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EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 deletions. However, during replication or due to irradiation several breaks might be induced simultaneously. To analyze the mutagenic potential of such a situation we established an experimental system in tobacco harboring two unlinked transgenes each carrying an I-SceI site. After transient expression of I-SceI a kanamycin resistance marker could be restored by joining two previously unlinked broken ends, either by homologous recombination (HR) or by non-homologous end-joining (NHEJ). Indeed, we were able to recover HR and NHEJ events with similar frequencies. Despite of the fact that no selection was applied for joining the two other ends, the respective linkage could be detected in most cases tested, demonstrating that the respective exchanges were reciprocal. The frequencies obtained indicate that DSB-induced translocation is up to two orders of magnitude more frequent in somatic cells than ectopic gene conversion. Thus, DSB induced reciprocal exchanges might play a significant role in plant genome evolution. The technique applied in this study may also be useful for the controlled exchange of unlinked sequences in plant genomes. ---------------------------------------------------------------------------------------------------------------------------------- P - 24. ATM-mediated transcriptional and developmental responses to ?-rays in Arabidopsis Lilian Ricaud, Caroline Proux, Jean-Pierre Renou, Olivier Pichon, Sylvain Fochesato, Phiippe Ortet, Marie-Hélène Montané. Direction des Sciences du vivant, CEA, 13108 St Paul-lez- Durance cedex, France (1) CEA, DSV, Institut de Biologie Environnementale et de Biotechnologie (iBEB), Service de biologie végétale et de microbiologie environnementales (SBVME), F-13108 Saint Paul-lez- Durance, France. (2) Unité de Recherche en Génomique Végétale, INRA, 2 rue Gaston Crémieux, F-91057 Evry, France (3) Present address: Plate-forme puces à ADN, Genopole, Institut Pasteur, 28 rue du Docteur Roux, F-75724 Paris cedex 15, France *E-mail: mariehelene.montane@cea.fr ATM (Ataxia Telangiectasia Mutated) is an essential checkpoint kinase that signals DNA double-strand breaks in eukaryotes. Its depletion causes meiotic and somatic defects in Arabidopsis and progressive motor impairment accompanied by several cell deficiencies in patients with ataxia telangiectasia (AT). To obtain a comprehensive view of the ATM pathway in plants, we performed a time-course analysis of seedling responses by combining confocal laser scanning microscopy studies of root development and genome-wide expression profiling of wild-type (WT) and homozygous ATM-deficient mutants challenged with a dose of ?-rays (IR) that is sublethal for WT plants. Early morphologic defects in meristematic stem cells indicated that AtATM, an Arabidopsis homolog of the human ATM gene, is essential for maintaining the quiescent center and controlling the differentiation of initial cells after exposure to IR. Results of several microarray experiments performed with whole seedlings and roots up to 5 h post- IR, which were compiled in a single table; sequence and function homology searches; import of cell cycling, and mutant-constitutive expression characteristics; and a simplified functional classification system were used to identify novel genes. The transcription burst was almost exclusively AtATM-dependent or weakly AtATRdependent, and followed two major trends of expression in atm: (i)-loss or severe attenuation and delay, and (ii)-inverse and/or stochastic, as well as specific, enabling one to distinguish IR/ATM pathway constituents. The hundreds of radiomodulated genes identified were not a random collection, but belonged to functional pathways such as those of the cell cycle; cell death and repair; DNA 3R; and transcription; translation; and signaling, indicating the strong cell reprogramming and double-strand break abrogation functions of ATM checkpoints. Accordingly, genes in all functional classes were either down or up-regulated concomitantly with downregulation of chromatin deacetylases or upregulation of acetylases and methylases, respectively. Determining the early transcriptional indicators of prolonged S-G2 phases that coincided with cell proliferation delay, or an anticipated subsequent auxin increase, accelerated cell differentiation or death, was used to link IR-regulated hallmark functions and tissue phenotypes after IR. Our data provide a large resource for studies on the interaction between plant checkpoints of the cell cycle, development, hormone response, and DNA repair functions, Page 40 sur 56
EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 because IR-induced transcriptional changes partially overlap with the response to environmental stress. Putative connections of ATM to stem cell maintenance pathways, and to non-DSB/DSBs repair mechanisms in plants after IR are discussed. ---------------------------------------------------------------------------------------------------------------------------------- P - 25. Cellular and molecular understanding of plant DNA damage response through ATM, ATR, H2AX, E2F and RNR proteins. Hélène Roa, Julien Lang, Lenin Sanchez-Calderon, Ondrej Smetana, Frédéric Lincker, Chamseddine Mediouni, Guy Houlné. plant cellular biology, IBMP/CNRS UPR2357 Strasbourg, 67084 Strasbourg, France Plants are continuously exposed to high levels of genotoxic stresses which can induce DNA damage, and according to its could lead to cellular death. So through evolution, plants have fixed efficient DNA repair mechanisms. Among key factors involved in DNA repair, Ribonucleotide reductase has to be highly regulated for providing the cell with dNTPs. In Arabidopsis the 3 AtRNR2 encoding the small R2 subunit present specific profile of gene induction, R2-3b is significantly induced in response to IR or the radiomimetic bleomycin (ATM-dependency) but not upon HU contrarily to R2-3 and R2-5 genes. These genes presented E2F-elements in their promoters and as described in tobacco, we demonstrate that E2F factor plays an important role to drive their gene induction upon DNA damage in Arabidopsis (replicative stress or DSB) under the control of ATM, ATR or alternative pathways. In addition GFP-NtE2F protein fusion presents a nuclear localization in both tobacco and Arabidopsis cells and forms some clear and discrete nuclear foci when these cells are submitted to bleomycin. Most of these NtE2F nuclear foci colocalized with Atgamma H2AX, a marker of DSBs, and their number was increasing in a time-dependent manner upon BLM treatment. Such NtE2F foci formation is ATM-dependent and occurs with a higher density in the root meristem. Interestingly, increasing time of BLM treatment leads also to increased cell death concomitant with strong gene induction of particular PCD marker genes. Works is in progress to understand the role of E2F in DNA repair and PCD cellular processes. ---------------------------------------------------------------------------------------------------------------------------------- P - 26. CHARACTERIZATION OF ARABIDOPSIS EXONUCLEASE 1 Max Roessler, Karel Riha , Austrian Academy of Sceinces, Gregor Mendel Institute, 1030 Vienna, Austria Exonuclease 1 (EXO1) is a multitasking nuclease, with 5' - 3' exonuclease activity, which is present in virtually all eukaryotic organism from yeast to mammals. Studies performed mainly in yeast have demonstrated that EXO1 is involved in mismatch DNA repair (MMR). It is also implicated in the resection of DNA double strand breaks, homologous recombination, telomere maintenance, meiosis, and DNA replication. However, these functions of EXO1 are less well understood. We found that the Arabidopsis genome encodes two paralogous proteins, which we refer to as AtEXO1A and AtEXO1B. Arabidopsis is the only known organism containing two putative EXO1 genes. The homology between the AtEXO1A and AtEXO1B proteins is limited to the N-terminal nuclease domain suggesting that the proteins have evolved different functions. Supporting this idea we found that expression levels of EXO1A and EXO1B transcripts differes in various tissues. We characterized mutant lines harbouring T-DNA insertions in the AtEXO1A and AtEXO1B genes. These lines showed no sensitivity to DNA damage inducing agents (Mitomycin C, Menadion, Hydroxy Urea, Methyl-Methane Sulfonate, Bleocin). Further experiments are underway to examine the function of the EXO1 paralogues at telomeres, in MMR and in homologous recombination (HR). ---------------------------------------------------------------------------------------------------------------------------------- Page 41 sur 56
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Page 56 and 57: Sean Mayes University of Nottingham
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