Identification and characterization of two DNA polymerase activities present in Trypanosoma brucei mitochondria

We have identified and partially purified two DNA polymerase activities from purified Trypanosoma brucei mitochondrial extracts. The DNA polymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCl (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg²⁺ in preference to Mn²⁺ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn²⁺ favored a ribonucleotide template with a deoxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata β-like mitochondrial polymerase. In activity gels the catalytic peptide migrated at an apparent molecular weight of 35 kDa. The DNA polymerase activity present in the 0.3 M KCl DNA agarose fraction (polymerase M2) exhibited optimum activity at 120-180 mM KCl, used both Mg²⁺ and Mn²⁺ as cofactors, and used deoxyribonucleotide templates primed with either deoxyribose or ribose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is ~80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia fasciculata β-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.