TWINKLE Has 5′ → 3′ DNA Helicase Activity and Is Specifically Stimulated by Mitochondrial Single-stranded DNA-binding Protein
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m306981200
ISSN1083-351X
AutoresJenny Korhonen, Martina Gaspari, Maria Falkenberg,
Tópico(s)Metabolism and Genetic Disorders
ResumoMutations in TWINKLE cause autosomal dominant progressive external ophthalmoplegia, a human disorder associated with multiple deletions in the mitochondrial DNA. TWINKLE displays primary sequence similarity to the phage T7 gene 4 primase-helicase, but no specific enzyme activity has been assigned to the protein. We have purified recombinant TWINKLE to near homogeneity and demonstrate here that TWINKLE is a DNA helicase with 5′ to 3′ directionality and distinct substrate requirements. The protein needs a stretch of 10 nucleotides of single-stranded DNA on the 5′-side of the duplex to unwind duplex DNA. In addition, helicase activity is not observed unless a short single-stranded 3′-tail is present. The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5′-triphosphate, with UTP being the optimal substrate. DNA unwinding by TWINKLE is specifically stimulated by the mitochondrial single-stranded DNA-binding protein. Our enzymatic characterization strongly supports the notion that TWINKLE is the helicase at the mitochondrial DNA replication fork and provides evidence for a close relationship of the DNA replication machinery in bacteriophages and mammalian mitochondria. Mutations in TWINKLE cause autosomal dominant progressive external ophthalmoplegia, a human disorder associated with multiple deletions in the mitochondrial DNA. TWINKLE displays primary sequence similarity to the phage T7 gene 4 primase-helicase, but no specific enzyme activity has been assigned to the protein. We have purified recombinant TWINKLE to near homogeneity and demonstrate here that TWINKLE is a DNA helicase with 5′ to 3′ directionality and distinct substrate requirements. The protein needs a stretch of 10 nucleotides of single-stranded DNA on the 5′-side of the duplex to unwind duplex DNA. In addition, helicase activity is not observed unless a short single-stranded 3′-tail is present. The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5′-triphosphate, with UTP being the optimal substrate. DNA unwinding by TWINKLE is specifically stimulated by the mitochondrial single-stranded DNA-binding protein. Our enzymatic characterization strongly supports the notion that TWINKLE is the helicase at the mitochondrial DNA replication fork and provides evidence for a close relationship of the DNA replication machinery in bacteriophages and mammalian mitochondria. The molecular mechanisms by which mtDNA is replicated in mammalian cells are of fundamental biological interest. Saccharomyces cerevisiae has served as a model system for studies of mammalian mtDNA replication, but there are significant differences between yeast and mammalian cells (1.Shadel G.S. Clayton D.A. Annu. Rev. Biochem. 1997; 66: 409-435Crossref PubMed Scopus (806) Google Scholar). Replication of the S. cerevisiae mtDNA is initiated from multiple sites of the ∼86-kb genome, and the mtDNA molecules frequently undergo recombination. In contrast, the smaller mammalian mtDNAs (∼16 kb) initiate DNA replication from two specific origins of replication, oriH and oriL, and recombination is a rare or possibly even non-existent phenomenon (2.Eyre-Walker A. Awadalla P. J. Mol. Evol. 2001; 53: 430-435Crossref PubMed Scopus (63) Google Scholar).Mammalian mtDNA contains two major promoters, the light and heavy strand promoters, which produce near genomic length transcripts that, after RNA processing, release individual mRNAs, tRNAs, and rRNAs. A separate transcription unit for the rRNA genes in mammalian mitochondria has also been reported (3.Montoya J. Christianson T. Levens D. Rabinowitz M. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 7195-7199Crossref PubMed Scopus (202) Google Scholar). Transcription from light strand promoters is not only necessary for gene expression but also produces the RNA primers required for initiation of mtDNA replication at oriH (1.Shadel G.S. Clayton D.A. Annu. Rev. Biochem. 