Progress 10/01/01 to 10/01/06
Outputs
Maize was found to have two Mre11 genes, termed Mre11A and Mre11B. In addition, both maize and Arabidopsis were found to have an Nbs1 homolog. mRNA and genomic DNA for all three genes have been cloned and sequenced. Antibodies to all three proteins have been raised and the maize Mre11A and Nbs1 homologs found to interact in yeast two-hybrid screens. In contrast, Mre11B does not interact with Nbs1 and has what appears to be a mitochondrial targeting peptide at its N-terminal end. Cellular localization of these proteins is in progress.
Impacts
Understanding this complex in its entirety, the other cellular components it interacts with and the molecular signaling events that are associated with it will now allow us to understand the signaling processes in how plants respond to DNA damage.
Publications
- Weil, C. F. and R. Monde (2006) Getting the point: mutations in maize. Plant Genome (in press)
- Yu, J.-H., K. Marshall, M. Yamaguchi, J.E. Haber and C.F. Weil. (2004) Microhomology-dependent end-joining and repair of transposon-induced DNA hairpins by host factors in yeast. Molec. Cell. Biol. 24:1351-1363
- Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, Young K, Bowers E, Codomo CA, Enns LC, Odden AR, Greene EA, Comai L, Henikoff S. (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol. 4(1):12
- Weil, C. F., R. Monde, B. Till, L. Comai and S. Henikoff (2005) Mutagenesis and functional genomics in maize. Maydica 50:415-424
- Yang, G., C. F. Weil and S. R. Wessler (2006) A rice Tc1/mariner-like element transposes in yeast. Plant Cell 18:2469-2478
- Waterworth, W., C. Altun. K. Young, S. Armstrong, C. Weil, C. Bray, C. West (2006) A plant homolog for Nbs1, the signaling component of the Mre11 DNA repair/recombination complex, (submitted to PNAS)
- Weil, C. (2002) Finding the crosswalks on DNA. Proc. Natl. Acad. Sci. (USA) 99:5763
- Hannah, L.C. and C.F. Weil (2004) Transposons and Allelic Diversity. In Encyclopedia of Plant and Crop Sciences. R. Goodman, ed. 1: 1-4, Marcel Dekker, New York
- C.F. Weil (2004) Recombination and Genetic Diversity. In Encyclopedia of Plant and Crop Sciences. R. Goodman, ed. 1: 54-57 Marcel Dekker, New York
- Weil, C. F., (2005) Transposons in the modern age. Maydica 50:339-348
- Weil, C. F. (2005) Single base hits score a home run in wheat. Trends in Biotech. 23:220-222
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Progress 10/01/04 to 09/30/05
Outputs
We have identified the signaling component Nbs1 of the major DNA repair complex Mre11/Rad50/Nbs1 ("MRN"). Nbs1 had not been identified outside animals until now. The gene is present in EST collections from ten species examined to date. All contain conserved forkhead associated, BRCT-association, Mre11-binding and ATM-interaction domains as well as a conserved and functionally important phosphorylation site. We have cloned and sequenced the gene from both maize and Arabidopsis, expressed the full length maize protein and shown that it interacts with at least one of the two maize Mre11 proteins in yeast two hybrid assays. Cloning of the entire Arabidopsis CDS is in progress. We have identified mutants defective in Nbs1 expression from both maize and Arabidopsis. These mutants are hypersensitive to the DNA damaging agent methylmethane sulfonate (MMS) confirming a role for this gene in DNA damage response.
Impacts
Understanding this complex in its entirety, the other cellular components it interacts with and the molecular signaling events that are associated with it will now allow us to understand the signaling processes in how plants respond to DNA damage.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
In the past year, this project has revealed and characterized the roles of two plant genes in the repair of broken DNA created when mobile elements move in the genome. The Ku80 protein is part of a complex thought to bind broken DNA ends and protect them while the cell goes about repairing and rejoining the ends. In Arabidopsis mutants that are deficient for ku80, there is a significant increase (compared to non-mutant siblings) in deletion of DNA surrounding a transposon insertion site when the transposon leaves that site and breaks the DNA. The genomic sequence of a ku80 gene in maize has now been determined and we are pursuing a reverse genetics strategy to obtain lines deficient in ku80 for further study. A second gene, MRE11, is also important in repair of transposon excision sites. In yeast cells, the gene is required to repair transposon excision sites, however its role in plants remains unclear. We have obtained mutant Arabidopsis lines deficient in Mre11 and
are testing the effects of these mutations. In addition, we have characterized the Mre11 genes of two different maize inbreds and find that they carry two different Mre11 genes that differ in their C-terminal sequences. One of these, Mre11A, is alternatively spliced and is predicted to produce both a full-length and a truncated protein. Mre11A is expressed in leaves, ears and tassel. In contrast, the second gene, Mre11B is expressed only in ears and tassel. We are further characterizing the expression patterns of these two genes to see if there are interactions between them that confer mitotic vs meiotic specificity to them.
