has been paid to the bouquet, a nearly universal event, during which the telomeres cluster in early prophase. It has been suggested that because the telomeres are in close proximity this would enhance the pairing of the homologues. We have shown in Arabidopsis that we do not observe a classical bouquet, rather the telomeres are organised already around the prophase nucleolus. The homologous telomeres are paired at the transition from G2 to leptotene during the assembly of the axial elements. As the chromosomes synapse during zygotene, the telomeres reveal only a loose clustering, which may represent a relic bouquet. To investigate the nature of chromosome pairing we are carrying out a functional analysis of pairing in recombination mutants. We have already shown in a range of Arabidopsis asynaptic meiotic mutants (spo11, asy1, mre11, dmc1, rad 51c) that the telomeres appear to be paired in early leptotene but this is not permanent and appears to be lost as meiotic prophase progresses. We are using FISH with unique chromosomal paints to find out if our observations are due purely to random associations or are we observing homologous telomeric pairing Our analysis will uncover whether initial homologous pairing requires DSB, recombination proteins MRE11, DMC1, RAD51C or structural meiotic components. ---------------------------------------------------------------------------------------------------------------------------------- T - 20. HR versus NHEJ at the chromosomal level Ingo Schubert, Koichi Watanabe, Ales Pecinka Cytogenetics and Genome Analysis, Leibniz- Institute of Plant Genetics and Crop Plant Research, D-06466 Gatersleben, Germany HR versus NHEJ at the chromosomal level Ingo Schubert, Koichi Watanabe, Ales Pecinka,IPK, D-06466 Gatersleben, Germany According to molecular studies, HR is the major mechanism for double-strand break (DSB) repair in yeast, while in somatic cells of vertebrates and of seed plants NHEJ is considered to be predominant. A low HR frequency is coincident with a low frequency of homologous gene targeting in seed plants. However, at least at the level of genotoxin-mediated structural rearrangements between freshly replicated chromatids, HR is remarkably frequent; apparently 100% of SCEs and up to 8-fold more chromatid-type aberrations than expected at random are the outcome of HR. Both phenomena can be quantified reliably only during the first mitotic division after the S-phase of their origin. Later such events are either not (unambiguously) detectable or the carrier cells are eliminated. Thus it could be possible that quite a proportion of HR is overlooked, the more so as it remains to be elucidated which proportion of the preclastogenic DSBs are processed via HR into SCE or chromatid-type aberrations. Therefore it is needed to determine i) the probability of DSB processing into the different endpoints, including restoration of the pre-break situation, and ii) how are the spatial requirements for HR achieved. As a prerequisite to address the first aim, a screening system to monitor the induction of DSBs per nucleus at a specific locus is being established. In chromatin mutants with a hyper-recombination phenotype and an up to 100-fold increase of intrachromosomal somatic HR (in cis), chromosome territory arrangement and homologous pairing frequencies are not significantly altered. Contrarily, clastogenic doses of X-rays cause a significant increase of the positional homologous pairing frequencies in A.thaliana that decays during 1h after irradiation. These data indicate that for homologous intrachromatid recombination chromatin relaxation might be sufficient, while interchromatid recombination likely requires an active search for homology. ---------------------------------------------------------------------------------------------------------------------------------- T - 21. AtRad1/AtErcc1 endonuclease and telomere stability in Arabidopsis. Jean-Baptiste Vannier, Annie Depeiges, Charles White, Maria Eugenia Gallego CNRS UMR 6547, Universite Blaise Pascal, 63177 Aubiere, France Telomeres are the specific chromatin structures present at the ends of chromosomes Page 16 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 serving to avoid chromosomal shortening at replication and recombination. Proteins known to be involved in DNA repair and recombination have been found associated to telomeres, however, their specific roles in the maintenance of functional chromosome ends are poorly understood. The XPF/Ercc1 complex, a structure-specific endonuclease that acts in nucleotide excision repair and recombination, has been shown to