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

Abstract

BackgroundDNA replication requires contributions from various proteins, such as DNA helicases; in mitochondria Twinkle is important for maintaining and replicating mitochondrial DNA. Twinkle helicases are predicted to also possess primase activity, as has been shown in plants; however this activity appears to have been lost in metazoans. Given this, the study of Twinkle in other organisms is required to better understand the evolution of this family and the roles it performs within mitochondria.ResultsHere we describe the characterization of a Twinkle homologue, Twm1, in the amoeba Dictyostelium discoideum, a model organism for mitochondrial genetics and disease. We show that Twm1 is important for mitochondrial function as it maintains mitochondrial DNA copy number in vivo. Twm1 is a helicase which unwinds DNA resembling open forks, although it can act upon substrates with a single 3′ overhang, albeit less efficiently. Furthermore, unlike human Twinkle, Twm1 has primase activity in vitro. Finally, using a novel in bacterio approach, we demonstrated that Twm1 promotes DNA replication.ConclusionsWe conclude that Twm1 is a replicative mitochondrial DNA helicase which is capable of priming DNA for replication. Our results also suggest that non-metazoan Twinkle could function in the initiation of mitochondrial DNA replication. While further work is required, this study has illuminated several alternative processes of mitochondrial DNA maintenance which might also be performed by the Twinkle family of helicases.Electronic supplementary materialThe online version of this article (10.1186/s12867-018-0114-7) contains supplementary material, which is available to authorized users.

Structural and dynamic basis of DNA capture and translocation by mitochondrial Twinkle helicase

“…The NTDs are always on the 5′ side of the DNA relative to the helicase domains to assist primer synthesis ( Supplementary Figure S5 ). Like gp4, Twinkle helicases from plants and lower eukaryotes can catalyze primer synthesis ( 62 , 63 ). However, the primase active site residues are mutated in vertebrate Twinkle ( 11 ).…”

Structural and Dynamic Basis of DNA Capture and Translocation by Mitochondrial Twinkle Helicase

“…The NTDs are always on the 5’-side of the DNA relative to the helicase domains to assist primer synthesis (Supplementary Figure 5). Like gp4, Twinkle helicases from plants and lower eukaryotes can catalyze primer synthesis (60,61). However, the primase active site residues are mutated in vertebrate Twinkle (11).…”

Mutation of TWNK Gene Is One of the Reasons of Runting and Stunting Syndrome Characterized by mtDNA Depletion in Sex-Linked Dwarf Chicken

“…Twinkle primase domain is composed of ZBD and RPD; the ZBD interacts with the DNA template during primer synthesis and the RPD forms RNA polymerases (Kato et al, 2003). However, the primase domain of metazoan Twinkle lacks the critical residues for primase function and does not have primase activity in vitro (Harman and Barth, 2018). It was reported that several recessive mutations in Twinkle primase domain can lead to MDS in human (Hakonen et al, 2007;Hartley et al, 2012;Vi et al, 2012).…”