Identification and Characterization of Two DNA Polymerase Activities Present in Trypanosoma brucei Mitochondria

Abstract

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.

Trypanosoma brucei Has Two Distinct Mitochondrial DNA Polymerase β Enzymes

“…The size of pol ␤-PAK, as well as its propensity for salt activation, raises the possibility that this enzyme corresponds to a second T. brucei mitochondrial polymerase activity observed previously (28). Although there are some differences (for instance, in optimal MgCl 2 concentration), it should be noted that these previous analyses were done on crudely fractionated mitochondrial extracts.…”

DNA Polymerase γ in Mitochondrial DNA Replication and Repair

“…In trypanosomes, mitochondrial DNA is wrapped up in kinetoplasts (kDNA), where it is replicated and/or repaired by a number of β-like DNA polymerases. − Interestingly, a γ-like DNA polymerase has never been isolated or identified in trypanosome mitochondria. In addition to earlier localization of pol β from trypanosomatid mitochondria, other DNA polymerases were detected in mitochondrial extracts from Trypanosoma brucei . Four different mitochondrial DNA polymerases, TbPOLIA, IB, IC, and ID, have been identified in T. brucei , raising the total number of mtDNA polymerases in trypanosome mitochondria to five .…”

“…In addition to earlier localization of pol β from trypanosomatid mitochondria, 22 other DNA polymerases were detected in mitochondrial extracts from Trypanosoma brucei. 23 Four different mitochondrial DNA polymerases, TbPOLIA, IB, IC, and ID, have been identified in T. brucei, raising the total number of mtDNA polymerases in trypanosome mitochondria to five. 24 None of the four new proteins are homologous to pol γ, although all four are related to bacterial pol I.…”

DNA Polymerase Beta Participates in Mitochondrial DNA Repair

“…The large spectrum of diseases associated with Pol␥ mutation is indirect evidence that Pol␤ is not able to compensate completely for Pol␥ (57)(58)(59). This is despite a homolog of Pol␤ being solely responsible for the repair and replication of mtDNA in the parasite Trypanosoma brucei (20,60). However, in humans the two polymerases are vastly different, with Pol␥ dwarfing Pol␤ in size and complexity and encoding an efficient proofreading function.…”

“…To date, research has been unable to definitively answer whether Pol␤ is in mammalian mitochondria. However, a number of parasitic organisms not only have Pol␤ in the mitochondria but use the enzyme for both replication and repair of mtDNA in the absence of Pol␥, evidence that a homolog of mammalian Pol␤ is able to function in the organelle (20)(21)(22). Data obtained by using bovine mitochondrial extracts have found a Pol␤-like enzyme in the heart (23); however, purified mitochondrial mouse liver extracts have been negative (24).…”