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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 suppresses recombination between divergent direct repeats in somatic cells or between homologs from different ecotypes during meiosis. We describe the involvement of other genes from the mismatch repair family on homologous recombination and discuss the implications of these results for plant improvement via gene transfer across species. ---------------------------------------------------------------------------------------------------------------------------------- P - 11. Transient inactivation of NHEJ proteins Sylvia de Pater, Vanessa Costa, Paul Hooykaas. Institute Biology Leiden, Leiden University, 2333 AL Leiden, Netherlands Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome and frequencies of gene targeting (GT) in plants are very low. It would be an advantage for analysis of gene function and for exploitation of genetically modified organisms if the frequency of targeted integration could be increased. Experiments with yeast mutants have shown that GT can be enhanced by mutations in the DNA repair pathway of non-homologous end joining (NHEJ)(1). Subsequently, Arabidopsis NHEJ T- DNA insertion mutants have been tested in GT experiments, and preliminary results show that GT frequency is improved somewhat in these mutants. Since constitutive inhibition of the NHEJ DNA repair pathway results in telomere lengthening (2,3) and maybe other DNA rearrangements, we applied the peptide aptamer technology (4) combined with an inducible promoter system for the temporary inactivation of NHEJ proteins. A peptide aptamer has been isolated that binds to AtKu80 and yKu80 in yeast (Y2H) and in vitro and expression in yeast and Arabidopsis results in more sensitivity for DNA damage inducing agents. 1. van Attikum H and Hooykaas PJJ (2003) Genetic requirements for targeted integration of Agrobacterium T-DNA in Saccharomyces cereviciae. Nucl Acid Res 31, 826-832. 2. Bundock P, van Atticum H, Hooykaas P (2002) Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutants. Nucleic Acids Res 30, 3395-3400. 3. Bundock P and Hooykaas P (2002) Severe developmental defects, hypersensitivity to DNA-damaging agents, and lengthened telomeres in Arabidopsis MRE11 mutants. Plants Cell 14, 2451-2462. 4. Colas P, Cohen B, Jessen T, Grishina, McCoy J, Brent R (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature 380, 548-550. ---------------------------------------------------------------------------------------------------------------------------------- P - 12. ADI IS A NOVEL COMPONENT OF THE ARABIDOPSIS SOMATIC DNA DOUBLE STRAND BREAK RESPONSE AND IS ESSENTIAL FOR MEIOSIS Philip Dean, Susan Armstrong, Christopher West. CENTRE FOR PLANT SCIENCES, UNIVERSITY OF LEEDS, UK, LS2 9JT Leeds, United Kingdom Here we describe ATM DEPENDENT INDUCTION (ADI), a plant specific gene which shows a large induction specifically in response to genotoxic stress. Quantitative PCR demonstrates that the induction of ADI is dependent on the protein kinase ATM, placing ADI in an ATM pathway. ADI is also essential for Arabidopsis meiosis as adi knockout mutant plants are both male and female sterile. Detailed cytological analysis of these mutants reveals extensive chromosome fragmentation during meiosis. Studies in an adi heterozygous background correlate with ADI also being required for pollen mitosis as there is a failure to complete mitotic cell divisions in approximately half the pollen. Preliminary yeast 2-hybrid interaction studies indicate ADI may interact with components required for both DNA double strand break repair and cell cycle progression. ---------------------------------------------------------------------------------------------------------------------------------- P - 13. A Cre::FLP fusion protein recombines FRT or loxP sites in transgenic maize plants Vesna Djukanovic, Brian Lenderts, Alex Lyznik. Crop Genetics Research, Pioneer Hi-Bred Page 34 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 International, Inc, IA 50131 Johnston, United States The coding sequences of Cre (site-specific recombinase from the bacteriophage P1) and FLP (the yeast 2mm plasmid site-specific recombinase) were fused in frame to produce a novel, dual-function