Abstract
BackgroundThe a2 mating type locus gene lga2 is critical for uniparental mitochondrial DNA inheritance during sexual development of Ustilago maydis. Specifically, the absence of lga2 results in biparental inheritance, along with efficient transfer of intronic regions in the large subunit rRNA gene between parental molecules. However, the underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved.Methodology/Principal FindingsWe have investigated the enzymatic activity of I-UmaI in vitro based on expression of a tagged full-length and a naturally occurring mutant derivative, which harbors only the N-terminal LAGLIDADG domain. This confirmed Mg2+-dependent endonuclease activity and cleavage at the LRII1 insertion site to generate four base pair extensions with 3′ overhangs. Specifically, I-UmaI recognizes an asymmetric DNA sequence with a minimum length of 14 base pairs (5′-GACGGGAAGACCCT-3′) and tolerates subtle base pair substitutions within the homing site. Enzymatic analysis of the mutant variant indicated a correlation between the activity in vitro and intron homing. Bioinformatic analyses revealed that putatively functional or former functional I-UmaI homologs are confined to a few members within the Ustilaginales and Agaricales, including the phylogenetically distant species Lentinula edodes, and are linked to group II introns inserted into homologous positions in the LSU rDNA.Conclusions/SignificanceThe present data provide strong evidence that intron homing efficiently operates under conditions of biparental inheritance in U. maydis. Conversely, uniparental inheritance may be critical to restrict the transmission of mobile introns. Bioinformatic analyses suggest that I-UmaI-associated introns have been acquired independently in distant taxa and are more widespread than anticipated from available genomic data.
Highlights
Homing endonuclease genes (HEGs) are widespread in microbial genomes
Expression of I-UmaI To express the I-UmaI gene in Escherichia coli, the mitochondrial codon usage of U. maydis was determined based on its annotated mitochondrial genome sequence
Except for the very rarely occurring triplets TTA/G and AGG, which were absent from the I-UmaI sequence, and the complete absence of the AGA and TGA codons, the mitochondrial code of U. maydis basically did not deviate from the standard code
Summary
Homing endonuclease genes (HEGs) are widespread in microbial genomes. They frequently exist in self-splicing group I and group II introns, and in archaeal introns, intein coding sequences and phage genomes [1,2,3]. On the basis of conserved amino acid motifs, at least five families of homing endonuclease (HE) proteins are distinguished [3,4,5,6,7]. LAGLIDADG motifs are restricted to homing endonucleases, and exists in other proteins, such as the HO endonuclease, which in yeast mediates the mating type switch [8,9]. The underlying role of the predicted LAGLIDADG homing endonuclease gene I-UmaI located within the group II intron LRII1 has remained unresolved
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