Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3α (TOP3α) functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3α in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3α-2), which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3α-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3α, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3α is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3α mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3α and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.

SPO11, a homolog of the subunit A of the archaebacterial topoisomerase VI, is essential for double-strand break (DSB)-induced initiation of meiotic recombination. In contrast with single homologs in animals and yeasts, three homologs are present in Arabidopsis thaliana and other higher plants. Whereas At SPO11-3 is involved in somatic endoreduplication, At SPO11-1 and, as recently shown, At SPO11-2 are essential for the initiation of meiotic recombination. Further defining the role of At SPO11-2, we were able to demonstrate that it is required for proper chromosome segregation, as its loss resulted in aneuploidy in the surviving progeny. The double mutant spo11-1 spo11-2 does not differ phenotypically from the single mutants, indicating that both proteins are required for the same step. Contrary to the observations for the At rad51-1 single mutant, the combination of spo11-2 and rad51-1 did not lead to chromosome fragmentation, indicating that SPO11-2, like SPO11-1, is required for DSB induction. As the meiotic phenotype of both single SPO11 mutants can be reversed by complementation using the full-length genes but not the same constructs mutated in their respective catalytically active Tyr, both proteins seem to participate directly in the DNA breakage reaction. The active involvement of two SPO11 homologs for DSB formation reveals a striking difference between plants and other eukaryotes in meiosis.

Summary• Mutations in the breast cancer susceptibility gene 2 (BRCA2) are correlated with hereditary breast cancer in humans. Studies have revealed that mammalian BRCA2 plays crucial roles in DNA repair. Therefore, we wished to define the role of the BRCA2 homologs in Arabidopsis in detail.• As Arabidopsis contains two functional BRCA2 homologs, an Atbrca2 double mutant was generated and analyzed with respect to hypersensitivity to genotoxic agents and recombination frequencies. Cytological studies addressing male and female meiosis were also conducted, and immunolocalization was performed in male meiotic prophase I.• The Atbrca2 double mutant showed hypersensitivity to the cross-linking agent mitomycin C and displayed a dramatic reduction in somatic homologous recombination frequency, especially after double-strand break induction. The loss of AtBRCA2 also led to severe defects in male meiosis and development of the female gametophyte and impeded proper localization of the synaptonemal complex protein AtZYP1 and the recombinases AtRAD51 and AtDMC1.• The results demonstrate that AtBRCA2 is important for both somatic and meiotic homologous recombination. We further show that AtBRCA2 is required for proper meiotic synapsis and mediates the recruitment of AtRAD51 and AtDMC1. Our results suggest that BRCA2 controls single-strand invasion steps during homologous recombination in plants.