site-specific recombinase gene. Transgenic maize plants containing the Cre::FLP-fusion expression vector were crossed to transgenic plants containing either loxP or FRT excision substrates. Complete and precise excisions of chromosomal fragments flanked by the respective target sites were observed in the F1 and F2 progeny plants. The episomal DNA recombination products were frequently lost. Some chromosomal recombination substrates survived in the F1 plants producing the chimeric F1 plants for the excision products. They became stabilized in the F2 generation after the cre::FLP gene segregated out. These observations may indicate that the efficiency of sitespecific recombination is affected by plant developmental stages with site-specific recombination being more prevalent in the developing embryos. Both FLP and Cre activities were comparable, producing clean and precise chromosomal excision products in maize. The crossing strategy proved to be suitable for genetic engineering of maize using the FLP or Cre site-specific recombination systems. ---------------------------------------------------------------------------------------------------------------------------------- P - 14. In search of a role for Dss1 in the model plant Arabidopsis thaliana Emeline Dubois, Fabien Delacote, Marie-Pascale Doutriaux. Institut de Biotechnologie des Plantes-UMR8618, Université Paris XI-CNRS, 91405 Orsay, France In human, mutations of the BRCA2 gene are responsible for a hereditary form of breast cancer predisposition (1,2). One important role of Brca2 is to act on homologous recombination by loading Rad51 onto ssDNA at the site of the double strand breaks. Dss1 is a partner of Brca2 in human and Ustilago maydis (3,4). In Ustilago, Dss1 is essential to the Brca2 function in somatic cells (5). In addition, Dss1 was shown to belong to the 26S proteasome in human and yeast (6,7), whereas there is no Brca2 in yeast. Two isoforms of Brca2 and two isoforms of Dss1 are encoded for in the Arabidopsis thaliana genome. A potential role for AtDss1 in the proteasome has been revealed by the functional complementation of sem1 (the Dss1 ortholog) mutants in yeast. We are now examining the role of Dss1 in relation to Brca2 in Arabidopsis. One of the two Brca2 isoforms interacts with both AtDss1 isoforms, whereas the other one interacts with only one (8). RNAi constructs have been established to inactivate Brca2 or Dss1 constitutively (p35S) or at meiosis (pDMC1). Plants transformed with the RNAi/BRCA2 constructs were sterile, due to aberrant meiosis (9). Plants transformed with the RNAi/DSS1 constructs are fertile. Thus, a role of AtDss1 at meiosis seems to be excluded. To further investigate the role of Brca2 and Dss1 in somatic DNA repair, we have transformed plants containing recombination substrates (10), with both RNAi constructs (under the control of the constitutive p35S promoter). Those plants are now being evaluated in terms of their sensitivity to genotoxic stresses and for their recombination rate. 1. Wooster et al (1995) Identification of the breast cancer susceptibility gene BRCA2. Nature 378: 789-792 2. Scully , Livingston (2000) In search of the tumour-suppressor functions of BRCA1 and BRCA2. Nature 408: 429-432 3. Marston, et al (1999) Interaction between the product of the breast cancer susceptibility gene BRCA2 and DSS1, a protein functionally conserved from yeast to mammals. Mol. Cell. Biol. 19 4633-42. 4. Kojic et al (2003) The BRCA2interacting protein DSS1 is vital for DNA repair, recombination, and genome stability in Ustilago maydis. Mol. Cell. 12:1043-9. 5. Kojic et al (2005) Brh2-Dss1 interplay enables properly controlled recombination in Ustilago maydis.Mol Cell Biol. 25:2547-57. 6. Funakoshi et al (2004) Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability. J Cell Sci. 117:6447-54. 7. Krogan, et al (2004) Proteasome involvement in the repair of DNA double-strand breaks. Mol. Cell. 16 : 1027-34. 8. Dray et al (2006) Interaction between Arabidopsis Brca2 and Its Partners Rad51, Dmc1, and Dss1. Plant Physiol. 140:1059-69.. 9. Siaud et al (2004) Brca2 is involved in meiosis in Arabidopsis thaliana as Page 35 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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