DNA REPAIR AND RECOMBINATION IN PLANTS
We are analysing the mechanisms of genomic changes in higher plants which is not only relevant in terms of basic science but also in terms of biotechnology. Our main interest focuses on two different ways to link DNA molecules by covalent bonds: homologous recombination (HR), in which the reaction partners have identical sequences and non-homologous end-joining (NHEJ), in which the partner molecules differ in their sequence. Whereas HR is the mechanism of meiotic recombination, in somatic cells NHEJ plays a major role in higher plants.
Double-strand break repair and gene technology
By expression of the restriction endonuclease I-SceI we were able to induce a specific double strand break (DSB) at its 18 bp recognition site in transgenic plants. Using this assay system in somatic cells we could demonstrate that DSBs are repaired only in about 1 out of 10 000 cases by the use of homology at allelic or ectopic positions in the plant genome. By contrast, if homology is present adjacent on the same chromosome, up to a third of the breaks can be repaired by homologous recombination. Based on these results a method for the efficient elimination of selectable marker genes was established. Within the transgene the sequence to be eliminated is flanked by recognition sites of I-SceI, the expression of the latter results in the elimination of the intervening DNA from the plant genome. Recently we were also able to induce reciprocal exchanges between unlinked sites in the plant genome (Figure 1). At the moment we are involved in several projects to set up efficient gene targeting techniques for plants.
Figure 1: I-SceI-induced reciprocal exchanges between unlinked sites in the tobacco genome. Using the rare-cutting endonuclease I-SceI two simultaneous double-strand breaks were introduced in unlinked positions in the genome of Nicotiana tabacum. Both transgenes (linkeage A—B and C—D, respectively contain an 18mer I-SceI recognition site (red line). Under appropriate selection it was possible to identify plants bearing the recombinant linkeage A—D. Surprisingly, most of these plants also harboured another new junction, namely C—B. Thus reciprocal translocation is one possible outcome of two double-strand breaks occurring simultaneously in the same genome.
Isolation of factors involved in DNA repair and recombination in Arabidopsis
The major focus of our current research concentrates on the identification of factors involved in DNA repair and recombination reactions in plants. By analysing homologies to proteins involved in these processes in other organisms we were able to isolate a set of factors from Arabidopsis thaliana. We are especially interested in plant specific peculiarities of the recombination machinery.
Factors involved in meiotic recombination
We were able to identify three homologues of Spo11, the protein responsible for the induction of DSBs during meiosis, which is encoded by a single gene in other eukaryotes. Spo11 descends from the subunit A of the archaebacterial topoisomerase VI. We were able to demonstrate that plants carry - in contrast to other eukaryotes - also a B subunit of this enzyme, indicating that plants harbour an extra topoisomerase of archaebacterial origin. Mutation analysis demonstrates that this subunit together with one of three Spo11 homologues constitutes a topoisomerase that is mainly required for DNA endoreduplication in somatic plant cells. Recently we were able to demonstrate that the two other Spo11 homologues are both actively involved in meiotic DSB induction.
Factors related to human genetic syndromes
In humans the genetic disorders Blooms and Werner Syndrome are caused by mutations in genes that are coding for RecQ DNA helicases. Patients with the Werner syndrome senesce at young age and show a high prevalence of cancer, as it is the case with Blooms patients. We were able to identify a surprising large family of seven RecQ homologues in Arabidopsis. By analysing insertion mutants we characterize their biological function in vivo. Our results indicate that at least some family members have unique even antagonistic functions. Two of them (AtRECQ4A and 4B) arose due to a recent gene duplication and are still nearly 70% identical on the protein level. Knock-out of these genes leads to antagonistic phenotypes: the AtRECQ4A mutant shows sensitivity to DNA damaging agents, enhanced homologous recombination (HR) and lethality in a mus81 background. Moreover, mutation of AtRECQ4A partially suppresses the lethal phenotype of an AtTOP3a mutant, a phenomenon that had only been demonstrated for RecQ homologues of unicellular eukaryotes previously. Together, these facts strongly suggest that in plants RECQ4A is functionally equivalent to the mammalian BLM protein. In stark contrast, mutants of the closely related AtRECQ4B are not mutagen sensitive, viable in a mus81 background, and are not able to suppress the induced lethality caused by loss of AtTOP3a. Moreover, they are strongly impaired in HR. Thus, AtRECQ4B is specifically required to promote but not to suppress cross over events, a role in which it differs from all eukaryotic RecQ homologues known. At the moment we are trying to define the role of the other RecQ helicases in plants as well as their interactions with other DNA repair pathways.
Figure 2: Compared to the wildtype Col-0 (A), the top3α mutant (B) develops only small seedlings which die within three weeks
when sowed on agar plates. Surprisingly, the lethal top3α phenotype can be reverted in a recq4A mutant background.
Double homozygous individuals of these crossings are viable but remain smaller (D) than the wildtype (C).
These plants show mitotic and meiotic defects resulting in a characteristic shape of the plants and sterility.
Factors homologous to genes involved in human breast cancer
We analyze the biological roles of homologues of genes that are involved in breast cancer in humans. Surprisingly homologues of BRCA1and BARD1 could be identified in the Arabidopsis genome. Mutations of these genes lead to a deficiency in homologous recombination. The factors seem to be involved in the regulation of DNA repair by signal transduction. Currently we are in the process to define by genetic and biochemical means interaction networks of these proteins to learn more about the reaction of plants to genotoxic stress.
Biochemical characterization of DNA processing enzymes
Besides analysing the biological role of RecQ helicases by genetic means we also characterize them biochemically. The human RecQ homologue WRN carries, besides the RecQ helicase domain,also an exonuclease domain. Plants have no homologue of this bifunctional protein, but surprisingly the Arabidopsis genome contains a small ORF (AtWRNexo) with homology to the exonuclease domain of hWRN. Expression of this ORF in E. coli revealed a WRN-like exonuclease activity for AtWRNexo. We were able to demonstrate that AtWRNexo can interact with AtRecQ2. By expression of AtRecQ2 we not only could demonstrate its 3'-5' helicase activity but also that the enzyme is able to dissolve recombination intermediates like D-loops and Holliday Junctions (Figure 3). Our analysis indicates that AtRecQ2 might be biochemically homologous to the helicase part of hWRN. Currently we are expressing and analysing other DNA helicases and also other enzymes involved in DNA repair of Arabidopsis.
Figure 3: On this autoradiography the processing of a Holliday-Junction by RECQ2 from Arabidopsis thaliana is shown. Holliday junctions are important intermediates in the Homologous Recombination pathway. On the left, the different possible structures are pictured. In the right lane (i) the Holliday junction is shown in the absence of the enzyme (substrate control). It is clearly visible in lanes d to h, that AtRECQ2 (in different concentrations) processes the Holliday junction - and the main product is a two-oligonucleotide structure, indicative to a branch migration activity of AtRECQ2. Expectedly, this product appears only in the presence of ATP (lane c), as helicases are fuelled by the hydrolysis of nucleotide-tri-phosphates. If ATP can not be hydrolysed, which is the case with a protein, in which the therefore required Walker A motif is mutated (lane b, K117M), the Holliday junction is not processed.
Enzyme-mediated assembly of DNA nanostructures
As part of a CFN-funded project C5 we contribute to the "bio-assembly of nanostructured crystals". By the programmable pairing of DNA strands bound to organic cores higher-ordered nanostructures should be built up. As nature developed during evolution a huge repertoire of highly specific enzymes for the manipulation of DNA, we are trying to use DNA processing enzymes for the production of regular three-dimensional structures.