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. 2012 Apr;40(8):3496-510.
doi: 10.1093/nar/gkr1272. Epub 2011 Dec 30.

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

Yasuko Kamisugi  1 , Didier G Schaefer, Jaroslav Kozak, Florence Charlot, Nathalie Vrielynck, Marcela Holá, Karel J Angelis, Andrew C Cuming, Fabien Nogué
Affiliations

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

Yasuko Kamisugi et al. Nucleic Acids Res. 2012 Apr.

Abstract

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.

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Figures

Figure 1.
Figure 1.
Targeted disruption of Physcomitrella MRN genes. (A) Structure of the PpMRE11, PpRAD50 and PpNBS1 genes. Exons are represented by shaded boxes, with 5′- and 3′-UTR sequences in darker grey. The region deleted by cre-lox excision of a selection cassette is shown as a line above each gene. For the replacement constructs (below each gene) the extent of targeting sequence homology is indicated by the line, and the P35S-nptII-g6ter selection cassette is shown as a white box, replacing the genomic region indicated by the lines joining the gene structure diagram and the replacement cassette. Arrows indicate position of primers used for RT-PCR analysis. (B) RT-PCR analysis of MRN transcripts in wild-type and mutant plants. RNA was isolated from protonemal tissue of wild-type and mutant lines for cDNA synthesis and PCR amplification using gene-specific primers (PpMRE11#1 + PpMRE11#2 for MRE11, PpRAD50#1 + PpRAD50#2 for RAD50, PpNBS1#1 + PpNBS1#2 for NBS1). The PpAPT transcript has been used as control (primers: PpAPT#14 + PpAPT#19). Primers are listed in Supplementary Figure S4.
Figure 2.
Figure 2.
Vegetative developmental phenotypes of mre11 and rad50 mutants. WT (A and B), Rad50 7-20 (C–F) and Mre11 1-195 (G–J) 30-day-old colonies grown on BCD (A, C and G) or BCDAT (B, D and H) medium, scale 1 cm. (E, F, I and J) aborted gametophores observed at the edge of 2-month-old colonies grown on BCDAT, scale bar 500 mm in E and I, 200 mm in F and J.
Figure 3.
Figure 3.
Hypersensitivity of the rad50 and mre11 mutants to UV-B treatment. Survival curves of wild type and rad50 and mre11 mutant protoplasts regenerating after exposure to UV-B treatment. Wild-type survival is represented with diamonds, rad50 mutant survival is represented with squares and mre11 mutant survival is represented with triangles. Error bars indicate SDs based on at least two independent experiments in all cases.
Figure 4.
Figure 4.
Hypersensitivity of the rad50 and mre11 mutants to bleomycin. (A) Wild-type and mutant plants were inoculated as six explants into quadrants of plates containing standard growth medium with or without bleomycin at 8 ngml−1. For the mutant strains, each inoculum represents an independent disruption line. The photograph illustrates the extent of growth 10 days following inoculation of the explants. (B) Survival curves of wild type and nbs1, mre11, rad50 and rad51 mutant protoplasts regenerating after exposure to bleomycin treatment. Error bars indicate SD based on at least two independent experiments in all cases.
Figure 5.
Figure 5.
Kinetics of DNA repair in wild-type and mutant plants. (A) Bleomycin dose-response. Protonemal tissue from wild-type and mutant lines was treated with bleomycin for 1 h at the indicated concentrations, prior to nuclear extraction and the analysis of DNA damage by single-cell electrophoresis (the ‘comet assay’). The extent of DNA damage is indicated by the proportion of DNA detected in the fragmented fraction (the ‘comet tail’). The background level of genomic DNA damage in all lines is similar, at between 20 and 30%, indicating that the mutations have no significant effect on natural levels of DNA fragmentation. (B) Repair kinetics in 1-day regenerated protonemata. In both wild-type and mutant lines, the fragmentation of DNA induced by bleomycin is repaired with rapid kinetics (t1/2 between 1 and 4 min). (C) Repair kinetics in relation to protonemal age. As protonemata are regenerated for longer periods (resulting in a concomitant reduction in the proportion of mitotically active apical cells), so the proportion of the rapid phase DNA repair declines. This occurs in both the wild-type and the rad50KO mutant lines.
Figure 6.
Figure 6.
Gene expression analysis of DNA repair genes in the MRN mutants. Quantitative determination of the relative abundances of transcripts encoding DNA repair genes (PpRad51-1, PpRad51-2, PpPARP-1, PpPARP-2, PpKu70, PpKu80 and PpCtIP) in 7-day-old wild-type, Ppmre11KO, PpRad50KO and Ppnbs1KO strains was done by quantitative real-time PCR. Relative transcript abundance was calculated using the ΔΔCt method and normalized to the wild-type value. Error bars indicate SD based on at least three independent experiments with two replicates for each sample in all cases.
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