The N‐terminal Domain of Twinkle, the Replicative Helicase in Human Mitochondria, Binds DNA and is Essential in Supporting Processive DNA Synthesis by the Mitochondrial DNA Polymerase

Abstract

Twinkle is the replicative helicase in human mitochondria, and is relatively poorly studied compared to its nuclear counterpart. Evolutionarily related helicases in the SF4 family, including T7 gp4 and Twinkle orthologs have an N‐terminal domain that serve as the primase for replication while Human Twinkle has been demonstrated to have lost this function, with several key metal binding residues being lost evolutionarily. Without the primase activity, the utility of the NTD is in question as Human Twinkle has been demonstrated to retain its helicase functionality without the NTD, with somewhat reduced efficiency. We generated and purified a novel Twinkle construct containing only the C‐terminal domain and linker region (CTD, amino acids 360‐684) alongside a full length (aa 43‐684) and a previously studied N‐terminal domain construct (NTD, aa 43‐372). The NTD does not oligomerize in gel filtration analysis or have any of the enzymatic activities of Twinkle. Interestingly, NTD displays a notable nonspecific binding affinity for nucleic acids longer than 16 nt. The CTD retains much of the activities of full‐length Twinkle including comparable DNA binding and, annealing, and somewhat reduced ATPase and helicase activities. CTD supports strand displacement synthesis by Polymerase γ on short replication forks but does not promote formation of multi‐kilobase length products in rolling circle DNA synthesis. Strikingly, CTD is able to support long stretch DNA replication when it is part of the reconstituted mitochondrial replisome with Polymerase γ and mtSSB, albeit with a lower efficiency than Human Twinkle. Thus, the C‐terminal domain retains the majority of the lower order component abilities of full length Twinkle but struggles to implement them for processive DNA synthesis. The results suggest that the role of the N‐terminal domain of Twinkle is in optimizing the motor activity of the C‐terminal domain for synthesizing biologically relevant lengths of DNA. The implications are foundational for understanding the mechanism of action for a subsection of sometimes debilitating mitochondrial genetic diseases which result from several single point mutations in the N‐terminal region which have previously been cryptic.