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EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 ---------------------------------------------------------------------------------------------------------------------------------- P - 19. Majority of DNA double strand breaks are in plant genome rapidly removed independently of NHEJ repair pathways. Jaroslav Kozak, Chris West, Charles White, Karel J Angelis . Molec. Farming and DNA Repair, Inst. Experimental Botany, 160 00 Praha 6, Czech Republic Plants developed during evolution repair capacity to protect genome integrity against environmental genotoxines like UV or ionizing radiation. Among DNA lesions induced by radiation, the most lethal are double strand breaks (DSB) when only few, if unrepaired, can cause cell death. Repair of DSB induced in plant DNA by ionizing radiation or radiomimetic mutagens has been shown earlier. Several repair mechanisms were proposed for DSB elimination and nonhomologous joining of DNA ends (NHEJ) suggested as the major repair pathway in mammals and higher plants. However, the role of individual genes on the kinetic and extent of DSB repair in genomic DNA have not been established yet. Here we show that genes of NHEJ pathway do not have key role in overall repair of DSB and other repair pathway that quickly removes majority of DSB is active in higher plants. DSB detected by electrophoretic single cell (comet) assay under neutral conditions are in NHEJ pathway mutants AtLig4 and AtKu80 repaired faster (t½ = 3.5 and 3.4 minutes respectively) than in w.t. Arabidopsis (t½ = 5 min) and AtLig1a (t½ = 9 min), which was tested as possible alternative ligase to complete DSB sealing. Also chromosome maintenance proteins AtMim and AtBru1 (t½ = 50 and 15 min. respectively) were found to have dramatic effect on kinetics of overall DSB repair. Our results demonstrate that role of individual genes, if mutated, could be substituted by other genes, e.g. Lig4 in ligation step by Lig1. We also show dominant role of genes involved in structural maintenance of DNA to repair DSB. ---------------------------------------------------------------------------------------------------------------------------------- P - 20. Zinc finger transcription factors to discover Arabidopsis mutants with enhanced homologous recombination Beatrice Lindhout, Johan Pinas, Paul Hooykaas, Bert van der Zaal. Molecular and developmental genetics, Institute of Biology, Leiden University, 2333 AL Leiden, Netherlands A library of genes for zinc finger artificial transcription factors (ZF-ATF) was generated by fusion of DNA sequences encoding three-fingered Cys2His2 ZF domains to the VP16 activation domain and put under control of the promoter of the ribosomal protein gene RPS5A of Arabidopsis thaliana. After introduction of this library into an Arabidopsis homologous recombination (HR) indicator line, we selected primary transformants exhibiting multiple somatic recombination events. After PCR-mediated rescue of ZF sequences, reconstituted ZF-ATFs were reintroduced in the target line. In this manner, a ZF-ATF was identified that led to a 200 to 1000-fold increase in somatic HR, also in an independent second target line. A mutant plant line, expressing the HR-inducing ZF-ATF exhibited increased resistance to the DNA-damaging agent bleomycin and was more sensitive to methyl methanesulfonate (MMS), a combination of traits not described before. Our results demonstrate that the use of ZF-ATF pools is highly rewarding when screening for novel dominant phenotypes in Arabidopsis. ---------------------------------------------------------------------------------------------------------------------------------- P - 21. Addressing the role of chromatin structure on gene targeting rates Marie Mirouze, Jon Reinders, Jerzy Paszkowski. Laboratory of Plant Genetics, Université de Genève Sciences III, 1211 Genève, Switzerland Addressing the role of chromatin structure on gene targeting rates Marie Mirouze, Jon Reinders and Jerzy Paszkowski Laboratory of Plant Genetics, University of Geneva, Page 38 sur 56
EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 Sciences III, Quai Ernest-Ansermet 30, CH-1211 Genève, Switzerland We describe an experimental system which is currently under development in our laboratory to address to which extent epigenetic modifications affect gene targeting rates in Arabidopsis thaliana. For this we use PPOX gene encoding protoporphyrinogen oxidase, an enzyme involved in chlorophyll biosynthesis. Inhibition of PPOX by the herbicide Butafenacil results in rapid plant death due to oxidative stress. However, the combination of two mutations in PPOX gene confers resistance towards Butafenacil. It was shown that the introduction of these mutations to the chromosomal copy of PPOX gene by gene targeting is possible and can be used to assess gene targeting frequencies. We first examined epigenetic marks at the PPOX locus. DNA methylation analyses indicate that in the wild type plants PPOX locus is associated with substantial levels of DNA methylation, therefore, using mutants impaired in maintenance of DNA methylation may alter the epigenetic status of this locus. We developed an EpiRILs population comprising F8 lines from a cross between Col WT and met1-3, a mutant affected in a methyltransferase responsible for CpG methylation maintenance. These EpiRILs have the same genetic Col background, but differ in their cytosine methylation profiles. We will describe the interest of using this epigenetic material to assess the role of chromatin structure in homologous recombination rates. This research is supported by the European Community in the frame of the TAGIP project (No. 018785). ---------------------------------------------------------------------------------------------------------------------------------- P - 22. Genetic modification of rice plants by using Zinc Finger technology. Keishi Osakabe, Kiyomi Abe, Masaki Endo, Hiroaki Saika, Seiichi Toki. Division of Plant Sciences, National Institute of Agrobiological Sciences, 305-8602 Tsukuba, Ibaraki, Japan Zinc finger nucleases (ZFNs) are synthetic nucleases consisting of a zinc finger DNAbinding domain fused to the cleavage domain of the restriction endonuclease, Fok I. ZFNs can be used to introduce double-strand DNA breaks (DSBs) in specific DNA sequences, and promote site-specific homologous recombination (gene targeting) and targeted genome modification at the desired genomic loci including deletion- and insertion-type mutations. To apply this technology to the genetic modification of rice, we made a system to identify potential zinc-finger target sites in a gene of interest with the FASTA program based on the finding by Barbas's group (1). We already established the efficient transformation system of rice (2) and the gene targeting system with the rice acetolactate synthase (OsALS) gene locus (3). Thus, we chose the OsALS gene for the initial gene targeting experiment with ZFNs. We identified a ZFN recognition sequence at the Cterminal region of OsALS using our searching system, and designed ZFNs (ZFNosals_L and ZFNosals_R). We also confirmed that the combination of ZFNosals_L and ZFNosals_R could digest its recognition site on the OsALS gene in in vitro. We are further confirming digestion in in vivo by the ZFNs, and testing whether the introduction of the ZFNs can increase the efficiency of gene targeting at the OsALS gene locus. References 1. Mandell, J.G. and Barbas III, C.F. (2006) Nucleic Acids Res., 34, W516-W523. 2. Toki, S., et al. (2006) Plant J., 46, 969-976. 3. Endo, M., et al. (2006) 8th International Congress of Plant Molecular Biology, Book of Abstracts, p153. ---------------------------------------------------------------------------------------------------------------------------------- P - 23. Two unlinked double-strand breaks can induce reciprocal exchanges in plant genomes via homologous recombination and non-homologous endjoining Michael Pacher, Waltraud Schmidt-Puchta, Holger Puchta. Molecular Biology & Biochmeistry of plants / Prof. Puchta, University of Karlsruhe / Institute of Botany II, 76128 Karlsruhe, Germany Using the rare-cutting endonuclease I-SceI we were able to demonstrate before that the repair of a single DSB in a plant genome can be mutagenic due to insertions and Page 39 sur 56
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