TWNK encodes Twinkle, the nuclear-encoded replicative DNA helicase of mitochondria. Twinkle is a superfamily 4, DnaB/T7 gp4-like ring helicase with an N-terminal zinc-binding-like and catalytically inactive primase-like (RNA polymerase-like) domain, a linker, and a C-terminal RecA-like helicase domain. It assembles into hexamers and heptamers that bind single- and double-stranded DNA and couple NTP hydrolysis (UTP and ATP) to 5'->3' translocation and unwinding of duplex DNA. Together with DNA polymerase gamma (POLG/POLG2) and the mitochondrial single-stranded DNA-binding protein SSBP1 it forms the minimal mtDNA replisome, unwinding the parental duplex ahead of the polymerase; it is the only replicative helicase in mammalian mitochondria and is required for nascent D-loop strand synthesis. Twinkle also anneals complementary single strands, dissociates D-loops and branch-migrates three-stranded structures. It localises to mitochondrial nucleoids in the matrix, associated with the inner membrane. Mutations cause autosomal dominant progressive external ophthalmoplegia with multiple mtDNA deletions, mtDNA depletion syndrome 7 (infantile-onset spinocerebellar ataxia/hepatocerebral) and Perrault syndrome 5. The splice isoform Twinky is monomeric and lacks ATPase, unwinding and DNA-binding activity.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000262 mitochondrial chromosome | IDA PMID:26253742 Regulation of DNA replication at the end of the mitochondria... | ACCEPT | Summary: ChIP in HeLa cells shows TWINKLE bound to mtDNA, with reversible loading at the coreTAS element at the 3′ end of the D-loop. Reason: Direct in vivo occupancy of the mitochondrial genome by ChIP supports is_active_in mitochondrial chromosome; consistent with the nucleoid location and replicative helicase role. Supporting Evidence: PMID:26253742 we demonstrate that the loading of the helicase TWINKLE at coreTAS is reversible PMID:18971204 While wild-type Twinkle co-localized with mtDNA in punctate nucleoids within mitochondria |
| GO:0002020 protease binding | IPI PMID:14739292 DNA and RNA binding by the mitochondrial lon protease is reg... | KEEP AS NON CORE | Summary: Co-immunoprecipitation of Twinkle with the mitochondrial Lon protease LONP1. Reason: The LONP1 interaction is reported in the abstract (abstract-only cache) and is consistent with Reactome, where Twinkle is a LONP1 substrate. Binding a protease reflects being a client of mitochondrial protein quality control, not part of the helicase function; retained as non-core. Supporting Evidence: PMID:14739292 Co-immunoprecipitation experiments show that Lon interacts with mtDNA polymerase gamma and the Twinkle helicase |
| GO:0003678 DNA helicase activity | IBA GO_REF:0000033 | MODIFY | Summary: DNA helicase activity; correct but less specific than the experimentally established 5′→3′ polarity. Reason: Twinkle is an SF4 DnaB-like helicase with demonstrated 5′→3′ polarity. GO:0043139 5′-3′ DNA helicase activity is an is_a descendant of GO:0003678 (checked with OAK) and the gene already carries it by many EXP/IDA rows, so the row should be refined to it. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000305614 SUPPORTS TRANSFER Ancestral node for the Twinkle/T7 gp4-like helicase clade; helicase activity is conserved and human Twinkle lies inside the clade. Only the granularity is refined. UniProtKB:Q96RR1 SUPPORTS TRANSFER Human Twinkle experimental helicase annotations are among the descendant evidence; not circular. Proposed replacements: 5'-3' DNA helicase activity Supporting Evidence: PMID:12975372 TWINKLE is a DNA helicase with 5' to 3' directionality and distinct substrate requirements PMID:27226550 Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates |
| GO:0003678 DNA helicase activity | IEA GO_REF:0000120 | MODIFY | Summary: DNA helicase activity; correct but less specific than the experimentally established 5′→3′ polarity. Reason: Twinkle is an SF4 DnaB-like helicase with demonstrated 5′→3′ polarity. GO:0043139 5′-3′ DNA helicase activity is an is_a descendant of GO:0003678 (checked with OAK) and the gene already carries it by many EXP/IDA rows, so the row should be refined to it. Proposed replacements: 5'-3' DNA helicase activity Supporting Evidence: PMID:12975372 TWINKLE is a DNA helicase with 5' to 3' directionality and distinct substrate requirements PMID:27226550 Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates |
| GO:0003697 single-stranded DNA binding | IBA GO_REF:0000033 | ACCEPT | Summary: Single-stranded DNA binding by Twinkle. Reason: EMSA and anisotropy assays show purified human Twinkle binds ssDNA, and the N-terminal region is required for efficient ssDNA binding; ssDNA engagement is intrinsic to the 5′→3′ translocating helicase mechanism. Supporting Evidence: PMID:18039713 the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA PMID:18971204 The binding of Twinkle to single-stranded DNA in vitro was evaluated by electromobility shift assay |
| GO:0003697 single-stranded DNA binding | IDA PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | ACCEPT | Summary: Single-stranded DNA binding by Twinkle. Reason: EMSA and anisotropy assays show purified human Twinkle binds ssDNA, and the N-terminal region is required for efficient ssDNA binding; ssDNA engagement is intrinsic to the 5′→3′ translocating helicase mechanism. Supporting Evidence: PMID:18971204 The binding of Twinkle to single-stranded DNA in vitro was evaluated by electromobility shift assay PMID:18039713 the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA |
| GO:0003697 single-stranded DNA binding | IEA GO_REF:0000002 | ACCEPT | Summary: Single-stranded DNA binding by Twinkle. Reason: EMSA and anisotropy assays show purified human Twinkle binds ssDNA, and the N-terminal region is required for efficient ssDNA binding; ssDNA engagement is intrinsic to the 5′→3′ translocating helicase mechanism. Supporting Evidence: PMID:18039713 the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA |
| GO:0005515 protein binding | IPI PMID:28514442 Architecture of the human interactome defines protein commun... | REMOVE | Summary: Bare protein binding from a high-throughput interactome screen (LGALS3BP). Reason: GO:0005515 protein binding is uninformative and is removed per the project protein-binding policy; the LGALS3BP hit has no functional follow-up for Twinkle. Supporting Evidence: file:human/TWNK/TWNK-deep-research-manual.md High-throughput Y2H hits (LGALS3BP) from HuRI/BioPlex-type screens (PMID:28514442, PMID:33961781) have no functional follow-up. |
| GO:0005515 protein binding | IPI PMID:33961781 Dual proteome-scale networks reveal cell-specific remodeling... | REMOVE | Summary: Bare protein binding from a high-throughput interactome screen (LGALS3BP). Reason: GO:0005515 protein binding is uninformative and is removed per the project protein-binding policy; the LGALS3BP hit has no functional follow-up for Twinkle. Supporting Evidence: file:human/TWNK/TWNK-deep-research-manual.md High-throughput Y2H hits (LGALS3BP) from HuRI/BioPlex-type screens (PMID:28514442, PMID:33961781) have no functional follow-up. |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: ATP binding inferred from the helicase domain. Reason: Twinkle binds and hydrolyses NTPs (ATP and, optimally, UTP) to drive unwinding. ATP binding is true but is a constituent of the ATP hydrolysis and helicase activities captured elsewhere; retained as non-core. Supporting Evidence: PMID:12975372 The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5'-triphosphate, with UTP being the optimal substrate. |
| GO:0005739 mitochondrion | HTP PMID:34800366 Quantitative high-confidence human mitochondrial proteome an... | ACCEPT | Summary: Mitochondrion. Reason: Twinkle is a mitochondrial protein; its nucleoid and matrix sub-compartment locations are captured by separate rows. For the HTP row, organelle proteomics cannot resolve the sub-compartment. For IBA/IEA, the root organelle claim holds: the IBD rests on mitochondrial orthologs (Drosophila, mouse, Dictyostelium) and the organelle-level term is sound. Supporting Evidence: file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid PMID:18971204 While wild-type Twinkle co-localized with mtDNA in punctate nucleoids within mitochondria |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Mitochondrion. Reason: Twinkle is a mitochondrial protein; its nucleoid and matrix sub-compartment locations are captured by separate rows. For the HTP row, organelle proteomics cannot resolve the sub-compartment. For IBA/IEA, the root organelle claim holds: the IBD rests on mitochondrial orthologs (Drosophila, mouse, Dictyostelium) and the organelle-level term is sound. Supporting Evidence: PMID:18971204 While wild-type Twinkle co-localized with mtDNA in punctate nucleoids within mitochondria |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Mitochondrion. Reason: Twinkle is a mitochondrial protein; its nucleoid and matrix sub-compartment locations are captured by separate rows. For the HTP row, organelle proteomics cannot resolve the sub-compartment. For IBA/IEA, the root organelle claim holds: the IBD rests on mitochondrial orthologs (Drosophila, mouse, Dictyostelium) and the organelle-level term is sound. Supporting Evidence: PMID:18971204 While wild-type Twinkle co-localized with mtDNA in punctate nucleoids within mitochondria |
| GO:0005743 mitochondrial inner membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Association with the mitochondrial inner membrane. Reason: Endogenous human Twinkle is reported to be firmly attached to the inner membrane even without mtDNA, and full-length human Twinkle binds cardiolipin-containing liposomes; it is a peripheral rather than integral membrane protein. Membrane tethering is relevant to nucleoid organisation but peripheral to the helicase function. Supporting Evidence: PMID:34950192 endogenous human mtDNA helicase is attached firmly to the MIM even in the absence of mtDNA file:human/TWNK/TWNK-uniprot.txt Preferentially associates with membranes enriched with cardiolipin, a lipid abundant in the mitochondrial inner membrane |
| GO:0005743 mitochondrial inner membrane | NAS PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | KEEP AS NON CORE | Summary: Association with the mitochondrial inner membrane. Reason: Endogenous human Twinkle is reported to be firmly attached to the inner membrane even without mtDNA, and full-length human Twinkle binds cardiolipin-containing liposomes; it is a peripheral rather than integral membrane protein. Membrane tethering is relevant to nucleoid organisation but peripheral to the helicase function. Supporting Evidence: PMID:34950192 endogenous human mtDNA helicase is attached firmly to the MIM even in the absence of mtDNA |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-1592239 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9838081 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9838093 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9913634 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914364 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914404 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914409 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914438 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914478 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914498 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0005759 mitochondrial matrix | TAS Reactome:R-HSA-9914858 | ACCEPT | Summary: Mitochondrial matrix (Reactome). Reason: Twinkle acts on mtDNA in nucleoids, which are part_of the mitochondrial matrix (GO:0042645 part_of GO:0005759, checked with OAK). Reactome reactions place TWNK in the matrix. Supporting Evidence: Reactome:R-HSA-9914438 TWINKLE (TWNK, PEO1) binds DNA as a hexamer encircling the replication origin and employs energy from ATP hydrolysis to unwind double stranded DNA in advance of the mitochondrial DNA polymerase POLgamma (POLG:POLG2) file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0006260 DNA replication | IEA GO_REF:0000002 | MODIFY | Summary: DNA replication inferred from the DnaB-like helicase domain. Reason: Correct but general: Twinkle is the replicative helicase specifically of mitochondrial DNA. Refine to GO:0006264 mitochondrial DNA replication, a descendant the gene already carries. Proposed replacements: mitochondrial DNA replication Supporting Evidence: PMID:23393161 TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria PMID:15167897 Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. |
| GO:0006261 DNA-templated DNA replication | IDA PMID:15167897 Reconstitution of a minimal mtDNA replisome in vitro. | MODIFY | Summary: In vitro reconstitution of a minimal mtDNA replisome with POLG, Twinkle and mtSSB. Reason: The reconstitution is of the mitochondrial replisome; the term is correct but the specific descendant GO:0006264 mitochondrial DNA replication (is_a GO:0006261, checked with OAK) is supported and already carried by the gene. Proposed replacements: mitochondrial DNA replication Supporting Evidence: PMID:15167897 In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. PMID:15167897 Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. |
| GO:0006264 mitochondrial DNA replication | IBA GO_REF:0000033 | ACCEPT | Summary: Twinkle is the replicative helicase of mtDNA replication. Reason: Reconstitution with POLG and mtSSB, the mouse conditional knockout showing it is the only replicative mtDNA helicase, and replication stalling with dominant human mutants all establish direct participation, by unwinding the parental duplex at the fork. Supporting Evidence: PMID:23393161 TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria PMID:15167897 Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. |
