mtDNA-helicase encodes the Drosophila ortholog of human TWINKLE, the hexameric superfamily 4 replicative DNA helicase of the mitochondrial replisome, related to the bacteriophage T7 gene 4 primase-helicase. Its C-terminal domain carries the NTPase and 5'-3' duplex-unwinding activity, whereas the N-terminal primase-like domain has lost primase activity but coordinates a 2Fe-2S cluster, binds single- and double-stranded DNA, and binds cardiolipin-rich membranes in vitro. The helicase acts with DNA polymerase gamma and mtSSB at mtDNA nucleoids: RNAi knockdown lowers mtDNA copy number about five-fold, overexpression raises it, and active-site or disease-analogous mutants deplete mtDNA in a dominant-negative manner.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000262 mitochondrial chromosome | ISS GO_REF:0000024 | ACCEPT | Summary: Mitochondrial chromosome (ISS, is_active_in). Reason: Transferred from human TWINKLE (Q96RR1), which colocalizes with mtDNA nucleoids; consistent with its role as replicative helicase. Supporting Evidence: file:DROME/mtDNA-helicase/mtDNA-helicase-uniprot.txt Colocalizes with mtDNA in mitochondrial nucleoids |
| GO:0003678 DNA helicase activity | IBA GO_REF:0000033 | MODIFY | Summary: DNA helicase activity (IBA/IEA). Reason: Correct but a less specific parent of GO:0043139 5'-3' DNA helicase activity, which the gene already carries (ISS/IEA) and which is the core function. IBA donors include human TWINKLE (Q96RR1), mouse Twnk and Arabidopsis/Dictyostelium homologs. Proposed replacements: 5'-3' DNA helicase activity Supporting Evidence: PMID:25023283 The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain |
| GO:0003678 DNA helicase activity | IEA GO_REF:0000002 | MODIFY | Summary: DNA helicase activity (IBA/IEA). Reason: Correct but a less specific parent of GO:0043139 5'-3' DNA helicase activity, which the gene already carries (ISS/IEA) and which is the core function. IBA donors include human TWINKLE (Q96RR1), mouse Twnk and Arabidopsis/Dictyostelium homologs. Proposed replacements: 5'-3' DNA helicase activity Supporting Evidence: PMID:25023283 The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain |
| GO:0003690 double-stranded DNA binding | IDA PMID:25023283 The N-terminal domain of the Drosophila mitochondrial replic... | KEEP AS NON CORE | Summary: Double-stranded DNA binding (IDA). Reason: The isolated N-terminal domain binds dsDNA oligomers; the term is as precise as the evidence allows, and peripheral to the core helicase activity. Supporting Evidence: PMID:25023283 the N-terminal domain binds both single- and double-stranded DNA oligomers |
| GO:0003697 single-stranded DNA binding | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Single-stranded DNA binding. Reason: The helicase binds ssDNA (via the N-terminal domain and the translocating C-terminal ring); correctly stated, but peripheral to the core 5'-3' DNA helicase activity. Supporting Evidence: PMID:25023283 the N-terminal domain binds both single- and double-stranded DNA oligomers |
| GO:0003697 single-stranded DNA binding | IDA PMID:25023283 The N-terminal domain of the Drosophila mitochondrial replic... | KEEP AS NON CORE | Summary: Single-stranded DNA binding. Reason: The helicase binds ssDNA (via the N-terminal domain and the translocating C-terminal ring); correctly stated, but peripheral to the core 5'-3' DNA helicase activity. Supporting Evidence: PMID:25023283 the N-terminal domain binds both single- and double-stranded DNA oligomers |
| GO:0003697 single-stranded DNA binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: Single-stranded DNA binding. Reason: The helicase binds ssDNA (via the N-terminal domain and the translocating C-terminal ring); correctly stated, but peripheral to the core 5'-3' DNA helicase activity. Supporting Evidence: PMID:25023283 the N-terminal domain binds both single- and double-stranded DNA oligomers |
| GO:0003697 single-stranded DNA binding | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Single-stranded DNA binding. Reason: The helicase binds ssDNA (via the N-terminal domain and the translocating C-terminal ring); correctly stated, but peripheral to the core 5'-3' DNA helicase activity. Supporting Evidence: PMID:25023283 the N-terminal domain binds both single- and double-stranded DNA oligomers |
| GO:0005524 ATP binding | IEA GO_REF:0000002 | KEEP AS NON CORE | Summary: ATP binding (IEA). Reason: SF4 helicases bind NTPs to translocate; ATP binding is as precise as the electronic evidence allows and is peripheral to the core helicase activity. Supporting Evidence: PMID:25023283 The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain |
