TWINKLE and Other Human Mitochondrial DNA Helicases: Structure, Function and Disease

Abstract

Mammalian mitochondria contain a circular genome (mtDNA) which encodes subunits of the oxidative phosphorylation machinery. The replication and maintenance of mtDNA is carried out by a set of nuclear-encoded factors-of which, helicases form an important group. The TWINKLE helicase is the main helicase in mitochondria and is the only helicase required for mtDNA replication. Mutations in TWINKLE cause a number of human disorders associated with mitochondrial dysfunction, neurodegeneration and premature ageing. In addition, a number of other helicases with a putative role in mitochondria have been identified. In this review, we discuss our current knowledge of TWINKLE structure and function and its role in diseases of mtDNA maintenance. We also briefly discuss other potential mitochondrial helicases and postulate on their role(s) in mitochondria.

Keywords: PEO; TWINKLE; mitochondria; mtDNA; replication.

Figure 1

Strand displacement model of mtDNA replication. Replication of mtDNA is initiated at the heavy strand origin of replication (O_H) by mtSSB, POLγ and TWINKLE and proceeds unidirectionally to produce the full-length nascent H-strand. When the replisome passes the light strand origin of replication (O_L), a stem-loop structure is formed from which POLRMT can initiate the synthesis of the lagging strand primer. Synthesis of the two strands proceeds in a continuous manner to produce two mtDNA molecules.

Figure 2

Structure and domain organization of TWINKLE and T7 gp4. (Left panel) Top and side views of the heptameric T7 gp4 protein (PDB ID: 5IKN). The helicase (green) and primase (blue) domains as well as the linker helix (orange) are shown. (Right panel) Homology model of a TWINKLE monomer comprising a non-functional primase-like domain (blue) connected to a conserved helicase domain (green) by a flexible linker helix (orange). The positions of disease-causing mutations for which in vitro biochemical data is available are shown as red spheres. Single-particle negative stain images are also shown for selected disease variants, highlighting the effects of these mutations on TWINKLE oligomerization.

Figure 3

Helicase activity of TWINKLE. TWINKLE is the sole replicative helicase in mitochondria. It is proposed that heptameric TWINKLE ejects a subunit upon binding to ssDNA and performs ATP-dependent 5ʹ–3ʹ unwinding of DNA. This enables POLRMT to synthesize RNA primers on which POLγ can extend, allowing for leading-strand replication. The displaced ssDNA is coated by mtSSB until it is displaced by the lagging-strand replication machinery.

Figure 4

Disease-causing mutations and polymorphisms in the TWINKLE helicase. The domain organization of TWINKLE containing the conserved primase (II–VI) and helicase (H1–H4) motifs is shown. MTS—mitochondrial targeting sequence. The list of mutations was generated based on reported mutations and polymorphisms in the online Mendelian inheritance in man/OMIM database [102]. The upper region shows all reported mutations associated with disease while the lower region shows reported mutations for which a disease phenotype is uncertain or has not yet been described. Note the clustering of disease-causing mutations in the primase-like domain and linker helix regions. Green—adPEO and other ataxia neuropathy spectrum disorders (MIRAS, SANDO). Blue—Perrault syndrome. Pink—mitochondrial hepatopathy. Orange—mtDNA depletion syndrome (including IOSCA). White—not specified/not yet linked to disease.

Figure 5

Disease-causing mutations in the NTP-binding and ssDNA-binding regions of TWINKLE. Disease-causing mutations in TWINKLE are often associated with defective ATPase and ssDNA-binding activities, leading to impaired helicase activity and subsequent stalling at the replication fork. The TWINKLE CTD (orange) was aligned to the T7 gp4 CTD (grey/blue; PDB ID: 6N9V). In cases where the T7 residues are shown, the equivalent residue number in TWINKLE is given in parentheses. (A) The residues involved in NTP binding/hydrolysis are shown, as are nearby (<10 Å) disease-causing mutations (red spheres). (B) Residues involved in ssDNA binding. Both T7 gp4 and TWINKLE possess a conserved set of positively-charged amino acids that form a ssDNA interaction interface. Nearby (<10 Å) disease-causing mutations are indicated (red spheres).

Figure 6

Potential DNA helicases in human mitochondria. In addition to the TWINKLE helicase, human mitochondria possess a number of additional helicases with unknown/poorly understood function. These include PIF1 (PDB ID: 6HPH), DNA2 (PDB ID: 5EAW), RECQL4 (PDB ID: 5LST) and SUV3 (PDB ID: 3RC3). Although some evidence exists for the presence of PIF1 and RECQL4 in human mitochondria, the mitochondrial localization and role of PIF1 and RECQL4 remains debated.