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EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 P - 3. Immunolocalization of Mre11 and Rad50 proteins during prophase I in tomato Lorinda Anderson, Leslie Lohmiller, Hildo Offenberg, Christa Heyting . Biology, Colorado State University, 80523-1878 Fort Collins, Colorado, United States Mre11 and Rad50 proteins act together in a protein complex that is involved in DNA double-strand break (DSB) repair, and the proteins also have roles in meiotic synapsis and recombination. mre11 and rad50 mutants in Arabidopsis thaliana lack homologous synapsis, suffer extensive chromosome fragmentation during prophase I, and are sterile. Current models predict the presence of both Mre11 and Rad50 in early recombination nodules (ENs) that are thought to represent sites of DSBs. Here, we used immunofluorescence to examine the meiotic behavior of these two proteins in Solanum lycopersicum (tomato) microsporocytes during early prophase I. Mre11 was present as numerous immunofluorescent foci that associated preferentially with the axes of tomato chromosomes before, during and immediately after synapsis. As many as 800 Mre11 foci were observed in leptotene nuclei (more than twice the maximum number of ENs observed at zygotene), and Mre11 foci were most common in distal euchromatic regions of chromosomes (as are ENs). Electron microscopic immunogold localization demonstrated that Mre11 is a component of only some ENs at mid-late zygotene while accumulations of Mre11 along the axial/lateral elements are common. In addition, Mre11 foci are far more numerous than ENs on asynapsed euchromatic segments of leptotene and early zygotene chromosomes. Like Mre11, Rad50 foci preferentially associate with chromosomal axes during early prophase I. Unexpectedly, the number of Rad50 foci was less than that of Mre11 foci (although more similar to EN numbers), and Rad50 and Mre11 foci did not often colocalize. The presence of Rad50 in ENs is still under investigation, but our results indicate that most Mre11 foci do not correspond to ENs. ---------------------------------------------------------------------------------------------------------------------------------- P - 4. Human Rad51 binding and the subsequent unwinding of duplex DNA requires different nucleotide cofactors kamakshi Balakrishnan, Rekha Govekar, Basuthkar J. Rao . Department of Biological sciences, Tata Institute of Fundamental Research (TIFR), 400006 Mumbai, India Homologous recombination is one of the central mechanisms for removing DNA double strand breaks in humans and other eukaryotes. Human Rad51 (hRad51) is a recombinase, which brings about this process in concert with other recombination mediator proteins such as Rad52, Replication Protein A (RPA), Rad54 and other accessory proteins by performing DNA homology search and pairing, followed by DNA strand exchange. With the aim of understanding the roles of different nucleotide cofactors such as ATP, ADP, ATP gammaS (a slow hydrolysable ATP analog), GTP and GDP on: (a) hRad51 binding to duplex DNA and (b) hRad51 mediated unwinding of duplex DNA , we carried out binding and topological assays. Our results suggest that binding of hRad51 to circular duplex DNA is efficient in the presence of nucleotide cofactors such as ATP, ATP gammaS or GTP, and results in the formation of higher order protein-DNA complexes. However, in absence of such nucleotide cofactors or in presence of ADP, lower order complexes result. We observe rapid unwinding only in the presence of ATP, whereas the unwinding kinetics slow down in presence of ATP gammaS. This indicates that hRad51 mediated duplex DNA unwinding depends on ATP hydrolysis, unlike binding. Although providing GTP as a cofactor enhances hRad51 binding to duplex DNA as much as ATP does, the unwinding kinetics mediated by GTP are poorer compared to ATP. ADP and GDP on the other hand, neither facilitate binding nor unwinding. Upon inclusion of hRad52 however, in the reaction system hRad51 unwinds dsDNA utilizing ADP as cofactor. Same is not true for GDP. Our results thus indicate that (a) hRad51 mediated unwinding of duplex DNA is kinetically favored when ATP can be hydrolyzed as against the slower unwinding observed with ATP gammaS, (b) A new role for hRad52 in the unwinding reaction carried out by hRad51 when ADP is provided as cofactor, (c) GTP can also serve as a cofactor for Page 30 sur 56