| GO:0006264 mitochondrial DNA replication | IEA GO_REF:0000120 | ACCEPT | Summary: Twinkle is the replicative helicase of mtDNA replication. Reason: Reconstitution with POLG and mtSSB, the mouse conditional knockout showing it is the only replicative mtDNA helicase, and replication stalling with dominant human mutants all establish direct participation, by unwinding the parental duplex at the fork. Supporting Evidence: PMID:23393161 TWINKLE is essential for mouse embryonic development and thus is the only replicative DNA helicase in mammalian mitochondria |
| GO:0006264 mitochondrial DNA replication | IMP PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | ACCEPT | Summary: Twinkle is the replicative helicase of mtDNA replication. Reason: Reconstitution with POLG and mtSSB, the mouse conditional knockout showing it is the only replicative mtDNA helicase, and replication stalling with dominant human mutants all establish direct participation, by unwinding the parental duplex at the fork. Supporting Evidence: PMID:18971204 We show that expression of dominant Twinkle mutations results in the accumulation of mtDNA replication intermediates in cell culture. |
| GO:0006264 mitochondrial DNA replication | NAS PMID:12975372 TWINKLE Has 5' -> 3' DNA helicase activity and is specifical... | ACCEPT | Summary: Twinkle is the replicative helicase of mtDNA replication. Reason: Reconstitution with POLG and mtSSB, the mouse conditional knockout showing it is the only replicative mtDNA helicase, and replication stalling with dominant human mutants all establish direct participation, by unwinding the parental duplex at the fork. Supporting Evidence: PMID:12975372 Our enzymatic characterization strongly supports the notion that TWINKLE is the helicase at the mitochondrial DNA replication fork |
| GO:0006390 mitochondrial transcription | IMP PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | MARK AS OVER ANNOTATED | Summary: Reduced mitochondrial transcript levels after expression of dominant adPEO Twinkle mutants. Reason: Full text read. The paper shows mutant expression lowers steady-state mitochondrial transcripts ahead of mtDNA depletion, but does not show that Twinkle performs any step of mitochondrial transcription; the effect is a downstream consequence of replication stalling and nucleoid disruption. No transcription-related activity of Twinkle is known. Supporting Evidence: PMID:18971204 our results in cell culture pointed to a problem of transcription that preceded the mtDNA depletion phenotype PMID:18971204 PEO mutations impair the helicase activity of Twinkle and lead to mtDNA replication stalling or pausing and reduced mitochondrial transcript levels |
| GO:0008289 lipid binding | IDA PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | KEEP AS NON CORE | Summary: Full-length human Twinkle binds cardiolipin-containing liposomes. Reason: Full text shows purified human Twinkle and its RPD bind CL-asolectin liposomes and that liposomes stimulate its ATPase. Lipid binding is a genuine but accessory property linked to inner-membrane tethering of nucleoids; the cardiolipin preference was shown for the Drosophila NTD, so the general lipid binding term is kept as is. Supporting Evidence: PMID:34950192 We observed that both the FL Hs mtDNA helicase and its RPD domain (the RNA polymerase-like subdomain of the NTD) bound to CL-asolectin liposomes in a dose-dependent manner in the vesicle cosedimentation assays (Figure 4A). PMID:34950192 ATPase activity of the human replicative mtDNA helicase is enhanced by binding to liposomes. |
| GO:0016887 ATP hydrolysis activity | EXP PMID:12975372 TWINKLE Has 5' -> 3' DNA helicase activity and is specifical... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:12975372 The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5'-triphosphate, with UTP being the optimal substrate. |
| GO:0016887 ATP hydrolysis activity | EXP PMID:17324440 Modular architecture of the hexameric human mitochondrial DN... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:17324440 Truncations of the N and C termini affect differentially DNA-dependent ATPase activity PMID:17324440 an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication |
| GO:0016887 ATP hydrolysis activity | EXP PMID:18039713 The N-terminal domain of TWINKLE contributes to single-stran... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:18039713 Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. |
| GO:0016887 ATP hydrolysis activity | EXP PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:18971204 Purified protein extracts of the various Twinkle mutations showed clearly reduced maximal unwinding activity in in vitro assays using an M13-based substrate. |
| GO:0016887 ATP hydrolysis activity | EXP PMID:22383523 Human mitochondrial DNA helicase TWINKLE is both an unwindin... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:22383523 The recombinant TWINKLE assembles into hexamers and higher oligomers |
