Isolation and characterization of "Arabidopsis" mutants with altered homologous recombination levels : a new function for an INO80 SWI/SNF ATPase

Abstract

Homologous recombination (HR) in eukaryotic organisms serves a dual role in providing genetic flexibility by creating novel sequence assortments upon meiosis and in maintaining genome integrity through DNA repair in somatic tissues. HR represents an alternative pathway to non-homologous end-joining (NHEJ) for the repair of double-strand breaks (DSB). The repair by NHEJ may not preserve the integrity of the genetic information whereas the HR pathway is more faithful. The choice of a pathway to repair DSBs is thus crucial for genome integrity and evolution, especially in plants where the germline is only determined late during development. Very little is known on what influences the choice of the pathway taken, but chromatin structure at the site of a lesion likely will play a major role in the recruitment of repair enzymes and thereby the choice of repair pathway. As a consequence, various proteins that are not part of the core of the recombination machinery may directly participate in the regulation of HR. At the time this work was initiated, no plant gene involved in the HR pathway or its regulation was characterized yet. As plants are powerful genetic tools especially for screening, we decided on designing and conducting a genetic screen to identify plant genes involved in HR. Here I describe a genetic screen in Arabidopsis thaliana for mutants with altered somatic recombination levels and the characterization of the resulting mutant candidates. For the screen, I used a stably integrated luciferase based intrachromosomal HR substrate and a T-DNA construct designed for activation tagging as a mutagenic agent. Out of 19520 individual transformants tested, 37 exhibited an altered HR phenotype. Nine of them were sterile and/or exhibited important developmental or growth phenotypes that precluded the formation of progeny seeds, which is more than the average number of sterile plants expected. However, in most cases the altered recombination phenotype was lost in the offspring. To characterize the mutations, I cloned all the T-DNA insertion sites by plasmid rescue and determined the potential target genes. I discuss the genes likely to be responsible of the observed phenotype. Here I report the thorough analysis of a mutant in the Arabidopsis INO80 ortholog of the SWI/SNF ATPase family, which shows a reduced frequency of HR. In contrast, sensitivity to genotoxic agents and efficiency of T-DNA integration remain unaffected. This suggests that INO80 is a positive regulator of HR, while not affecting other repair pathways. Further, transcriptionaly silent TSI loci are not reactivated in absence of INO80, suggesting that Arabidopsis INO80 function is independent of transcriptional silencing. Using whole genome expression studies by microarray profiling I show evidence that INO80 regulates a small subset of Arabidopsis genes, suggesting a dual role for INO80 in transcription and repair by HR. Moreover, the recombination-promoting function of INO80 is not likely to involve general transcriptional regulation, and the transcriptional regulation of repair related genes is unaffected in the mutant. This is the first report of INO80 function in a higher eukaryote. Mononucleosome binding studies support the suggestion that INO80 positively regulates HR through modification of chromatin structure at sites of DNA repair by HR. Finally, I provide evidence for the existence and/or connectivity of INO80 with other INO80 complex partners in Arabidopsis.