Genetics of DNA replication and homologous recombination in arabidopsis

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.