1997; 66: 409-435Crossref PubMed Scopus (806) Google Scholar, 4.Chang D.D. Clayton D.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 351-355Crossref PubMed Scopus (240) Google Scholar). DNA synthesis from oriH is unidirectional and proceeds to displace the parental heavy strand. The nascent H strands frequently terminate 700 bp downstream of oriH, giving rise to 7 S DNA (D-loop strand). This termination event produces a characteristic triple-stranded structure, called the D-loop (5.Kasamatsu H. Robberson D.L. Vinograd J. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2252-2257Crossref PubMed Scopus (219) Google Scholar). The function of the D-loops is unknown, but they presumably play a role in regulating mtDNA replication.The mitochondrial DNA polymerase γ is a heterodimer comprising catalytic (A) and accessory (B) subunits of 140 and 54 kDa, respectively. The accessory subunit, polymerase γ B, which is not present in yeast, has been characterized as a processivity factor for the polymerase (6.Carrodeguas J.A. Kobayashi R. Lim S.E. Copeland W.C. Bogenhagen D.F. Mol. Cell. Biol. 1999; 19: 4039-4046Crossref PubMed Google Scholar, 7.Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 38197-38203Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Polymerase γ B increases the affinity of the polymerase for DNA and promotes tighter nucleotide binding, increasing the polymerization rate (7.Lim S.E. Longley M.J. Copeland W.C. J. Biol. Chem. 1999; 274: 38197-38203Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 8.Johnson A.A. Tsai Y. Graves S.W. Johnson K.A. Biochemistry. 2000; 39: 1702-1708Crossref PubMed Scopus (122) Google Scholar). The processivity of DNA polymerase γ is specifically stimulated by the mitochondrial single-stranded DNA-binding protein, mtSSB 1The abbreviations used are: mtSSBmitochondrial single-stranded DNA-binding proteinntnucleotidesadPEOautosomal dominant progressive external ophthalmoplegiaMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightATPγSadenosine 5′-O-(thiotriphosphate).1The abbreviations used are: mtSSBmitochondrial single-stranded DNA-binding proteinntnucleotidesadPEOautosomal dominant progressive external ophthalmoplegiaMALDI-TOFmatrix-assisted laser desorption/ionization time-of-flightATPγSadenosine 5′-O-(thiotriphosphate). (9.Mignotte B. Marsault J. Barat-Gueride M. Eur. J. Biochem. 1988; 174: 479-484Crossref PubMed Scopus (24) Google Scholar, 10.Farr C.L. Wang Y. Kaguni L.S. J. Biol. Chem. 1999; 274: 14779-14785Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).The TWINKLE gene was originally identified in a search for mutations associated with chromosome 10q24-linked autosomal dominant progressive external ophthalmoplegia (adPEO) (11.Spelbrink J.N. Li F.Y. Tiranti V. Nikali K. Yuan Q.P. Tariq M. Wanrooij S. Garrido N. Comi G. Morandi L. Santoro L. Toscano A. Fabrizi G.M. Somer H. Croxen R. Beeson D. Poulton J. Suomalainen A. Jacobs H.T. Zeviani M. Larsson C. Nat. Genet. 2001; 28: 223-231Crossref PubMed Scopus (679) Google Scholar), which is a human disorder with exercise intolerance, muscle weakness, peripheral neuropathy, deafness, ataxia, cataracts, and hypogonadism. Homology searches revealed a striking sequence similarity between TWINKLE and the bacteriophage T7 gene 4 protein, which contains both the DNA helicase and the primase activities needed at the bacteriophage replication fork. Primary sequence analysis revealed that TWINKLE contains sequence motives typically found in DNA helicases, whereas no obvious similarities could be identified with known primases.Interestingly, adPEO is characterized by the presence of multiple mtDNA deletions, and the disorder has also been linked to mutations in DNA polymerase γ (12.Suomalainen A. Kaukonen J. Am. J. Med. Genet. 