be associated to telomeres. Arabidopsis plants mutated for either Xpf (AtRad1) or Ercc1 (AtErcc1) orthologs develop normally and show wild-type telomere length. However in the absence of telomerase, mutation of either of these two genes induces an earlier onset of chromosomal instability. End-to-end chromosome fusions are detected at generation 1 as compared to generation 5 in telomerase mutant plants. Furthermore a significantly higher number of anaphase bridges per cell are observed in telomerase mutant plants lacking either AtRad1 or AtErcc1 as compared to single telomerase mutants. Plants showing fertility defects are observed at generation 2 both in AtRad1 and AtErcc1 telomerase double mutants. This early appeareance of uncapped telomeres is not related to a general acceleration of telomeric repeat loss. Fluorescent in situ hybridization show the presence of extrachromosomal DNA fragments, containing both subtelomeric and telomeric repeat signals, which are not observed in telomerase single mutant plants presenting a similar numbers of anaphase bridges. Anaphase bridges present in nuclei with extrachromosomal fragments never show both subtelomeric and telomeric signals. These data and the possible roles of AtRad1/AtErcc1 in Arabidopsis telomere stability will be discussed. ---------------------------------------------------------------------------------------------------------------------------------- T - 22. A role of ATM in telomere length regulation in Arabidopsis Laurent Vespa, Ross Warrington, Jiri Siroky, Petr Mokros, Dorothy Shippen Biochemistry and Biophysics, Texas A&M University, TX 77840 College Station, United States Telomeres are the indispensable nucleoprotein complexes that protect linear chromosomes from replicative attrition and from the DNA damage response machinery. Without them, chromosome ends would be recognized as double-strand breaks and fused to each other, leading to deleterious chromosomal rearrangements. This function has led to investigations of the relationship between telomeres and the DNA damage response machinery. Paradoxically, several of these factors have an essential role in the physical integrity of telomeres. We have taken advantage of the outstanding resistance of Arabidopsis to genomic instability to study the role of several DNA damage response factors at telomeres, including the central sensor/activator protein kinase ATM. We have shown that a combined deficiency in ATM and TERT, the reverse transcriptase subunit of telomerase, leads to a premature onset of telomere uncapping. However the nature of the fusion events in atm/tert are different from the single tert mutants. Our telomere-fusion amplification assay mostly failed to detect products in the double atm tert mutant, while cytogenetic experiments showed very high number of anaphase bridges, a hallmark for chromosome fusions. Therefore, we have characterized telomere fusions arising in atm tert mutants by FISH analysis using telomeric probes and an extensive series of subtelomeric BAC probes. Our results indicate that one particular chromosome end is involved in most of the fusions. We also revealed that about 40% of the fusions in atm tert (versus ~5% in tert) do not involve chromosome ends, which suggests that ATM may prevent multiple breakage-fusion-breakage events as a cell cycle checkpoint controller. A precise telomere length analysis of individual chromosome ends was then performed by PETRA (Primer Extension Telomere Repeat Amplification). These data showed that the chromosome end involved in the fusions was much shorter than its counterparts, and that this shortening event happened in early generations (G2-G3). This finding suggests that a particularly large Telomere Rapid Deletion (TRD) event occurred in the double mutant. We then addressed the putative role of ATM in controlling TRD by performing parent/progeny PETRA analysis on a large number of tert and atm tert plants. We found that large size telomeres are more prone to TRD in the double mutant, which can lead to the stochastic generation of critically shortened telomeres. We conclude that ATM has a role in preventing TRD or in preventing cells that have undergone large deletions from Page 17 sur 56
Page 1: PLANT DNA Repair & Recombination In
Page 4 and 5: EMBO Plant DNA Repair and Recombina
Page 6 and 7: 1400-1700 SESSION V: Recombination
Page 20 and 21: eing propagated. EMBO Plant DNA Rep
Page 54 and 55: Cyril Charbonnel CNRS UMR6547 Unive
Page 56 and 57: Sean Mayes University of Nottingham
Page 58: Wanda Melody Waterworth University

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