Impacts
This work is expected to begin unravelling how plants repair broken DNA ends that do not share homologous sequences. These occur frequently in plant cells in response to many environmental challenges. A better understanding of how plants fix DNA breaks will help us understand how they undergo recombination, create genetic diversity and incorporate transgenes.
Publications
- J. Yu, K. Marshall, M. Yamaguchi, J. E., Haber and C. F. Weil. (2004) Microhomology-dependent end-joining and repair of transposon-induced DNA hairpins by host factors in yeast. Molec and Cell Biol. 24: 1351-1360
- Till, B. J., S. H. Reynolds, C. Weil, N. Springer, C. Burtner, C. A. Codomo, L. C. Enns, A. R. Odden, K. Young, E. Bowers, E. A. Greene, L. Comai and S. Henikoff (2004) Discovery of induced point mutations in maize genes by TILLING, BMC Plant Biol. 2004 Jul 28;4(1):12
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Progress 10/01/02 to 09/30/03
Outputs
Progress was made in characterizing the maize mre11 gene as well as an apparent paralog of mre11 we have named mre11B located in the 19kD zein cluster. It is not yet clear whether mre11B is expressed. However, it has a strikingly well-conserved predicted protein sequence and intron position. Interestingly, it is shorter than mre11 and seems to end at the same general position as the bacterial homolog to mre11 gene, sbcD. We have evidence now that the mre11 gene apparently undergoes alternative splicing into at least two alternative forms, one of which is terminated prematurely and the other of which contains the C-teminal portion of the protein that, at least by analogy to yeast, may be important for its DNA binding. It remains unclear whether the multiple forms are expressed in different tissues or at different times in development. We have also identified potential transposon insertions into the maize rad50 gene. In the model plant, Arabidopsis, we have identified
T-DNA insertions in several available collections that disrupt several potentially important DNA repair genes, such as mre11, rad50, Ku80, Ku70, rad54, XPB1 and others. We are presently examining their effects on the repair of Ac transposon excisions.
Impacts
This work is expected to begin unravelling how plants repair broken DNA ends that do not share homologous sequences. These occur frequently in plant cells in response to many environmental challenges. A better understanding of how plants fix DNA breaks will help us understand how they undergo recombination, create genetic diversity and incorporate transgenes.
Publications
- No publications reported this period
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Progress 10/01/01 to 09/30/02
Outputs
In the past year we have crossed transposon containing lines of Arabidopsis to more than 30 different T-DNA insertion mutants defective for likely homologs of DNA repair genes. We have also identified alleles of the mre11 gene through the Arabidopsis TILLING Project. These have all been selfed and we arre screening them for homozygous mutant lines that contain Ac. We have begun to characterize the effects of these mutations on repair of DNA breaks induced by transposition. The first of these, mutant for the AtKu80 gene, has been shown to have a repair defect by Anne Britt's lab at UC Davis. We have found that this mutation reduces the amount of excision site repair we can recover in a PCR based assay as compared to either a heterozygous or homozygous wild type sibling. Either the frequency of transposition has been decreased in the ku80 mutants or there is deletion of DNA from the transposon excision site that extends greater than 100 bp and removes the binding site
for one of the primers in our PCR assay. This second hypothesis would be consistent with data from ku80 mutants in other systems. Either result is interesting and we are currently testing both hypotheses. In addition, we have collaborated with Pioneer Hi-Bred to obtain corn lines mutant for the mre11 gene and the rad50 gene. These are Mutator transposon alleles and we are currently backcrossing them to isolate the mutations in each. They will then be crossed to Ac containing lines for analysis.
Impacts
Identification of the genes involved in transposition may begin to explain why maize suffers less additional damage when repairing transposon excisions than most other higher eukaryotes. Our hypothesis is that these adaptations by the DNA repair system are in response to the presence of active transposons in maize for the past 10,000 years--a situation found in no other higher eukaryote we are aware of.
Publications
- Kunze, R and C. F. Weil. (2002) The hAT and CACTA element families in plants. Invited chapter for Mobile DNA II, N. Craig, R. Craigie, M Gellert, A Lambowitz eds., ASM Press, Washington, DC, pp 565-610
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