2001; 106: 53-61Crossref PubMed Scopus (93) Google Scholar, 13.Ponamarev M.V. Longley M.J. Nguyen D. Kunkel T.A. Copeland W.C. J. Biol. Chem. 2002; 277: 15225-15228Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Taken together, these data demonstrate a functional relationship between TWINKLE and the DNA polymerase and suggest that TWINKLE may be a DNA helicase active in mammalian mitochondrial DNA replication. A molecular characterization of TWINKLE would therefore not only be of interest for a molecular understanding of adPEO but would also reveal important insights into the molecular mechanisms of mtDNA replication.We have purified TWINKLE in recombinant form and studied its enzymatic functions. We demonstrate here that TWINKLE is a 5′ → 3′ DNA helicase, which is specifically stimulated by mtSSB. Our findings strongly support the notion that TWINKLE is involved in mammalian mtDNA replication and further demonstrate the remarkable structural and functional similarities between the DNA replication machinery in bacteriophages and mammalian mitochondria.MATERIALS AND METHODSRecombinant Proteins—Spodoptera frugiperda (Sf9) cells were maintained and propagated in suspension in SFM 900 medium (Invitrogen), containing 5% fetal calf serum, at 27 °C. DNA fragments encoding TWINKLE and mtSSB were PCR-amplified from human cDNAs and cloned into the pBacPAK9 vector (Clontech). The TWINKLE expression construct encoded the mitochondrial form of the protein lacking the import signal (1–42 amino acids). A His6 tag was introduced at the N terminus. The construct for mtSSB also encoded the mitochondrial form of the protein without the import signal (1–16 amino acids) but had no affinity tag. Autographa californica nuclear polyhedrosis viruses recombinant for the individual expression constructs were prepared as described in the BacPAK™ manual (Clontech). Protein expression was performed by growing 400 ml of Sf9 cells to a density of 2 × 106 cells/ml in suspension. The cells were infected with 10 plaque-forming units/cell of recombinant baculovirus and harvested 72 h after infection. Infected cells were frozen in liquid nitrogen and thawed at 4 °C in 20 ml of lysis buffer containing 25 mm Tris-HCl, pH 8.0, 10 mm β-mercaptoethanol, and 1× protease inhibitors (for all purifications, a 100× stock of protease inhibitors contained 100 mm phenylmethlsulfonyl fluoride, 200 mm pepstatin A, 60 mm leupeptin, and 200 mm benzamidine in 100% ethanol). The cells were incubated on ice for 20 min, transferred to a Dounce homogenizer, and disrupted using 20 strokes of a tight-fitting pestle. Next, NaCl was added to a final concentration of 1.0 m, and the homogenate was swirled gently for 45 min at 4 °C. The extract was cleared by centrifugation at 36,000 rpm for 30 min at 4 °C using a Beckman TLA 100.3 rotor.Protein Purification—TWINKLE was purified by diluting the protein extracts with an equal volume of buffer A (50 mm Tris-HCl, pH 8.0, 0.6 m NaCl, 20% glycerol, 10 mm β-mercaptoethanol, and 1× protease inhibitors) containing 20 mm imidazole. The extract was then added to 1 ml of Ni2+-agarose Superflow beads (Qiagen) and incubated for 1 h at +4 °C. Ni2+-agarose beads were collected by centrifugation (JA-17, 2,500 rpm, 10 min, +4 °C), washed once with 15 ml of buffer A containing 40 mm imidazole, again collected by centrifugation, and finally loaded into a column. The column was washed with 10 column volumes of buffer A containing 40 mm imidazole and eluted with 15 column volumes of buffer A containing 250 mm imidazole. The recombinant protein was identified by SDS-PAGE and Coomassie Brilliant Blue staining, and the peak fractions were pooled and diluted with an equal volume of buffer B (10 mm K·PO4, pH 7.2, 10% glycerol, 1 mm dithiothreitol, 100 mm NaCl, and 1× protease inhibitors). TWINKLE was further purified on a 2-ml hydroxyapatite column (Bio-Rad) equilibrated in buffer B. The column was washed with 3 volumes of buffer B, and the proteins were eluted with a linear gradient (20 ml) of buffer B to buffer B containing 400 mm K·PO4. The TWINKLE protein eluted at a concentration of ∼300 mm K·PO4. The peak fractions were diluted with 2 volumes of buffer C (20 mm Tris-HCl, pH 7.7, 10% glycerol, 1 mm dithiothreitol, and 0.5 mm EDTA, pH 8.0) and loaded on a 1-ml HiTrap SP column (Amersham Biosciences), which had been equilibrated with buffer C containing 150 mm NaCl. The TWINKLE protein eluted in the flow-through. The flow-through was collected and loaded on a 1-ml HiTrap heparin-Sepharose column (Amersham Biosciences), which had been equilibrated with buffer C