The COM1/SAE2 homologue of Arabidopsis thaliana: Identification and characterization of a DNA repair protein essential for meiosis

Central to the formation of gametes is the process of meiosis. It includes two successive cell divisions that follow a single round of DNA replication. Thereby, the chromosome number is reduced by half. During the first division, called meiosis I, pairs of replicated homologous chromosomes segregate, while during the second division, meiosis II, sister chromatids are separated. During meiosis I, reciprocal recombination between homologous chromosomes occurs before they are segregated. The process of homologous recombination contributes to the genetic diversity of the meiotic products, provides an opportunity for the repair of DNA damage and is a pre-requisite for the correct segregation of homologous chromosomes. Homologous recombination requires the controlled formation of a DNA double strand break (DSB). This process is mediated by the topoisomerase-related protein Spo11, together with other protein factors. Spo11 is found covalently linked to the 5` termini of DNA in an intermediate of the DNA cleavage reaction. Further processing of DNA requires release of Spo11 from DNA. This is mediated by ssDNA nick formation next to the DSB site, thereby liberating the Spo11 protein attached to a few nucleotides. In budding yeast this release requires Rad50, Mre11 and Com1/Sae2. This grant proposal describes the first characterisation of a COM1/SAE2 homologue in a higher eukaryote, the model plant Arabidopsis thaliana (AtCOM1). Preliminary data demonstrate that homologues exist in various higher eukaryotes, and that AtCOM1 is essential for meiosis in A. thaliana. The outlined experiments concentrate on seven clearly-defined issues and will (1) answer what underlies the fertility defect observed in A. thaliana Atcom1 mutants, (2) clarify the epistatic relation of AtCOM1 to other meiotic genes, (3) establish the spatial and temporal distribution of AtCOM1 during meiosis, (4) clarify whether meiotic DSBs are processed aberrantly in Atcom1 mutants, (5) identify interaction partners of AtCOM1, (6) establish the role of AtCOM1 in somatic plant development and (7) reveal the modes of AtCOM1 protein regulation.

The FWF funded project "Characterization of COM1/SAE2 in Arabidopsis thaliana" (P19307-B12) was implemented by Peter Schlögelhofer and his co-workers (University of Vienna, Max F. Perutz Laboratories). The project aimed at the characterization of a novel plant protein, AtCOM1 (A. thaliana completion of meiosis 1), which is essential for meiosis. In case this protein is missing, no viable generative cells and hence no offspring can be formed. Meiosis is a specialized cell division prior to the formation of generative cells (in plants these are pollen and egg cells). A single round of DNA replication is followed by two subsequent divisions, thereby halving the genome content. In the case of diploid organisms, like Arabidopsis or humans, haploid generative cells are formed. In a later fertilization, two generative cells are fused and in the newly formed organism the diploid set of chromosomes is restored. Meiosis serves as well the important aspect of mixing genetic information. During the first meiotic division, the chromosomes are broken in a well-controlled manner by molecular tools and repaired in such a way, that parts of the original chromosomes are recombined into unique and new constellations. Our individuality (as those of plants) and evolutionary changes greatly depend on this process. AtCOM1 is one of these molecular tools needed for meiotic DNA repair. In a bio-informatic screen we identified a sequence in the genome of the model plant Arabidopsis thaliana to be distantly related to the yeast Com1 gene. We then established that corresponding mutant plants are sterile and sensitive to DNA damaging agents. Furthermore, we could show that AtCOM1 promotes the same meiotic step as its yeast counterpart. We provided a detailed characterization of the plant gene and corresponding mutant phenotypes. Most importantly, our findings helped to identify the mammalian Com1 homologue (CtIP) and thereby contributed considerably to deepen the general understanding of meiotic and somatic DNA repair. Even so, the project has been successfully terminated and afore mentioned findings published, two more publications are in the pipeline, that will provide further insights in the regulation of AtCOM1 on a transcriptional and post-transcriptional level. Furthermore, knowledge obtained and tools generated during the funding period provided the foundation for two follow-up projects. One projects aims at identifying all proteins that are phosphorylated by two important DNA damage activated kinases (ATM and ATR), with AtCOM1 being one of the targets. The other projects aims at identifying meiotic DNA double strand break sites genome-wide, to gain a deeper understanding how genetic traits are recombined during meiosis. Atcom1 mutant plants can be utilized to stabilize meiotic recombination intermediates and thereby facilitate their analysis.