TbPIF5 Is a Trypanosoma brucei Mitochondrial DNA Helicase Involved in Processing of Minicircle Okazaki Fragments
Figure 2
ATPase and helicase assays of recombinant TbPIF5.
(A) Coomassie-stained SDS-PAGE gel and Western blot of purified recombinant TbPIF5. (B) Assay of TbPIF5 ATPase activity. The substrates and products were separated by polyethyleneimine thin layer chromatography; arrow shows origin. The [32P]Pi standard in the left-hand lane was prepared from [γ-32P] ATP by boiling 5 min in 1 M HCl. (C) Assay of TbPIF5 helicase activity. Substrates and products were separated by 12% polyacrylamide gel electrophoresis. (D) Helicase activity was measured at various concentrations of Mg++-ATP. (E) Assay of polarity of TbPIF5 helicase activity. Helicase substrates are diagrammed in Panels C (strand lengths are not to scale) and E (strand lengths for oligonucleotides a, b and c are 21, 21 and 90 nucleotides). * indicates 5′ 32P end label.