Genetics of DNA replication and homologous recombination in arabidopsis

Abstract

The integrity of genetic information of each cell is constantly subjected to various threats, originating from environmental and endogenous sources. Metabolic byproducts and complex DNA-involving molecular processes such as transcription and replication comprise a high intrinsic mutagenic potential. Although these DNA sequence alterations contribute substantially to the evolution of species, they may primarily be detrimental to biological functions and the survival of a cell or, as a cause of mammalian cancer, even to the whole organism. Many evolutionarily conserved molecular machineries control, orchestrate and execute faithful repair of the damaged DNA, ensuring the integrity of the genome prior to its transmission into the next cellular or sexual generation. Among those machineries, homologous recombination repairs one of the most deleterious DNA lesions - the double-strand breaks - in an accurate fashion, engaging a homologous sequence as template. Alternatively, these breaks are sealed by nonhomologous end-joining, which is an error-prone pathway but nevertheless used preferentially in somatic cells of plants and other higher eukaryotes. Thus, the employment of either repair mechanism greatly impacts the genome integrity of cells and is regulated by factors such as cell cycle phase, chromatin structure and availability of the respective repair proteins. Although homologues for most of the repair and replication proteins can be identified in plants, the current knowledge about these molecular pathways and their contribution to genome stability of plant lags far behind other model organisms. In recent years, several repair-related Arabidopsis genes were characterised by reverse genetics, whose outcome suggested a functional conservation of these pathways. This approach could not elucidate the reasons for the prominent exploitation of end-joining to repair double-strand breaks in somatic cells; this may result in substantial alteration of the genetic information in cells, which potentially form the germline of plants. The development of an artificial reporter system facilitates the in planta assessment of the rare homologous recombination events. This allows the genomewide screening for plant factors that influence the frequency of somatic homologous recombination. The application of this genetic tool resulted in the isolation of an Arabidopsis thaliana mutant plant with a moderately increased frequency of intramolecular homologous recombination. In this mutant line the structure of multiple genes is altered: among them, genes predicted to be a DNA polymerase and a DNA- dependent ATPase. By genetic means, the dominant mutation responsible for the increased homologous recombination level could be assigned to the DNA polymerase gene: the analysis of allelic mutations and the suppression of the phenotype by the ectopic expression of the polymerase gene confirm the causality between this mutation and the homologous recombination phenotype. The mutated gene encodes for the catalytic subunit of the DNA polymerase δ holoenzyme (POLδ1), which is implicated in multiple aspects of DNA metabolism such as genome replication and most of the DNA repair pathways. The inhibition of cell division in embryos with homozygous polδ1 mutations underlines the essential function of POLδ1 in replicative DNA synthesis. Moreover, lowered expression of POLδ1 results in severe developmental aberrations and in genomic instabilities, which are reflected by the frequencies of homologous recombination. Stalled and collapsed replication forks due to DNA lesions or lack of replication factors trigger cell cycle arrest and apoptosis, avoiding an unbalanced cellular division with deleterious sequence loss. In order to prevent this, molecular mechanisms have evolved, which stabilise the replication fork and promote the resuming of DNA synthesis by a homology-dependent interaction of parental and nascent DNA strands, mediated by proteins of the recombination machinery. Little was known about such mechanisms in Arabidopsis but findings presented in this work provide evidence for an evolutionary conserved function of these processes in plant genome replication. Interfering with S-phase DNA synthesis by chemical inhibition results in an increase of intra-molecular but not of inter-molecular homologous recombination frequency and a similar specificity is observed for polδ1 mutant alleles. This suggests that hampered or slowed down DNA replication leads to arrested replication forks and the formation of aberrant DNA structures. In order to continue DNA replication, fork reversal and recombination between homologous sequences of the sister-chromatids are engaged, presumably leading to the increased homologous recombination frequencies observed in the Arabidopsis polδ1 mutant plants.