RAD51 loss of function abolishes gene targeting and de-represses illegitimate integration in the moss Physcomitrella patens.

Gene targeting (GT) is a major tool for basic and applied research during which the transforming DNA, which shares sequence homology with a chromosomal target, integrates at the corresponding locus by homologous recombination (HR). In eukaryotes, GT recruits enzymes from the HR-mediated double strand break repair pathway. Different mechanisms of HR have been described which depend on the Rad52 epistasis group of genes, but which specific mechanism is used by the cell for GT remains unclear. In Saccharomyces cerevisiae, the RAD52 protein is essential for GT, and the RAD51 protein plays a minor role. In filamentous fungi and animal cells, however, GT depends on RAD51 and is weakly affected by suppression of RAD52. Genetic evidence also indicates that the non-homologous end-joining pathway of DSB repair has a negative impact on GT efficiencies, but how the balance between these two pathways is controlled is poorly understood. Here, we have examined the role of RAD51 in the only plant that exhibits high GT frequencies, the model bryophyte Physcomitrella patens. Our results show that the two RAD51 proteins have partially redundant functions in the maintenance of genome integrity and resistance to ionizing radiation. Furthermore, we demonstrate that loss of function of the two RAD51 proteins completely abolishes GT and strongly increases illegitimate integration rates in this moss. These findings demonstrate for the first time in plant the critical role of RAD51 in controlling the balance between targeted and random integration events observed upon transgenesis, and confirm that P. patens is a particularly interesting tool for studying GT in higher eukaryotes.

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency.

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.

Four mismatch repair paralogues coexist in Arabidopsis thaliana: AtMSH2, AtMSH3, AtMSH6-1 and AtMSH6-2.

By using degenerate oligonucleotides based on the sequence homology between known MutS homologues, three MSH cDNAs belonging to the MSH2, MSH3 and MSH6 families, as defined in eukaryotes, have been isolated from Arabhidopsis thaliana (ecotype Columbia). Genomic sequences for two of these genes (AtMSH2 and AtMSH6-2) were also isolated and determined, whereas the genomic sequence of AtMSH3 was obtained through the Arabidopsis sequencing project, as was the sequence of a second, distinct AtMSH6 homologue (AtMSH6-1). Comparative analysis of the AtMSH2 Landsberg erecta genomic sequence (reported here) and the previously described AtMSH2 Columbia allele revealed several polymorphisms, including the presence of a small, transposon-like element in the 3' untranscribed region of the former allele. Arabidopsis is the first organism to show such divergence of two AtMSH6 genes; the divergence is strongly supported by sequence data and phylogenetic analysis. Southern analysis revealed that the three genes we have isolated exist as single copies, and genetic mapping indicated that AtMSH2 and AtMSH6-2 both reside on chromosome III. Finally, expression of these three genes could only be observed in suspensions of A. thaliana cells. Such a cell suspension divides actively after subculture, and the AtMSH genes are most strongly expressed at this stage.

Random chromosome segregation without meiotic arrest in both male and female meiocytes of a dmc1 mutant of Arabidopsis.

In yeast, the DMC1 gene is required for interhomolog recombination, which is an essential step for bivalent formation and the correct partition of chromosomes during meiosis I. By using a reverse genetics approach, we were able to identify a T-DNA insertion in AtDMC1, the Arabidopsis homolog of DMC1. Homozygotes for the AtDMC1 insertion failed to express AtDMC1, and their residual fertility was 1.5% that of the wild type. Complete fertility was restored in mutant plants when a wild-type copy of the AtDMC1 gene was reintroduced. Cytogenetical analysis points to a correlation of the sterility phenotype with severely disturbed chromosome behavior during both male and female meiosis. In this study, our data demonstrate that AtDMC1 function is crucial for meiosis in Arabidopsis. However, meiosis can be completed in the Arabidopsis dmc1 mutant, which is not the case for mouse or some yeast mutants.

Is arcA3 a possible mediator in the signal transduction pathway during agonist cell cycle arrest by salicylic acid and UV irradiation?