with 150 mm NaCl. The column was washed with 3 volumes of buffer C with 0.3 m NaCl. The peak fractions were collected and eluted with a linear gradient (6 ml) of buffer C with 0.3–1 m NaCl. The TWINKLE protein eluted ∼700 mm NaCl. The peak fractions were collected and dialyzed against buffer C with 0.2 m NaCl, frozen in liquid nitrogen, and finally stored at –80 °C. The yield of TWINKLE protein was ∼2 mg from a 400-ml culture. The purity of the protein was at least 95% as estimated by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining (Fig. 1A).The mitochondrial SSB was purified by dialyzing the clarified Sf9 lysate against buffer C with 0.1 m NaCl. The lysate was loaded onto a 5-ml CM Superose column (Amersham Biosciences), which had been equilibrated with buffer C with 0.1 m NaCl. MtSSB eluted in the flow-through was collected and loaded onto a 5-ml HiTrap heparin-Sepharose column (Amersham Biosciences), which had been equilibrated with buffer C (0.1 m NaCl). MtSSB was eluted with a linear gradient (50 ml) of buffer C (0.1–1 m NaCl), and the peak fractions of mtSSB were found at ∼300 mm NaCl. The mtSSB fractions were collected and loaded on a 1-ml hydroxyapatite column, which had been equilibrated with buffer B (10 mm K·PO4). The column was washed by 3 volumes of equilibration buffer, and mtSSB was eluted with a linear gradient (10 ml) of buffer B (10–400 mm K·PO4). MtSSB, eluted at ∼100 mm K·PO4, and the peak fractions were collected and dialyzed against buffer C (0.2 m NaCl), frozen in liquid nitrogen, and stored at –80 °C. The yield of mtSSB was ∼20 mg from a 400-ml starting culture, and the purity was at least 95% as judged by SDS-PAGE and Coomassie Brilliant Blue staining (see Fig. 5A).Fig. 5The TWINKLE helicase is specifically stimulated by mtSSB. In A, mtSSB (2 μg) purified over hydroxyapatite was separated by SDS-PAGE (14–20%) and revealed with Coomassie Brilliant Blue staining. The faint smear below the protein is an artifact in the SDS-PAGE analysis. The protein migrated as one single peak in mass determination with MALDI-TOF mass spectrometry. In B, DNA helicase assays were performed as described under "Material and Methods" using the MG1/M13mp18 helicase substrate. The reactions contained 60 nm TWINKLE protein when indicated and increasing amounts of mtSSB and E. coli SSB (EcSSB). The reactions were incubated for 30 min. The unwinding reaction was unaffected by EcSSB but stimulated 2.6- (lane 8) and 2.3-fold (lane 9) with mtSSB. Lane 1, substrate heated to 100 °C before loading; lane 2, untreated substrate; lane 4, 475 nm mtSSB; lane 5, 475 nmE.Coli SSB; lane 7, 47.5 nm mtSSB; lane 8, 133 nm mtSSB; lane 9, 475 nm mtSSB; lane 11, 47.5 nmE.Coli SSB; lane 12, 133 nmE.Coli SSB; lane 13, 475 nmE.Coli SSB. S, double-stranded substrate; P, single-stranded product.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Helicase Substrates—A 60-nt oligonucleotide (MG1, 5′-ACATGATAAGATACATGGATGAGTTTGGACAAACCACAACGTAAAACGACGGCCAGTGCC-3′) was labeled with 32P at its 5′-terminus with T4 polynucleotide kinase (Stratagene) and annealed to M13mp18 single-stranded DNA (Amersham Biosciences) to generate a 20-bp double-stranded region with a 40-nucleotide 5′-tail. The annealed DNA was purified from unannealed oligonucleotide by Centricon 100 (Amicon) using a buffer containing 20 mm Tris-HCl (pH 7.6), 100 mm NaCl, and 0.1 mm EDTA. The helicase substrate used to decide the tail length necessary for unwinding was made by annealing a 60-nt-long oligonucleotide (MG2, 5′-GCCCTGATCACGGTACTCGGTTTTTTTTTTTTTTTTTTTTGGCTCCTCTAGACTCGACCG-3′) to three different oligonucleotides labeled with 32P at their 5′-termini. This procedure gave rise to three different substrates containing a common 40-nt 5′-tail and no 3′-tail (MG3, 5′-CGGTCGAGTCTAGAGGAGCC-3′), a 10-nt 3′-tail (MG4, 5′-CGGTCGAGTCTAGAGGAGCCTTTTTTTTTT-3′), or a 15-nt 3′-tail (MG5, 5′-CGGTCGAGTCTAGAGGAGCCTTTTTTTTTTTTTTT-3′) (see Fig. 3B). The helicase substrates used in the directionality assays were made by annealing the radiolabeled MG2 oligonucleotide to either of two different 60-nt-long oligonucleotides (MG6, 5′-CGGTCGAGTCTAGAGGAGCCCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTCAG-3′, or MG7, 5′-CTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTCAGCCGAGTACCGTGATCAGGGC-3′). The MG1/MG6 and the MG1/MG7 annealing reactions also included an additional 40-nt oligonucleotide (MG8, 5′-CTGACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAG-3′) to generate the two different fork substrates used in the directionality assays (see Fig.4, A and B). DNA helicase substrates were purified by polyacrylamide gel electrophoresis as described (14.