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Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria
Author(s) -
Fuenmayor Jahely,
Zhang Jun,
Ruyechan William,
Williams Noreen
Publication year - 1998
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.1998.tb05091.x
Subject(s) - crithidia fasciculata , biology , microbiology and biotechnology , dna polymerase , polymerase , biochemistry , deoxyribose , deoxyribonucleotide , dna clamp , trypanosoma brucei , enzyme , dna , polymerase chain reaction , oligonucleotide , reverse transcriptase , gene
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 KCI (polymerase MI) was significantly inhibited by salt concentrations greater than 100 mM, utilized Mg 2+ in preference to Mn 2+ as a cofactor on deoxyribonucleotide templates with deoxyribose primers, and in the presence of Mn 2+ 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 KCI DNA agarose fraction (polymerase M2) exhibited optimum activity at 120‐180 mM KCI, used both Mg 2+ and Mn 2+ 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 MI shows similarities to the Crithidia fasciculata β‐like mitochondrial polymerase while polymerase M2 is a novel, salt‐activated enzyme of higher molecular weight.