| GO:0016887 ATP hydrolysis activity | EXP PMID:25824949 The hexameric structure of the human mitochondrial replicati... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:25824949 DNA unwinding activity of the three forms of Twinkle, expressed in SF9 insect cells |
| GO:0016887 ATP hydrolysis activity | EXP PMID:27226550 Biochemical Characterization of the Human Mitochondrial Repl... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:27226550 Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates |
| GO:0016887 ATP hydrolysis activity | EXP PMID:35914129 Structural insight and characterization of human Twinkle hel... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:35914129 Twinkle, a Superfamily 4 (SF4) helicase, catalyzes nucleoside 5′-triphosphate (NTP) hydrolysis and unwinds DNA substrates with 5′–3′ polarity |
| GO:0016887 ATP hydrolysis activity | IDA PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:34950192 ATPase activity of the human replicative mtDNA helicase is enhanced by binding to liposomes. |
| GO:0016887 ATP hydrolysis activity | IEA GO_REF:0000116 | ACCEPT | Summary: NTP/ATP hydrolysis by Twinkle. Reason: Twinkle has its own DNA-dependent NTPase (ATP/UTP) activity, mechanistically coupled to 5′→3′ translocation and unwinding; this activity is placed in molecular_function alongside the helicase activity in core_functions. Supporting Evidence: PMID:12975372 The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5'-triphosphate, with UTP being the optimal substrate. |
| GO:0034214 protein hexamerization | IDA PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | KEEP AS NON CORE | Summary: Twinkle self-assembles into hexamers. Reason: Purified Twinkle forms hexamers (and heptamers) without nucleotide; hexamerization is a structural prerequisite for its ring-helicase activity but is self-assembly rather than a biological role, so retained as non-core. Supporting Evidence: PMID:18971204 wild-type Twinkle forms hexamers even under high ionic-strength conditions and in the absence of any nucleotide PMID:18039713 TWINKLE forms stable hexamers even in the absence of Mg(2+) or NTPs |
| GO:0042645 mitochondrial nucleoid | IDA PMID:18063578 The layered structure of human mitochondrial DNA nucleoids. | ACCEPT | Summary: Mitochondrial nucleoid. Reason: Twinkle co-localises with mtDNA in punctate nucleoids and co-fractionates with mtDNA; this is where the replicative helicase acts. The PMID:18063578 cache is abstract-only and the abstract does not name Twinkle, so this row defers to the curator; the location is independently established. Supporting Evidence: PMID:18971204 While wild-type Twinkle co-localized with mtDNA in punctate nucleoids within mitochondria |
| GO:0042645 mitochondrial nucleoid | IDA PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | ACCEPT | Summary: Mitochondrial nucleoid. Reason: Twinkle co-localises with mtDNA in punctate nucleoids and co-fractionates with mtDNA; this is where the replicative helicase acts. Supporting Evidence: PMID:18971204 While wild-type Twinkle co-localized with mtDNA in punctate nucleoids within mitochondria |
| GO:0042645 mitochondrial nucleoid | IDA PMID:23275553 Alternative translation initiation augments the human mitoch... | ACCEPT | Summary: Mitochondrial nucleoid. Reason: Twinkle co-localises with mtDNA in punctate nucleoids and co-fractionates with mtDNA; this is where the replicative helicase acts. Supporting Evidence: PMID:23275553 The majority of the signal from proteins known to be involved in mtDNA metabolism, such as TWINKLE, TFAM, SSBP1 and POLG1, resolved in the same fractions as mtDNA |
| GO:0042645 mitochondrial nucleoid | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrial nucleoid. Reason: Twinkle co-localises with mtDNA in punctate nucleoids and co-fractionates with mtDNA; this is where the replicative helicase acts. Supporting Evidence: file:human/TWNK/TWNK-uniprot.txt Mitochondrion matrix, mitochondrion nucleoid |
| GO:0042802 identical protein binding | IPI PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | KEEP AS NON CORE | Summary: Homo-oligomerisation of Twinkle. Reason: Twinkle forms homohexamers; identical protein binding is true and reflects ring assembly, a prerequisite for helicase activity, but is not itself the function. Supporting Evidence: PMID:18971204 wild-type Twinkle forms hexamers even under high ionic-strength conditions and in the absence of any nucleotide PMID:22383523 The recombinant TWINKLE assembles into hexamers and higher oligomers |
