DNA double strand break repair in the context of chromatin. – RecInChromatin

The overall aim of this project is to deepen the understanding of the factors determining DNA repair pathway choice and the outcomes of recombinational repair at somatic and meiotic DNA double strand break (DSB) sites in plants. It thus focuses on understanding the fundamental genetic mechanisms ensuring the stability and transmission of the genome. Failure to repair damaged DNA seriously compromises the fitness of any organism. Therefore, DNA breaks need to be repaired efficiently, using one of multiple available pathways. Cell cycle state, the availability of different repair templates and other factors like chromatin context influence repair pathway choice and thereby the repair outcome. While multiple repair pathways during somatic and meiotic DNA repair have been identified, their interplay and regulation and especially the influence of DNA sequence, chromatin and chromosomal contexts on the nature of DSB repair remains poorly understood. Two leading plant recombination and molecular genetics research groups from Austria and France will combine their tools and expertise in this collaborative project to synergistically address this open question.

Novel genome engineering tools will be used to induce somatic and meiotic DNA breaks at specifically chosen sites. For meiotic analysis, assays to directly measure DSB formation and recombination will be employed. Analyses of somatic repair events will be performed on F1 Arabidopsis hybrids, introducing the breaks on only one of the two homologous chromosomes thereby greatly enhancing the resolution of the analyses and mimicking the natural situation. Sequencing of the DNA polymorphisms that distinguish the ecotypes will allow for determining the exact repair outcome. The experiments will yield a coherent analysis of the roles of chromatin structure and chromosomal context on recombination pathway choice and outcome at selected sites throughout the genome in somatic and meiotic cells. Implication of cohesins, small diRNAs and recombination mediator proteins in DNA break repair will be integrated in this analysis. The use of several mutant lines, cytological analyses and in vivo imaging of fluorescent fusion proteins will also shed light on the recruitment of key factors at selected chromosomal DSB sites.

Avoiding the pitfalls of artifical recombination-tester loci, possibly pleiotropic phenotypes of certain mutants and dependence upon spontaneous mitotic DNA breakage patterns, we will build an integrated analysis of the influence of DNA break context on the choice of repair pathways and outcome of the repair to ensure genome stability. In addition to the biological interest of understanding these essential and highly conserved processes, efficient plant breeding is of great importance to society and the urgency of understanding plant genome maintenance mechanisms should not be underestimated in a world faced with ever-increasing populations and changing climates.