Figure 4.

Helicase activities of purified Twinkle protein fractions. (A) An example of a helicase activity assay using the various Twinkle variants purified to near homogeneity. As indicated on the left-hand site, the substrate consisted of M13 plasmid with an annealed 60-nt 5′end-labeled oligo with a long (40 nt) single-stranded 5′ overhang. (−) indicates the purified substrate whereas the denatured substrate (95°C) indicates the released labeled product, which is also released when a purified protein contains helicase activity. (B) This panel shows the averaged results of six independent measurements, using several independent protein preparations. Except for the ▵70–343 mutant, which still had activity though significantly (Student's t-test) reduced, all other mutants had essentially lost all helicase activity.