Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybeaegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was chara cterized in the mitochondrial genome of the edible basidiomycete Agrocybe a egrita. The complete gene, named Aa-polB, and its flanking regions were clo ned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5' region) and a gene for tRNA(Asn) (3' regio n), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5' region) and 3864 (3' region) nucleotides, which lack repeated seq uences of mitochondrial or plasmid origin. The deduced Aa-POLB protein show s extensive sequence similarity with the family B DNA polymerases encoded b y genomes that rely on protein-primed replication (invertrons). The domains involved in the 3'-->5' exonuclease (Exo I to III) and polymerase (Pol I t o Pol V) activities were localized on the basis of conserved sequence motif s. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-te rminal region preceding fro I is short, suggesting a close relationship wit h the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB g ene was shown to be present in all wild strains examined, which were collec ted from a wide range of locations in Europe. As shown by RT-PCR, the Aa-po lB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol gamma in the A. aeger ita mitochondrion is discussed in the light of recent reports showing the c onservation of the nucleus-encoded Pol gamma from yeast to human.