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Vol. 1. Section 6.46–6.47, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar).Fig. 3Co-factor and substrate requirements for the TWINKLE helicase. In A, helicase assays were performed as described under "Materials and Methods" in the presence of 15 fmol of substrate and 30 nm TWINKLE protein. The ATP concentration was kept constant at 5 mm, and increasing amounts of ATPγS were added. Lane 1, substrate heated to 100 °C before loading; lane 2, untreated substrate; lane 3, no ATPγS; lane 4, 50 μm ATPγS; lane 5, 100 μm ATPγS; lane 6, 500 μm ATPγS. S, double-stranded substrate; P, single-stranded product. In B, helicase assays were performed as described under "Materials and Methods" except that the ATP was changed to indicated nucleoside 5′-triphosphates (3 mm). Reactions were incubated for 40 min in the presence of 15 fmol of substrate and 20 nm TWINKLE protein. C, helicase assays with varying lengths of the 3′-tail. An increasing amount of TWINKLE protein was added to 20 fmol of template and incubated for 30 min. A schematic representation of the templates used is included under the figure. Lanes 1, 6, and 11, untreated substrates; lanes 2, 7, and 12, substrates heated to 100 °C before loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Unwinding directionality assay for the TWINKLE protein. The assays were performed as described under "Materials and Methods." Increasing amounts of TWINKLE protein was added to 20 fmol of template and incubated for 40 min. As shown in A, TWINKLE cannot unwind the double-stranded template with a 3′-single-stranded tail. S, double-stranded substrate; P, single-stranded product. As shown in B, TWINKLE can efficiently unwind a double-stranded template containing a 5′-single-stranded tail. Lane 1, untreated substrate; lane 2, substrate heated to 100 °C before loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT)ATPase Assay—ATPase assays were performed in 20 μl of 20 mm Tris·HCl (pH 7.8), 10 mm NaCl, 1.5 mm MgCl2, 17.4% glycerol, 0.3 mg/ml bovine serum albumin, 0.7 mm ATP, 250–300 nCi of [γ32P]ATP (Amersham Biosciences), and 10 μg/ml activated calf thymus DNA. TWINKLE was added as indicated in the figure legends. Incubation was for 60 min at 37 °C. The reaction was stopped by the addition of 400 μl of a suspension of Norit A (12% in 0.1 m HCl, 10 mm K·PO4). The mixture was briefly vortexed and then centrifuged for 3 min at 7,000 × g. Two hundred μl of the supernatant was mixed in 3 ml of scintillation mixture (Ready Safe; Beckman-Coulter), and the radioactivity was measured in a liquid scintillation counter. The enzyme-dependent release of phosphate was calculated by subtracting the release of phosphate in samples without enzyme.Helicase Assay—The reaction mixture (15 μl) contained 15 fmol of DNA substrate (DNA concentrations in this report are expressed in moles of molecules), 20 mm Tris-HCl (pH 7.6), 10% glycerol, 10 mm dithiothreitol, 4.5 mm MgCl2, 3 mm ATP, 100 μg/ml bovine serum albumin, 40 mm NaCl, and the indicated amounts of TWINKLE, mtSSB, and Escherichia coli SSB. The reactions were incubated at 32 °C for the times indicated and stopped by the addition of 2 μl of stop solution (90 mm EDTA (pH 8.0), 6% SDS, 30% glycerol, 0.25% bromphenol, 0.25% xylene cyanol). We did not observe any significant levels of spontaneous reannealing of unwound DNA at the assay conditions used. The products were separated by electrophoresis through a 15% non-denaturing polyacrylamide gel, which was dried onto DE81 (Whatman) and autoradiographed overnight at –80 °C with an intensifying screen. Intensities of the bands were quantified by densitometry using the program NIH Image (rsb.info.nih.gov/nih-image/).RESULTSThe finding that TWINKLE mutations are associated with mtDNA deletions suggested that the protein might be involved in mtDNA replication, and we therefore characterized the enzymatic activities of the TWINKLE in vitro. We generated a recombinant baculovirus encoding the human TWINKLE gene to obtain sufficient quantities of the protein for studies of its associated biochemical activities. TWINKLE was expressed in insect cells and purified over Ni2+-agarose, hydroxyapatite, SP-Sepharose, and heparin-Sepharose to near homogeneity (Fig. 1A). Recombinant TWINKLE migrated as a doublet with an apparent molecular mass of about 70 kDa during SDS-polyacrylamide gel electrophoresis, corresponding to the predicted molecular mass of 72 kDa. The weaker, lower band is a shorter form of the protein, lacking 15 amino acids at the very C terminus as demonstrated by mass analysis with MALDI-TOF mass spectrometry (data not shown). The truncation is probably due to translational pausing since the relative levels of the two forms are unaffected by protease inhibitors. A strong contaminating exonuclease activity co-purified with TWINKLE over the first two columns, but it was lost at the SP-Sepharose step. Heparin column-purified TWINKLE co-migrated with a strong ATPase activity (Fig. 1B), supporting primary sequence analysis predictions that TWINKLE is a Walker-type ATPase.TWINKLE Is a DNA Helicase—To investigate whether TWINKLE is an active DNA helicase in vitro, we annealed a 32P-labeled 60-nt oligonucleotide to the complementary region of M13mp18 single-stranded DNA to form a helicase substrate with a 20-bp double-stranded region and a 40-nt 5′-single-stranded tail. Examination of the purified protein showed that it indeed possessed a strong DNA unwinding activity, which coincided perfectly with the peak of TWINKLE protein eluting from heparin-Sepharose (Fig. 1C). The helicase activity was not found when mock-infected insect cell extracts were purified in a similar way (data not shown). We next analyzed DNA unwinding as a function of TWINKLE protein concentration and found that addition of increasing concentrations of protein to our helicase substrate revealed a linear increase of displaced oligonucleotide (Fig. 2, A and B). Extensive DNA unwinding (>25%) was observed at protein concentrations above 30 nm of the protein, demonstrating that TWINKLE is a potent helicase in vitro. A time course experiment (Fig. 2C) demonstrated that TWINKLE initiates unwinding of the substrate without any apparent lag phase. The unwinding reaction is dependent on NTP hydrolysis and was inhibited at low concentrations of the non-hydrolyzable ATP analogue, ATPγS (Fig. 3A), TWINKLE was next incubated with a variety of different nucleoside 5′-triphosphates to analyze their ability to act as co-effectors for the helicase activity (Fig. 3B). UTP efficiently supported DNA unwinding, and to a lesser extent, so did ATP, GTP, and dTTP. CTP was a very poor co-effector.Fig. 2Helicase activity of TWINKLE. Assays were performed as described under "Materials and Methods" in the presence of the indicated amounts of TWINKLE. A, increasing amounts of TWINKLE incubated for 30 min. In B, helicase reactions were performed as in panel A and then analyzed with phosphorimaging, and the fractional amounts of base-paired substrate and single-stranded DNA product were determined. Each data point is the average of at least three experiments. Error bars represent the S.D. C, time course of DNA unwinding reactions. A reaction mixture (150 μl) was prepared in the presence of 30 nm TWINKLE. At the times indicated, 15-μl aliquots were removed and analyzed by 15% non-denaturing polyacrylamide gel electrophoresis. The gel was scanned and quantified as in panel B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TWINKLE Has 5′ → 3′ Helicase Directionality—We next investigated the efficiency by which TWINKLE could unwind different DNA substrates. To this end, we made a series of constructs with a 60-nt-long oligonucleotide annealed to complementary oligonucleotides to form helicase substrates with a 20-bp double-stranded region, a 40-nt single-stranded 5′-tail, and 3′-tails of varying