Progression of BY-2 tobacco cells through the cell cycle was followed after treatments with ultra violet (UV) and salicylic acid (SA) used as a potent inhibitor of the octadecanoid pathway which can mediate response to UV irradiation. Cells in S phase were more sensitive than G0/G1 or G2 cells to UV irradiation. Although SA efficiently blocked cells in G0/G1 or G2, it did not block S phase synchronized cells. UV and SA applied simultaneously to cells in G0/G1 delayed the cell cycle progression more than each one separately. Therefore UV irradiation and SA act as agonists to arrest BY-2 cells at cell cycle entry. To further investigate the signalling pathway mediating UV response, we complemented a UV-sensitive Escherichia coli strain with a Nicotiana xanthi cDNA expression library. A cDNA (arcA3) whose coding sequence is identical to the 2,4-D induced arcA cDNA cloned by Ishida et al. (1993) was isolated. We show that arcA3 transcription is induced at cell cycle entry but not directly by the 2,4-D treatment. Moreover, arcA3 transcription is induced prior to the restriction point as shown with the CDK inhibitor roscovitine. The arcA3 transcription level is increased by UV irradiation but prevented by SA. Indeed, addition of SA prior to UV irradiation blocks the induction of arcA3 transcription. This suggests that arcA3 gene is modulated in both UV and SA responses, the SA effect preceding the UV step. Since arcA3 is 67% similar to RACK1 (functional homology), a rat intracellular receptor for protein kinase C, and possesses identical PKC fixation motifs, it is hypothesised that the arcA3 gene is involved in UV and SA cell cycle arrest.

Isolation and characterisation of the RAD51 and DMC1 homologs from Arabidopsis thaliana.

By using RT-PCR and degenerate oligonucleotides based on the sequence homology between the yeast RAD51 and DMC1 genes, two genes belonging to the RAD51 and DMC1 families were isolated from Arabidopsis thaliana ecotype Columbia. A RAD51 genomic DNA was also sequenced which is almost identical to its Landsberg erecta counterpart, except for a few translationally silent substitutions and for the presence of a 527-bp element downstream of the polyadenylation site. This element is repeated in the genome of Arabidopsis. Northern analyses were conducted to characterize the expression pattern of both these genes. AtRAD51 and AtDMC1 are expressed in flower buds, but also in the mitotically active cells from a suspension culture. AtRAD51, but not AtDMC1, transcript level increases after gamma irradiation of the cells. Finally, a synchronisation experiment conducted with the suspension culture indicated that not only AtRAD51 but also AtDMC1 are regulated during the cell cycle, with S-phase-specific induction. Since DMC1 genes have always been regarded as being specifically meiotic, we discuss the significance of this mitotic transcriptional regulation in Arabidopsis.

Saturation of mismatch repair in the mutD5 mutator strain of Escherichia coli.

The mutD (dnaQ) gene of Escherichia coli codes for the proofreading activity of DNA polymerase III. The very strong mutator phenotype of mutD5 strains seems to indicate that their postreplicational mismatch repair activity is also impaired. We show that the mismatch repair system of mutD5 strains is functional but saturated, presumably by the excess of DNA replication errors, since it is recovered by inhibiting chromosomal DNA replication. This recovery depends on de novo protein synthesis.

Mismatch-stimulated killing.

DNA duplexes with or without mismatches and with or without adenine-methylated GATC sequences were prepared from separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli. Unmethylated heteroduplexes containing one or more repairable mismatches transfect cells with a functioning mismatch repair system less efficiently than they transfect cells deficient in mismatch repair. No difference is observed when the duplexes contain no mismatch or a poorly repaired mismatch or when the heteroduplexes are fully or hemimethylated. These results and the phenotypes of E. coli dam mutants suggest that the E. coli mismatch repair system may introduce double-strand breaks in unmethylated DNA at or near repairable mismatches.

Processing of mispaired and unpaired bases in heteroduplex DNA in E. coli.

Bacteriophage lambda and phi X 174 DNAs, carrying sequenced mutations, have been used to construct in vitro defined species of heteroduplex DNA. Such heteroduplex DNAs were introduced by transfection, as single copies, into E. coli host cells. The progeny of individual heteroduplex molecules from each infective center was analyzed. The effect of the presence of GATC sequences (phi X 174 system) and of their methylation (lambda system) was tested. The following conclusions can be drawn: some mismatched base pairs trigger the process of mismatch repair, causing a localized strand-to-strand information transfer in heteroduplex DNA: transition mismatches G:T and A:C are efficiently repaired, whereas the six transversion mismatches are not always readily recognized and/or repaired. The recognition of transversion mismatches appears to depend on the neighbouring nucleotide sequence; single unpaired bases (frameshift mutation "mismatches") are recognized and repaired, some equally efficiently on both strands (longer and shorter), some more efficiently on the shorter (-1) strand; large non-homologies (about 800 bases) are not repaired by the Mut H, L, S, U system, but some other process repairs the non-homology with a relatively low efficiency; full methylation of GATC sequences inhibits mismatch repair on the methylated strand: this is the chemical basis of strand discrimination (old/new) in mismatch correction; unmethylated GATC sequences appear to improve mismatch repair of a G:T mismatch in phi X 174 DNA, but there may be some residual mismatch repair in GATC-free phi X 174, at least for some mismatches.(ABSTRACT TRUNCATED AT 250 WORDS)