Cloning and initial characterization of a family A DNA polymerase from Entamoeba histolytica : A putative mitochondrial DNA Polymerase

Entamoeba histolytica is a unicellular eukaryotic organism that was located in theaamitochondriate branch. However, recent experiments reveal that presence of organelles and mitochondrial proteins in E. histoytica. Several protozoa parasites like Trypanosome cruzy and Plasmodium falciparium contain family A DNA polymerases that replicate their organelles. We have performed a “in silico” search for Family A DNA polymerases in the genome data base of E. histolytica using as bait the arquetype of family A DNA polymerases: Klenow Fragment. We identified an open reading frame that corresponds to a protein of 67 kDa with high homology to Klenow Fragment, bacteriophage T7 DNA polymerase and mitochondrial polymerases. We amplified this ORF using PCR reaction and subcloned it in a pCOLD vector. The expression of the recombinant polymerase was carried out at 16 C and its purification was facilitated by the addition of N‐terminal histidine tag. The recombinant polymerase is active in primer extension experiments and displays very low processivity. We are in the process to obtain antibodies against this polymerase to observe its cellular localization. We hypothesize that this DNA polymerase may target to the organelles present in E. histolytica. This would be evidence that E. histolytica may have a primitive mitochondrial‐like organelle.