EMBO Plant DNA Repair and Recombination Workshop, Presqu'île de Giens, France 2007 unwinding reaction although not as efficient as ATP. ---------------------------------------------------------------------------------------------------------------------------------- P - 5. Towards plant gene targeting – Enhancing homologous recombination frequency in tobacco. Abdellah Barakate, Claire Halpin. based at Scottish Crop Research Institute, University of Dundee , DD2 5DA Dundee, United Kingdom Gene targeting (GT) is a powerful tool that allows a homology-based replacement of an endogenous gene by an in vitro manipulated copy. Developing this technology has proved particularly difficult in plants where foreign DNA is integrated predominantly by nonhomologous recombination. The main competing pathways of recombination repair(1) could be manipulated to increase homologous recombination (HR) efficiency. One strategy to stimulate HR is to express recombinases involved in this pathway. We have expressed multiple bacterial (RecA, RecG, RuvC) and human (hRad51, hDMC1 and hRad52) recombinases in a tobacco line (N1DC4) that already contains a transgene as an artificial substrate for intrachromosomal recombination(2) (ICR). The transgene for ICR consists of a direct repeat of two defective b-glucoronidase (GUS) genes that can recombine to generate a functional GUS, which can be detected by histochemical staining of plant material. In order to co-ordinate the expression of multiple recombinases in a single plant, we have used an artificial self-dissociating polyprotein system. This unique and novel function was taken from the 2A region of foot-and-mouth disease virus(3). Several tobacco transgenic lines expressing multiple recombinases were selected and their ICR assessed both in pollen and six-week-old seedlings. While ICR stimulation in seedlings remained modest, a huge increase (up to 1000 fold) was detected in pollen of some transgenic lines. In addition, most transgenic lines showed various degrees of sterility and their meiosis will be analysed. This plant material will also be used to determine whether ICR stimulation will translate into efficient gene targeting using our newly designed vectors. References: 1) Trends Genet. (2005) 21, 172-181. 2) EMBO J. (1994) 13, 484-489. 3) Plant Physiol. (2004) 135, 16-24. Acknowledgements: Barbara Hohn (Basel) for N1DC4 line, Stephen West (London) for human recombinases. Funding: BBSRC, Scottish Enterprise Tayside, The Leverhulme Trust. ---------------------------------------------------------------------------------------------------------------------------------- P - 6. Repair of G:T mismatches in plants Joke Baute, Gert Van der Auwera, Ann Depicker. Plant Systems Biology, VIB - Ghent University, 9052 Ghent, Belgium G:T mismatches are the result of replication errors or of hydrolytic deamination of 5methylcytosine that occurs spontaneously in the cell. In mammals, G:T mismatches caused by deamination of methylated cytosines, are repaired by base excision repair. Two DNA glycosylases recognize this mismatch: thymine DNA glycosylase (TDG) and methylbinding domain 4 protein (MBD4). Both enzymes specifically convert G:T mismatches to G:C. In plants, the methylation level is higher than in animals, suggesting that plants also developed systems to repair G:T mismatches caused by deamination of 5-methylcytosine. To analyze this in plants, we are performing a functional analysis of MBD4 homologs in Arabidopsis thaliana. We identified only one annotated coding sequence in Arabidopsis thaliana that shows sequence homology to the glycosylase domain of MBD4. The human MBD4 consists of two DNA binding domains: a methyl-CpG-binding domain and a mismatch specific DNA glycosylase domain, while the Arabidopsis protein (AtMBD4) has the glycosylase domain but lacks the methyl-CpG-binding domain. Using Y2H, we are currently investigating with which proteins AtMBD4 interacts. We have found different splicing variants of AtMBD4 in different tissues, what is also the case for human MBD4 and for many other DNA glycosylases, and these will be presented. To learn more on the function of the AtMBD4 gene, we analyzed the AtMBD4 promotor activity by using Page 31 sur 56
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Page 6 and 7: 1400-1700 SESSION V: Recombination
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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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