| GO:0043139 5'-3' DNA helicase activity | EXP PMID:17324440 Modular architecture of the hexameric human mitochondrial DN... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:17324440 an enzyme that uses the energy of nucleotide hydrolysis to unwind duplex DNA during mitochondrial DNA replication |
| GO:0043139 5'-3' DNA helicase activity | EXP PMID:18039713 The N-terminal domain of TWINKLE contributes to single-stran... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:18039713 Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. |
| GO:0043139 5'-3' DNA helicase activity | EXP PMID:22383523 Human mitochondrial DNA helicase TWINKLE is both an unwindin... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:22383523 TWINKLE, although a helicase, has an antagonistic activity of annealing two complementary ssDNAs |
| GO:0043139 5'-3' DNA helicase activity | EXP PMID:25824949 The hexameric structure of the human mitochondrial replicati... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:25824949 DNA unwinding activity of the three forms of Twinkle, expressed in SF9 insect cells |
| GO:0043139 5'-3' DNA helicase activity | EXP PMID:27226550 Biochemical Characterization of the Human Mitochondrial Repl... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:27226550 Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates |
| GO:0043139 5'-3' DNA helicase activity | EXP PMID:35914129 Structural insight and characterization of human Twinkle hel... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:35914129 Twinkle, a Superfamily 4 (SF4) helicase, catalyzes nucleoside 5′-triphosphate (NTP) hydrolysis and unwinds DNA substrates with 5′–3′ polarity |
| GO:0043139 5'-3' DNA helicase activity | IDA PMID:12975372 TWINKLE Has 5' -> 3' DNA helicase activity and is specifical... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:12975372 TWINKLE is a DNA helicase with 5' to 3' directionality and distinct substrate requirements |
| GO:0043139 5'-3' DNA helicase activity | IDA PMID:15167897 Reconstitution of a minimal mtDNA replisome in vitro. | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:15167897 Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. |
| GO:0043139 5'-3' DNA helicase activity | IDA PMID:18971204 Twinkle mutations associated with autosomal dominant progres... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:18971204 Purified protein extracts of the various Twinkle mutations showed clearly reduced maximal unwinding activity in in vitro assays using an M13-based substrate. |
| GO:0043139 5'-3' DNA helicase activity | IDA PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. This paper measured DNA- and liposome-stimulated ATPase as a proxy for helicase activity rather than unwinding itself; the 5′→3′ activity is established by the other references. Supporting Evidence: PMID:34950192 ATPase activity of the human replicative mtDNA helicase is enhanced by binding to liposomes. |
| GO:0043139 5'-3' DNA helicase activity | IEA GO_REF:0000120 | ACCEPT | Summary: 5′→3′ DNA helicase activity, the core activity of Twinkle. Reason: Purified human Twinkle unwinds duplex DNA with 5′→3′ polarity in an NTP-hydrolysis-dependent manner; this is its defining activity at the mtDNA replication fork. Supporting Evidence: PMID:12975372 TWINKLE is a DNA helicase with 5' to 3' directionality and distinct substrate requirements |
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Download this section (compressed HTML)Q: What loads the closed Twinkle ring onto circular mtDNA in vivo, given that it forms stable hexamers without nucleotide and no dedicated helicase loader has been identified?
Q: Do the strand-annealing, D-loop dissociation and branch-migration activities of Twinkle act in mtDNA repair or recombination-dependent replication in vivo?
Q: Is the reduced mitochondrial transcript level seen with adPEO Twinkle mutants solely secondary to replication stalling, or does Twinkle at coreTAS affect transcription termination directly?
Experiment: Engineer separation-of-function Twinkle variants that keep unwinding but lose annealing in vitro, express them in TWNK-depleted cells, and quantify mtDNA deletions, replication intermediates and recovery after damage.
Hypothesis: Twinkle strand-annealing and branch-migration activities contribute to the formation or resolution of mtDNA deletions.
Experiment: Map lipid-binding residues in the RPD, test liposome binding and ATPase of mutants, and assess nucleoid membrane association and mtDNA copy number in cells expressing them.
Hypothesis: Inner-membrane tethering via the RPD is required for Twinkle function in vivo.
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