| GO:0005739 mitochondrion | IBA GO_REF:0000033 | ACCEPT | Summary: Mitochondrion. Reason: The helicase localizes to mitochondria. Supporting Evidence: file:DROME/mtDNA-helicase/mtDNA-helicase-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:17272269} |
| GO:0005739 mitochondrion | IDA PMID:17272269 Differential phenotypes of active site and human autosomal d... | ACCEPT | Summary: Mitochondrion. Reason: The helicase localizes to mitochondria. Supporting Evidence: file:DROME/mtDNA-helicase/mtDNA-helicase-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:17272269} |
| GO:0005739 mitochondrion | IEA GO_REF:0000120 | ACCEPT | Summary: Mitochondrion. Reason: The helicase localizes to mitochondria. Supporting Evidence: file:DROME/mtDNA-helicase/mtDNA-helicase-uniprot.txt SUBCELLULAR LOCATION: Mitochondrion {ECO:0000269|PubMed:17272269} |
| GO:0005743 mitochondrial inner membrane | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Mitochondrial inner membrane. Reason: The N-terminal domain binds cardiolipin-rich liposomes in vitro and the authors propose inner-membrane association to recruit mtDNA. mtDNA nucleoids are tethered to the inner membrane, so peripheral association is plausible, but the primary location is the nucleoid. Supporting Evidence: PMID:34950192 We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication |
| GO:0005743 mitochondrial inner membrane | NAS PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | KEEP AS NON CORE | Summary: Mitochondrial inner membrane. Reason: The N-terminal domain binds cardiolipin-rich liposomes in vitro and the authors propose inner-membrane association to recruit mtDNA. mtDNA nucleoids are tethered to the inner membrane, so peripheral association is plausible, but the primary location is the nucleoid. Supporting Evidence: PMID:34950192 We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication |
| GO:0006260 DNA replication | IEA GO_REF:0000002 | MODIFY | Summary: DNA replication (IEA). Reason: Correct but less specific than GO:0006264 mitochondrial DNA replication, which the gene already carries by IMP. Proposed replacements: mitochondrial DNA replication |
| GO:0006264 mitochondrial DNA replication | IBA GO_REF:0000033 | ACCEPT | Summary: Mitochondrial DNA replication (IBA/IEA/ISS). Reason: Consistent with direct fly evidence; IBA donors include human TWINKLE and fly mtDNA-helicase itself. Supporting Evidence: PMID:25023283 The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome |
| GO:0006264 mitochondrial DNA replication | IEA GO_REF:0000117 | ACCEPT | Summary: Mitochondrial DNA replication (IBA/IEA/ISS). Reason: Consistent with direct fly evidence; IBA donors include human TWINKLE and fly mtDNA-helicase itself. Supporting Evidence: PMID:25023283 The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome |
| GO:0006264 mitochondrial DNA replication | IMP PMID:17272269 Differential phenotypes of active site and human autosomal d... | ACCEPT | Summary: Mitochondrial DNA replication. Reason: RNAi knockdown reduces mtDNA copy number and active-site mutants are dominant-negative for mtDNA maintenance. Supporting Evidence: PMID:17272269 RNA interference knockdown of d-mtDNA helicase decreases the copy number of mitochondrial DNA (mtDNA) approximately 5-fold PMID:17272269 Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype file:DROME/mtDNA-helicase/mtDNA-helicase-deep-research-falcon.md In the original CG5924 study, RNAi lowering helicase to undetectable levels reduced mtDNA to approximately |
| GO:0006264 mitochondrial DNA replication | IMP PMID:18593709 Physiological and biochemical defects in carboxyl-terminal m... | ACCEPT | Summary: Mitochondrial DNA replication. Reason: C-terminal residues are essential for helicase function in vivo, as shown by dominant-negative mtDNA depletion. Supporting Evidence: PMID:18593709 identify critical residues between amino acids 572 and 596 of the 613 amino acid polypeptide that are essential for mitochondrial DNA helicase function in vivo |
| GO:0006264 mitochondrial DNA replication | ISS GO_REF:0000024 | ACCEPT | Summary: Mitochondrial DNA replication (IBA/IEA/ISS). Reason: Consistent with direct fly evidence; IBA donors include human TWINKLE and fly mtDNA-helicase itself. Supporting Evidence: PMID:25023283 The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome |
| GO:0008289 lipid binding | IDA PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | KEEP AS NON CORE | Summary: Lipid binding (IDA). Reason: The N-terminal domain binds phospholipid liposomes, with greater specificity as cardiolipin content increases. The physiological significance is proposed rather than established, so this is kept as a non-core property. Supporting Evidence: PMID:34950192 We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions |
