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<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 the occurrence of the segregating trait identified in the said SDR-I progeny population. This method provides a reliable method for the mapping of complex traits and is a very efficient alternative to the development and use of heterogeneous inbred families. WO 2006/094774. ---------------------------------------------------------------------------------------------------------------------------------- P - 39. A functional analysis of DNA ligases from Arabidopsis thaliana Wanda M. Waterworth, Ghzaleh Masnavi, Claire Provost, Paul Sunderland, Christopher E. West, Clifford M. Bray. Molecules to Cells, University of Manchester, UK, M13 9PT Manchester, United Kingdom Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, U.K.; Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK Eukaryotic organisms possess multiple DNA ligases with distinct roles in DNA metabolism. In plants, DNA ligases are crucial for the maintenance of nuclear, mitochondrial and plastid genome stability and both the levels and their intracellular distribution are tightly regulated. The three DNA ligase genes expressed in Arabidopsis (DNA ligases I, IV and VI; AtLIG1, AtLIG4 and AtLIG6) have been functionally characterized in our laboratory. AtLIG1 has roles in both DNA repair and replication, and possibly recombination, and is indispensable for cell viability. AtLIG1 expresses one major and two minor mRNA transcripts differing only in the length of the 5’ untranslated leader sequences preceding a common ORF. Intracellular targeting of DNA ligase 1 is controlled via an evolutionarily conserved posttranscriptional mechanism that involves the use of alternative start codons to translate multiple ligase activities from a single transcript. A further level of control involves hierarchical dominance of the different targeting sequences when more than one targeting sequence is present in the same ligase isoform. The maintenance of genomic integrity involves the function of all three DNA ligases in Arabidopsis. As with many DNA repair factors, the ligases are likely to form dynamic multi protein complexes to repair DNA damage. Ongoing studies are concerned with the identification and characterisation of interacting protein partners for AtLig1 and the physiological functions and importance of the novel plant specific ligase 6. ---------------------------------------------------------------------------------------------------------------------------------- P - 40. I-SceI-stimulated intrachromosomal homologous recombination in maize Sophie Wehrkamp-Richter, Wyatt Paul, Julien Levy, Samuel LeGoff, Jean-Baptiste Laffaire, Fabienne Degroote, Pascual Perez, Georges Picard. Gene Function and Maize Traits Group, Biogemma, 63000 Clermont-Ferrand, France Gene targeting (GT) by homologous recombination (HR) allows the precise modification of any gene. GT has become an indispensable tool in yeast and mouse, however, this tool is still missing in higher plants since the frequency of homologous recombination in plants is too low (10-4 to 10-5) compared to the frequency of illegitimate recombination (NHEJ). However, in model plants, induction of a double strand break at the target locus by the expression of the meganuclease I-SceI results in a dramatic stimulation of HR. In collaboration with Cellectis (who develop meganuclease technologies) we are applying these results to maize in order to develop an efficient gene targeting system for a major crop plant. We have generated maize plants which contain a reporter of intrachromosomal HR and plants with constitutive I-SceI expression. In this assay the frequency of HR is estimated by the reconstitution by HR, of GUS in pollen. Preliminary data suggests that I-SceI -induced DSBs can stimulate HR in maize. We are now developing an inducible meganuclease in order to control HR and develop vectors that both measure RH and NHEJ in maize. ---------------------------------------------------------------------------------------------------------------------------------- Page 48 sur 56
<strong>EMBO</strong> Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 P - 41. Programmed response to IR-induced genomic stress Kaoru Yoshiyama, Clare Robertson, Kevin Culligan, Anne Britt. Plant Biology, University fo California, Davis, 95616 Davis, United States Cellular responses to DNA damage are many and varied. Many of these responses are programmed, and the complete response requires the presence of multiple signal transduction pathways. The genetic requirements for these responses can be difficult to sort out in mammals, as many defects in damage repair or response lead to the induction of apoptosis. Plants, fortunately, are not as exquisitely sensitive to mismanaged damage, and so make excellent model systems for the study of the roles of these proteins both in response to acute damage, and in the general maintenance of unperturbed mitotic and meiotic cells. Whole-genome analysis indicates that plants, unlike yeasts and mammals, exhibit a robust transcriptional response to DNA damage that includes many genes relevant to DNA repair and cell cycle regulation. However, this response is governed, at least in part, by the same signal transduction components that are required for other types of damage response in yeasts and mammals. Thus plants may make an excellent model system for the genetic analysis of DNA damage response. In this presentation we will focus on our efforts, suing both classical genetic and reverse genetic techniques, to identify components of the plant DNA damage response pathway. ---------------------------------------------------------------------------------------------------------------------------------- P - 42. Identification of genes required for DNA-damage-induced cell cycle arrest in Arabidopsis Kaoru Yoshiyama, Anne Britt. Plant Biology, University of California Davis, 95616 Davis, United States The genome of a living organism is subjected to a wide variety of genotoxic stresses of both endogenous and environmental origin. DNA-damage-induced cell cycle checkpoints are a crucial component of genome maintenance. These checkpoints sense DNA damage, pause the cell division cycle to provide time for repair, and finally release the cell cycle from arrest. The mammalian ATM and ATR protein kinases are key proteins involved in the cell cycle arrest, and Arabidopsis has ATM and ATR homologs. However, in contrast to yeast or mammalian cells, little is known about DNA damage checkpoint in plants. AtXPF is a homolog of human repair endonuclease XPF. The xpf mutant of Arabidopsis can’t form true leaves after irradiation as seeds, showing sensitivity to gamma-radiation. We screened for suppressor mutants in the xpf background using EMS and isolated sog (suppressor of gamma radiation) mutants that can form true leaves following radiation, suggesting that this cell cycle arrest is due to a signaling mechanism rather than to inhibition of cell cycle by physically blocking the cellular machinery. We have observed that xpf sog1-1 seeds are hypersensitive to HU (hydroxyurea), an inhibitor of ribonucleotide reductase. They could germinate on the HU plates, but after that, they were dead. Surprisingly, when the seeds are grown on “no HU” plates for 5 days, and the seedlings are transferred to HU plates, the plants can grow as well as wild type. These results suggest that SOG might be important during or immediately after germination. We reported that in wild-type Arabidopsis, hundreds of genes are induced in response to gamma-radiation, and the induction is dependent on ATM. BRCA1, RAD51 and CYCB1;1 transcriptions increased rapidly and strongly. Interestingly, the transcription inductions of these genes were compromised severely in the xpf sog1-1 mutant, suggesting that the SOG gene plays an important role in upstream of signal transduction like ATM. (The xpf mutation does not affect the induction of the genes.) To identify the SOG gene, we’re currently mapping the mutation, and we know that the SOG gene lies in the vicinity of CIW12 marker on chromosome I. We are hoping that identifying the function of SOG gene is a good way to clarify the mechanism of checkpoint mechanism in plants and, perhaps, in animals. ---------------------------------------------------------------------------------------------------------------------------------- Page 49 sur 56
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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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