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Fig. 8 | BMC Molecular Biology

Fig. 8

From: The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication

Fig. 8

In vitro primase activity of purified D. discoideum Twm1. a Recombinant Twm1 was tested for primase activity in vitro using radiolabelled nucleotides and a circular ssDNA template (M13mp18). Following incubation, samples were run under non-denaturing conditions in order to visualise any synthesized RNA primers annealed to the template (P). Disassociated primers were too small to be differentiated from unincorporated nucleotides (U) under denaturing conditions. b The primase assay was repeated with the addition of Klenow DNA polymerase to determine whether any generated primer could be used for DNA synthesis. Signals were observed above the unincorporated nucleotides (U) when both enzymes (Twm1 and Klenow) were included, indicating incorporation of the radiolabelled primer into larger synthesized DNA (SD). All reactions lacking Twm1 were prepared using the empty vector purification as a negative control

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