lengths (0, 10, 15 nt). No unwinding was observed with substrate lacking a 3′-tail, low levels of unwinding were observed with the 10-nt 3′-tail, whereas the 15-nt 3′-tail template was efficiently unwound (Fig. 3C). Similar experiments were also performed with a 40-nt 3′-tail substrate and varying lengths of the 5′-tail (0, 10, 15 nt). We found that unwinding was dependent on a free 5′-tail as well. No unwinding was observed on the template lacking a 5′-tail. TWINKLE displayed a moderate activity on the 10-nt 5′-tail substrate and efficiently unwound the 15-nt 5′-tailed template (data not shown).TWINKLE needs a fork-like structure with both a 5′- and a 3′-single-stranded stretch of DNA to efficiently initiate DNA unwinding, similar to what had been shown previously for the T7 gp4 helicase (15.Matson S.W. Richardson C.C. J. Biol. Chem. 1983; 258: 14009-14016Abstract Full Text PDF PubMed Google Scholar). We could not use common DNA substrates for a directionality assay, due to this specific substrate requirement. Specific DNA substrates have been developed to circumvent this problem for the T7 gp4 protein. We used a directionality assay with substrates containing a 20-bp double-stranded region with one single-stranded and one double-stranded tail (Fig. 4, A and B) (16.Ahnert P. Patel S.S. J. Biol. Chem. 1997; 272: 32267-32273Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). TWINKLE requires a single-stranded stretch of DNA to initiate unwinding, and we could therefore determine the directionality of the enzyme by introducing a single-stranded 5′- or 3′-tail. We found that TWINKLE could only unwind the substrate with a 5′-single-stranded tail, thus demonstrating that TWINKLE unwinds DNA in the 5′ to 3′ direction, as does the T7 gp4 helicase.The TWINKLE Helicase Is Stimulated by mtSSB—We determined whether human mtSSB had any stimulatory effect on the TWINKLE helicase activity by using the same substrate as in Fig. 1C. We expressed recombinant mtSSB in insect cells and purified the protein to near homogeneity (Fig. 5A). We found that mtSSB had a strong stimulatory effect on the unwinding activity of the TWINKLE protein (Fig. 5B). The stimulation by mtSSB was specific because no such effect was observed with the E. coli SSB.DISCUSSIONThe mechanisms of mammalian mtDNA replication have not yet been fully defined (1.Shadel G.S. Clayton D.A. Annu. Rev. Biochem. 1997; 66: 409-435Crossref PubMed Scopus (806) Google Scholar). We have initiated a project aimed at reconstituting mtDNA replication in vitro, which we hope will generate new insights into this fundamental cellular process. According to the generally accepted model, mammalian mtDNA replication is continuous on both strands and takes place in a strand-asymmetric mode. DNA synthesis is initiated from two different sites, one for each strand (17.Clayton D.A. Cell. 1982; 28: 693-705Abstract Full Text PDF PubMed Scopus (915) Google Scholar). Activation of oriL and the heavy strand origin oriH are physically and temporally distinct. DNA synthesis first commences at oriH, which is localized in the non-coding region of mtDNA. After leading strand synthesis has reached two-thirds of the genome, it comes to oriL, which is activated, and the DNA synthesis then initiates in the opposite direction. Recently, however, this strand-asymmetric model for mtDNA replication has been challenged by two-dimensional gel electrophoresis analysis demonstrating the presence of conventional duplex mtDNA replication intermediates, indicative of coupled leading and lagging-strand DNA synthesis (18.Holt I.J. Lorimer H.E. Jacobs H.T. Cell. 2000; 100: 515-524Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 19.Yang M.Y. Bowmaker M. Reyes A. Vergani L. Angeli P. Gringeri E. Jacobs H.T. Holt I.J. Cell. 2002; 111: 495-505Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). A detailed biochemical analysis of these processes in vitro may help to clarify the molecular mechanisms of mtDNA replicationWe demonstrate here that the mammalian TWINKLE protein displays the classical features of a DNA helicase: it catalyzes the ATP-dependent unwinding of a DNA dupl
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