MSH2 is essential for the preservation of genome integrity and prevents homeologous recombination in the moss Physcomitrella patens

Abstract

MSH2 is a central component of the mismatch repair pathway that targets mismatches arising during DNA replication, homologous recombination (HR) and in response to genotoxic stresses. Here, we describe the function of MSH2 in the moss Physcomitrella patens, as deciphered by the analysis of loss of function mutants. Ppmsh2 mutants display pleiotropic growth and developmental defects, which reflect genomic instability. Based on loss of function of the APT gene, we estimated this mutator phenotype to be at least 130 times higher in the mutants than in wild type. We also found that MSH2 is involved in some but not all the moss responses to genotoxic stresses we tested. Indeed, the Ppmsh2 mutants were more tolerant to cisplatin and show higher sensitivity to UV-B radiations. PpMSH2 gene involvement in HR was studied by assessing gene targeting (GT) efficiency with homologous and homeologous sequences. GT efficiency with homologous sequences was slightly decreased in the Ppmsh2 mutant compared with wild type. Strikingly GT efficiency with homeologous sequences decreased proportionally to sequence divergence in the wild type whereas it remained unaffected in the mutants. Those results demonstrate the role of PpMSH2 in the maintenance of genome integrity and in homologous and homeologous recombination.

Figure 1

Molecular analysis of Ppmsh2 mutants. (A) Schematic representation of PpMSH2 genomic locus, schematic representation of PpMSH2 disrupted locus with a double crossover insertion and schematic representation of PpMSH2 disrupted locus without resistance gene. Black open boxes represent exons, black lines represent introns, gray open boxes are homology regions used to disrupt the gene, black boxes are Lox sites for Cre recombination and arrows show the primers used for PCR. Ppmsh2Ø is mutant without the resistance gene, resulting from Ppmsh2#1 after Cre recombinase expression. (B) PCR of the recombinant borders with a primer external to the targeting sequences and one on the resistance gene [primers: PpMSH2#4/Uc for left border (LB) and Lc/PpMSH2#5 for right border (RB)]. (C) PCR with primers flanking recombination sites (primers: PpMSH2#4/2), (D) RT–PCR for PpMSH2 transcripts (primers: PpMSH2#4/2), PpAPT was used as control.

Figure 2

Comparison of wild type and Ppmsh2 mutants apparent phenotypes: (A) Comparison of the length of the last three cells of protonema filaments; arrows show the cell walls. (B) Number of cell divisions after 8 days protoplast regeneration; arrows show the cell walls. (C) Wild-type and Ppmsh2Δ mutant colonies after 3 weeks development. (D) Male antheridia of wild-type and of Ppmsh2Δ mutant, (E) female archegonia of wild-type and of Ppmsh2Δ mutant. (F) Wild-type, Ppmsh2#1, Ppmsh2#3 and Ppmsh2#2 mutant colonies after 12 weeks development, a representative leafy shoot from those colonies and enlargement of the top of the same gametophore.

Figure 3

Survival fraction after different genotoxic stresses. (A) γ-radiation, (B) UV-B, (C) Cisplatin, (D) MNU. Wild-type survival is represented with triangles and dashed line, and Ppmsh2Δ mutant survival is represented with squares and solid line. Error bars indicate SD based on at least two independent experiments in all cases.

Figure 4

Gene targeting PpAPT fragments used for homologous and homeologous recombination studies. The hygromycin gene resistance cassette is represented between both PpAPT fragments by a horizontal interrupted line. (A) PpAPT fragments in the PpAPT-KO cassette in which the sequences are exactly identical to the locus PpAPT used as reporter gene. (B) Alignments of PpAPT fragments in homeologous recombination cassettes; each vertical line indicates mismatch between mutated PpAPT fragments and the original locus.