Functional Expression of a DNA-Topoisomerase IB from Cryptosporidium parvum
Figure 2
Expression and activity of CpTopIB and mutants into an EKY3 Top-IB-deficient yeast strain. (a) SDS-PAGE from the purified yeast extracts expressing wild type CpTopIB and the point mutations carried out into catalytic tetrad amino acids (R318A, K358A, R416A, and H489A) as well as the active site (Y600F). (b) Dilution assay 2-fold serial dilutions of wild type CpTopIB were assayed in a plasmid DNA relaxation assay for 30 minutes at 37 (as described under “Materials and Methods"). (c) Equal concentrations of CpTopIB were incubated stepwise for 10 seconds to 30 minutes in a plasmid DNA relaxation assay. Reaction products were resolved in agarose gel and subsequently visualized by ethidium bromide staining. The reactions were incubated at 30 for 30 minutes and then stopped with a mixture of 1% SDS and 6.1 g of proteinase K. The reaction products were resolved in a 1% agarose gel and visualized by ethidium bromide staining. N: nicked; Sc: supercoiled DNA, R: relaxed DNA.
