Related Experiment Videos
Identification and characterization of two DNA polymerase activities present in Trypanosoma brucei mitochondria
J Fuenmayor1, J Zhang, W Ruyechan
1Department of Microbiology, State University of New York, Buffalo 14214, USA.
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.
Insights
Two distinct DNA polymerase enzymes were identified in Trypanosoma brucei mitochondria. Polymerase M1 resembles known enzymes, while novel polymerase M2 is salt-activated and larger.
Area of Science:
- Molecular Biology
- Parasitology
- Biochemistry
Background:
- Mitochondrial DNA replication is crucial for kinetoplastid parasites like Trypanosoma brucei.
- Understanding the enzymes involved, particularly DNA polymerases, is key to elucidating replication mechanisms.
Purpose of the Study:
- To identify and characterize DNA polymerase activities within Trypanosoma brucei mitochondria.
- To differentiate and describe the properties of these identified enzymes.
Main Methods:
- Purification of mitochondrial extracts from Trypanosoma brucei.
- Single-stranded DNA agarose chromatography to separate DNA polymerase activities.
- Biochemical assays to determine cofactor preferences, salt optima, and substrate utilization.
- Western blot analysis using antisera against Crithidia fasciculata polymerase.
- Activity gel electrophoresis to estimate peptide sizes.
Main Results:
- Two distinct DNA polymerase activities, M1 and M2, were isolated.
- Polymerase M1 showed characteristics similar to Crithidia fasciculata beta-like mitochondrial polymerase, with a 35 kDa catalytic peptide.
- Polymerase M2 was a novel, salt-activated enzyme with optimal activity at 120-180 mM KCl and an estimated native catalytic peptide size of 80 kDa.
- Differential inhibitor sensitivities were observed between M1 and M2.
Conclusions:
- Trypanosoma brucei mitochondria possess at least two distinct DNA polymerases with differing biochemical properties.
- Polymerase M1 appears homologous to known beta-like mitochondrial polymerases.
- Polymerase M2 represents a novel mitochondrial DNA polymerase potentially involved in parasite DNA replication.