Abstract
The helicase and primase activities of the hexameric ring-shaped T7 gp4 protein reside in two separate domains connected by a linker region. This linker region is part of the subunit interface between monomers, and point mutations in this region have deleterious effects on the helicase functions. One such linker region mutant, A257T, is analogous to the A359T mutant of the homologous human mitochondrial DNA helicase Twinkle, which is linked to diseases such as progressive external opthalmoplegia. Electron microscopy studies show that A257T gp4 is normal in forming rings with dTTP, but the rings do not assemble efficiently on the DNA. Therefore, A257T, unlike the WT gp4, does not preassemble on the unwinding DNA substrate with dTTP without Mg(II), and its DNA unwinding activity in ensemble assays is slow and limited by the DNA loading rate. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. Interestingly, once loaded, A257T has almost WT-like translocation and DNA unwinding activities. Strikingly, A257T preassembles stably on the DNA in the presence of T7 DNA polymerase, which restores the ensemble unwinding activity of A257T to ∼75% of WT, and the rescue does not require DNA synthesis. The DNA loading rate of A257T, however, remains slow even in the presence of the polymerase, which explains why A257T does not support T7 phage growth. Similar types of defects in the related human mitochondrial DNA helicase may be responsible for inefficient DNA replication leading to the disease states.
Highlights
Helicases are molecular motors that are involved in almost all of the metabolic processes of the nucleic acids in the living cell
Twinkle contains a C-terminal helicase and N-terminal primase-like domain separated by a linker region that is highly homologous to the linker region of T7 gp4 [20]
The similarity between T7 gp4 and human Twinkle indicates that the detailed characterization of the linker region mutant of T7 gp4 can provide important insights into the mechanisms that lead to mitochondrial deletions in humans
Summary
Helicases are molecular motors that are involved in almost all of the metabolic processes of the nucleic acids in the living cell. Single molecule assays measured a 45 times slower rate of A257T loading on DNA compared with WT gp4. T7 gp4 WT or A257T (100 nM hexamer) was preincubated with the fluorescein-labeled fork DNA (10 nM) in the presence of dTTP (without Mg2ϩ), and the reaction was initiated by rapid mixing with MgCl2 (Fig. 2A).
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