Molekulargenetische und biochemische Charakterisierung der pflanzlichen SRS2 und RecQ Helikasen
Zusammenfassung der Projektergebnisse
RECQ-helicases as well as the helicase SRS2 are involved in the processing of DNA structures arising during replication, recombination and repair. Mutations of different RecQ- homologs are responsible for severe human diseases, such as Blooms (BLM) or Werner (WRN) syndrome. Loss of RECQ helicases often leads to a hyperrecombination phenotypes and a defect in DNA repair. In Arabidopsis thaliana seven different RECQ genes are present as well as a single homologue of SRS2, the helicase was originally found to be involved in the suppression of homologous recombination in yeast. With the project we were able to contribute significantly to a better understanding of the biochemical properties and the biological role of individual helicases homologs in plants. On one side biochemical studies were performed. We could characterize in detail SRS2, which is able to unwind the elongated invading strand from a donor strand. For the first time, we demonstrated strand annealing activity of an SRS2 homolog which might help in annealing of the elongated strand later in the recombination process. In case of AtRECQ2 and AtRECQ3 we could show that the enzymes differ in their biochemical properties: Both are 3’ to 5’ helicases with similar activities on partial duplex DNA. However, AtRECQ2 catalyzes Holliday junction branch migration while AtRECQ3 can not act on intact Holliday junctions. Both enzymes can process replication forks and nicked Holliday junctions, however with different outcomes. Additionally, AtRECQ3 is much more efficient at catalyzing DNA strand annealing. More recently we started to characterize the biochemical activites of the closely related but phenotypically antagonistic helicases RECQ4A and RECQ4B, a work that is still in progress. We also analyzed the function of RECQ4A in vivo in detail by complementing a respective T-DNA insertion mutant with cDNA constructs that were mutated in different domains of the protein. We were able to demonstrate that the N-terminus (where the protein interacts with Toposiomerase 3a) as well as its helicase activity is absolutely required for the repair of DNA cross-links. In contrast both domains are also involved in the suppression of homologous recombination however, here we could see only partial complementation. Interestingly, replacing the N-terminus of RECQ4A by the respective sequence of RECQ4B resulted in the same phenotype as the deletion of the N-terminus. In contrast, exchanging the C-terminus of RECQ4A with RECQ4B sequences had no effect, the respective DNA was still able to fully complement the hyperrecombination phenotype as well as the DNA repair defect. It has been reported before that the unique RECQ homloge sgs1 of yeast is involved in the control of recombination between similar but not identical sequences (homeologous recombination). This is a very important feature for avoiding recombination between closely related sequences. We set up a transgenic marker system to analyze homeologous recombination in different genetic backgrounds in Arabidopsis. Applying the system we found that in plants several RECQ helicase contribute to different extend in the regulation of homeologous recombination indicating an important function of the RECQ helicases for keeping the often large and highly repetitive plant genomes stable.
Projektbezogene Publikationen (Auswahl)
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(2008). AtRECQ2, a RecQ helicase homologue from Arabidopsis thaliana, is able to disrupt varios recombinogenic DNA structures in vitro. The Plant Journal 55, 397-405
Kobbe D., Blanck S., Demand K., Focke M. and Puchta H.
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(2009). A SRS2 homolog from Arabidopsis thaliana disrupts recombinogenic DNA intermediates and facilitates single strand annealing. Nucleic Acids Res. 37, 7163-76
Blanck, S., Kobbe, D., Hartung, F., Fengler, K., Focke, M. and Puchta, H.
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(2009). Biochemical characterization of AtRECQ3 reveals significant differences relative to other RecQ helicases. Plant Phys. 151, 1658-66
Kobbe, D., Blanck, S., Focke, M., Puchta, H.
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(2010). Purification and characterization of RecQ helicases of plants. Methods Mol Biol.; 587, 195-209
Kobbe, D., Focke, M. and Puchta, H.
