Characterization of mitochondrial DNA polymerase beta enzymes of trypanosomatid parasites

In higher eukaryotes, DNA polymerase β (pol β) is a nuclear enzyme involved in DNA repair. The insect parasite Crithidia fasciculata is an exception, instead importing this protein into its mitochondrion. Here it is thought to fill gaps between Okazaki fragments during the replication of this organism's unusual mitochondrial DNA, known as kinetoplast DNA (kDNA) which is a giant network consisting of thousands of interlocked DNA circles condensed in vivo into a disk-shaped structure near the base of the cell's flagellum. The mammalian nuclear pol β is known to have deoxyribose phosphate (dRP) lyase activity in addition to its nucleotidyl transferase, thus carrying out sequential steps of the base excision repair pathway. We demonstrate that the mitochondrial pol β from C. fasciculata also has this second enzymatic activity, supporting a possible role for this protein in kDNA repair. Interestingly, while the K _m for a dRP-containing substrate is similar between the mitochondrial pol β and its rat nuclear counterpart, the catalytic turnover rate (k _cat) for the C. fasciculata enzyme is severely impaired in comparison. We show this is due to the enzyme's altered ability to release the unsaturated dRP product following its cleavage from the DNA.; We also identified two genes encoding pol β proteins in the related organism Trypanosoma brucei, the parasite that causes African sleeping sickness. One is a clear homolog of the C. fasciculata mitochondrial pol β. While we anticipated the second protein would be nuclear, cloning of the full-length gene revealed a putative mitochondrial targeting signal as well as a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins are mitochondrial and display different localizations with respect to the kDNA disk. Additionally, both proteins when expressed recombinantly, are active as both DNA polymerases and dRP lyases, but the buffer conditions supporting maximal polymerase activity in vitro differ significantly. These data suggest these proteins may have distinct roles in kDNA maintenance.