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 M1) 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 beta-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 approximately 80 kDa in size. The two polymerases showed different sensitivities to several inhibitors: polymerase M1 shows similarities to the Crithidia fasciculata beta-like mitochondrial polymerase while polymerase M2 is a novel, salt-activated enzyme of higher molecular weight.
Fuenmayor
J
J
Department of Microbiology, State University of New York, Buffalo 14214, USA.
Zhang
J
J
Ruyechan
W
W
Williams
N
N
D000180:Acrylic Resins; D000818:Animals; D015153:Blotting, Western; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008274:Magnesium; D008345:Manganese; D008928:Mitochondria / Q000201:enzymology*; D013698:Templates, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D000180:Acrylic Resins; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004791:Enzyme Inhibitors; C016680:polyacrylamide gels; D008345:Manganese; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium