The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.

These authors contributed equally. SUMMARYTelomeres and ribosomal RNA genes (rDNA) are essential for cell survival and particularly sensitive to factors affecting genome stability. Here, we examine the role of RAD51 and its antagonist, RTEL1, in the moss Physcomitrella patens. In corresponding mutants, we analyse their sensitivity to DNA damage, the maintenance of telomeres and rDNA, and repair of double-stranded breaks (DSBs) induced by genotoxins with various modes of action. While the loss of RTEL1 results in rapid telomere shortening, concurrent loss of both RAD51 genes has no effect on telomere lengths. We further demonstrate here the linked arrangement of 5S and 45S rRNA genes in P. patens. The spacer between 5S and 18S rRNA genes, especially the region downstream from the transcription start site, shows conspicuous clustering of sites with a high propensity to form quadruplex (G4) structures. Copy numbers of 5S and 18S rDNA are reduced moderately in the pprtel1 mutant, and significantly in the double pprad51-1-2 mutant, with no progression during subsequent cultivation. While reductions in 45S rDNA copy numbers observed in pprtel1 and pprad51-1-2 plants apply also to 5S rDNA, changes in transcript levels are different for 45S and 5S rRNA, indicating their independent transcription by RNA polymerase I and III, respectively. The loss of SOL (Sog One-Like), a transcription factor regulating numerous genes involved in DSB repair, increases the rate of DSB repair in dividing as well as differentiated tissue, and through deactivation of G2/M cell-cycle checkpoint allows the cell-cycle progression manifested as a phenotype resistant to bleomycin.

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