| GO:0016887 ATP hydrolysis activity | ISS PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | KEEP AS NON CORE | Summary: ATP hydrolysis activity (ISS). Reason: NTP hydrolysis powers translocation and unwinding; carboxyl-terminal mutants analogous to arginine-finger residues lose nucleotide hydrolysis. Correctly stated but peripheral to the core helicase activity. Supporting Evidence: PMID:18593709 representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis |
| GO:0042645 mitochondrial nucleoid | IEA GO_REF:0000044 | ACCEPT | Summary: Mitochondrial nucleoid. Reason: Replicative helicase colocalizing with mtDNA nucleoids (by similarity to TWINKLE). Supporting Evidence: file:DROME/mtDNA-helicase/mtDNA-helicase-uniprot.txt Colocalizes with mtDNA in mitochondrial nucleoids |
| GO:0042645 mitochondrial nucleoid | ISS GO_REF:0000024 | ACCEPT | Summary: Mitochondrial nucleoid. Reason: Replicative helicase colocalizing with mtDNA nucleoids (by similarity to TWINKLE). Supporting Evidence: file:DROME/mtDNA-helicase/mtDNA-helicase-uniprot.txt Colocalizes with mtDNA in mitochondrial nucleoids |
| GO:0043139 5'-3' DNA helicase activity | IEA GO_REF:0000120 | ACCEPT | Summary: 5'-3' DNA helicase activity. Reason: The mtDNA helicase is the SF4 replicative helicase of the mitochondrial replisome (TWINKLE ortholog), which unwinds duplex DNA with 5'-3' polarity. Fly-specific evidence for unwinding is indirect (active-site mutants are dominant-negative in vivo), and the polarity is transferred from human TWINKLE. Supporting Evidence: PMID:25023283 The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain PMID:17272269 Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype |
| GO:0043139 5'-3' DNA helicase activity | ISS GO_REF:0000024 | ACCEPT | Summary: 5'-3' DNA helicase activity. Reason: The mtDNA helicase is the SF4 replicative helicase of the mitochondrial replisome (TWINKLE ortholog), which unwinds duplex DNA with 5'-3' polarity. Fly-specific evidence for unwinding is indirect (active-site mutants are dominant-negative in vivo), and the polarity is transferred from human TWINKLE. Supporting Evidence: PMID:25023283 The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain PMID:17272269 Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype |
| GO:0043139 5'-3' DNA helicase activity | ISS PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | ACCEPT | Summary: 5'-3' DNA helicase activity. Reason: The mtDNA helicase is the SF4 replicative helicase of the mitochondrial replisome (TWINKLE ortholog), which unwinds duplex DNA with 5'-3' polarity. Fly-specific evidence for unwinding is indirect (active-site mutants are dominant-negative in vivo), and the polarity is transferred from human TWINKLE. Supporting Evidence: PMID:25023283 The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain PMID:17272269 Overexpression of helicase active site mutants K388A and D483A results in a severe depletion of mtDNA and a dominant negative lethal phenotype |
| GO:0051536 iron-sulfur cluster binding | IDA PMID:34950192 Implications of Membrane Binding by the Fe-S Cluster-Contain... | MODIFY | Summary: Iron-sulfur cluster binding (IDA). Reason: Correct, but a less specific parent of GO:0051537 2 iron, 2 sulfur cluster binding, which the gene already carries (IDA, PMID:25023283). Proposed replacements: 2 iron, 2 sulfur cluster binding Supporting Evidence: PMID:34950192 we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase |
| GO:0051537 2 iron, 2 sulfur cluster binding | IDA PMID:25023283 The N-terminal domain of the Drosophila mitochondrial replic... | KEEP AS NON CORE | Summary: 2 iron, 2 sulfur cluster binding (IDA). Reason: The N-terminal primase-like domain coordinates a 2Fe-2S cluster through conserved cysteines; the cluster stabilizes the protein. A real cofactor-binding property, but structural rather than the core helicase function. Supporting Evidence: PMID:25023283 the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro |
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Download this section (compressed HTML)Q: Does the 2Fe-2S cluster in the N-terminal domain of the fly mtDNA helicase sense redox or DNA-mediated charge transfer to regulate replication, beyond stabilizing the protein?
Experiment: Purify full-length fly mtDNA helicase and measure unwinding polarity and rate, with and without an intact 2Fe-2S cluster, alone and with Pol gamma and mtSSB.
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Download this section (compressed HTML)mitochondrial DNA helicase (mtDNA-helicase) encodes the replicative mitochondrial DNA helicase, which is responsible for NTPase-dependent double-stranded DNA unwinding during mitochondrial DNA replication. It has roles in mitochondrial genome maintenance, and mitochondrial function.
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