Topoisomerase 3α and RMI1 Suppress Somatic Crossovers and Are Essential for Resolution of Meiotic Recombination Intermediates in Arabidopsis thaliana

Abstract

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.

The Fanconi Anemia Ortholog FANCM Ensures Ordered Homologous Recombination in Both Somatic and Meiotic Cells in Arabidopsis

“…At-FANCM Defines an RECQ4A-Independent Pathway for the Suppression of Spontaneous HR Previously, we have shown that the RecQ family helicase At-RECQ4A, a homolog of human BLM (for Bloom syndrome helicase) and yeast Sgs1 helicases, suppresses spontaneous HR events in somatic cells together with its partners At-RMI1 and At-TOP3A (Hartung et al, 2007a(Hartung et al, , 2008. To perform a similar analysis, we crossed fancm-1 mutant plants with the HR reporter line IC9 (Molinier et al, 2004).…”

“…Topoisomerase 3a and RMI1 are essential during meiosis in Arabidopsis, where they are thought to dissolve a subset of dHJs into NCO products (Chelysheva et al, 2008;Hartung et al, 2008). In the At-rmi1-1 mutant, extensive chromosome fragmentation occurs at the metaphase I to anaphase I transition when the unrepaired recombination intermediates that have accumulated break under division spindle tension.…”

“…Recently, AtTOP3a (for Topoisomerase3a) and At-RMI1 (for RecQ-mediated genome instability1) have been shown to be essential for meiosis in Arabidopsis. It is proposed they act by dissolution of dHJs and account for some NCO products (Chelysheva et al, 2008;Hartung et al, 2008).…”

In Planta Somatic Homologous Recombination Assay Revisited: A Successful and Versatile, but Delicate Tool

“…Helicases and their interaction partners are involved in the suppression of certain HR reactions in Arabidopsis, as is known for other eukaryotic organisms as well. Insertion mutants of RECQ4A, RMI1, Top3alpha, and FANCM revealed a hyperrecombination phenotype (Hartung et al, 2007;Hartung et al, 2008;Knoll and Puchta, 2011;Knoll et al, 2012). A similar phenotype of hyperrecombination was also seen in plants with reduced expression of the gene coding for the catalytic subunit of the DNA polymerase (Schuermann et al, 2009), indicating that replication stress enhances HR.…”

The translesion polymerase ζ has roles dependent and independent of the nuclease MUS81 and the helicase RECQ4A in DNA damage repair in Arabidopsis

“…This regression mechanism is thought to be accomplished by helicases, such as the RecQ helicase Bloom Syndrome Protein (BLM) in humans (Croteau et al, 2014). AtRECQ4A is the respective BLM homolog in Arabidopsis (Arabidopsis thaliana), and this enzyme has the ability to regress replication forks in vitro (Hartung et al, 2007(Hartung et al, , 2008Schröpfer et al, 2014b). The second error-free subpathway entails invasion of the newly synthesized strand on the blocked sister chromatid into the complementary newly replicated strand on the other sister chromatid.…”