IDENTIFICATION AND CHARACTERIZATION OF 2 DNA-POLYMERASE ACTIVITIES PRESENT IN TRYPANOSOMA-BRUCEI MITOCHONDRIA

We have identified and partially purified two DNA polymerase activitie s from purified Trypanosoma brucei mitochondrial extracts. The DNA pol ymerase activity eluted from the single-stranded DNA agarose column at 0.15 M KCl (polymerase M1) was significantly inhibited by salt concen trations greater than 100 mM, utilized Mg2+ in preference to Mn2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, a nd in the presence of Mn2+ favored a ribonucleotide template with a de oxyribose primer. A 44 kDa peptide in this fraction crossreacted with antisera against the Crithidia fasciculata beta-like mitochondrial pol ymerase. In activity gels the catalytic peptide migrated at an apparen t molecular weight of 35 kDa. The DNA polymerase activity present in t he 0.3 M KCI DNA agarose fraction (polymerase M2) exhibited optimum ac tivity at 120-180 mM KCI, used both Mg2+ and Mn2+ as cofactors, and us ed deoxyribonucleotide templates primed with either deoxyribose or rib ose oligomers. Activity gel assays indicate that the native catalytic peptide(s) is similar to 80 kDa in size. The two polymerases showed di fferent sensitivities to several inhibitors: polymerase M1 shows simil arities to the Crithidia fasciculata beta-like mitochondrial polymeras e while polymerase M2 is a novel, salt-activated enzyme of higher mole cular weight.