ABSTRACT
Genetic recombination pathways and genes are well studied, but relatively little is known in plants, especially in lower plants. To study the recombination apparatus of a lower land plant, a recombination gene well characterized particularly in yeast, mouse, and man, the RAD51 gene, was isolated from the moss Physcomitrella patens and characterized. Two highly homologous RAD51 genes were found to be present. Duplicated RAD51 genes have been found thus far exclusively in eukaryotes with duplicated genomes. Therefore the presence of two highly homologous genes suggests a recent genome duplication event in the ancestry of Physcomitrella. Comparison of the protein sequences to Rad51 proteins from other organisms showed that both RAD51 genes originated within the group of plant Rad51 proteins. However, the two proteins form a separate clade in a phylogenetic tree of plant Rad51 proteins. In contrast to RAD51 genes from other multicellular eukaryotes, the Physcomitrella genes are not interrupted by introns. Because introns are a common feature of Physcomitrella genes, the lack of introns in the RAD51 genes is unusual and may indicate the presence of an unusual recombination apparatus in this organism. The presence of duplicated intronless RAD51 genes is unique among eukaryotes. Studies of further members of this lineage are needed to determine whether this feature may be typical of lower plants.
Subject(s)
Bryopsida/genetics , DNA-Binding Proteins/genetics , Animals , Base Sequence , DNA, Plant , DNA-Binding Proteins/classification , Eukaryotic Cells , Gene Expression , Genes, Plant , Humans , Mice , Molecular Sequence Data , Phylogeny , Rad51 Recombinase , Sequence Homology, Nucleic AcidABSTRACT
Blue light inhibits the formation of asexual cycle spores (conidia) and stimulates the development of the sexual (female) reproductive structures (protoperithecia) in the nitrogen-starved mycelium of Neurospora crassa. The DNA methylation inhibitor, 5-azacytidine (3-300 microM), opposed the effect of light by suppressing the protoperithecia formation and stimulating a conidiation. The addition of 300 microM 5-azacytidine inhibited protoperithecia formation in the dark-cultivated mycelium by about two orders of magnitude and activated conidiation in the light-exposed mycelium by almost three orders of magnitude. Both in the dark-cultivated and the irradiated mycelium treated with various 5-azacytidine concentrations, the yield of conidia and protoperithecia demonstrated an inverse relationship. We suggest that DNA methylation and blue light are involved in the organism's selection of sexual or asexual reproductive cycle.
Subject(s)
Azacitidine/pharmacology , Neurospora crassa/drug effects , Neurospora crassa/radiation effects , DNA Methylation , DNA, Fungal/metabolism , Light , Neurospora crassa/growth & development , Photobiology , Reproduction , Spores, Fungal/drug effects , Spores, Fungal/growth & development , Spores, Fungal/radiation effectsABSTRACT
The moss Physcomitrella patens, which is a land plant with efficient homologous recombination, encodes two Rad51 proteins (PpaRad51.1 and PpaRad51.2). The PpaRad51.1 and PpaRad51.2 proteins, which share 94 % identity between them, interact with themselves and with each other. Both proteins bind ssDNA and dsDNA in a Mg(2+) and pH-dependent manner, with a stoichiometry of one PpaRad51.1 monomer per 3(+/-1) nt or bp and one PpaRad51.2 monomer per 1(+/-0.5) nt or bp, respectively. At neutral pH, a 1.6-fold excess of both proteins is required for ssDNA and dsDNA binding. PpaRad51.1 and PpaRad51.2 show ssDNA-dependent ATPase activity and efficiently promote strand annealing in a nucleotide-independent but in a Mg(2+)-dependent manner. Both proteins promote joint-molecule formation, DNA strand invasion and are able to catalyse strand exchange in the presence of Mg(2+) and ATP. No further increase in the activities is observed when both proteins are present in the same reaction. None of the PpaRad51 gene products complement the DNA repair and recombination phenotype of Saccharomyces cerevisiae rad51delta mutants. However, PpaRad51.1 confers a dominant-negative DNA repair phenotype, and both PpaRad51 proteins reduce the levels of double-strand break-induced recombination when overexpressed in S. cerevisiae wt cells. These results suggest that both PpaRad51 proteins are bona fide Rad51 proteins that may contribute, in a different manner, to homologous recombination, and that they might replace ScRad51 in a hypothetical yeast protein complex inactivating different functions required for recombinational repair.