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• W2016370670 abstract "Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and 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. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general. Mitochondrial DNA helicase, also called Twinkle, is essential for mtDNA maintenance. Its helicase domain shares high homology with helicases from superfamily 4. Structural analyses of helicases from this family indicate that carboxyl-terminal residues contribute to NTP hydrolysis required for translocation and DNA unwinding, yet genetic and biochemical information is very limited. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying a series of deletion and alanine substitution mutations in the carboxyl terminus and 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. Likewise, amino acid substitution mutants K574A, R576A, Y577A, F588A, and F595A show dose-dependent dominant-negative phenotypes. Arg-576 and Phe-588 are analogous to the arginine finger and base stack of other helicases, including the bacteriophage T7 gene 4 protein and bacterial DnaB helicase, respectively. We show here that representative human recombinant proteins that are analogous to the alanine substitution mutants exhibit defects in nucleotide hydrolysis. Our findings may be applicable to understand the role of the carboxyl-terminal region in superfamily 4 DNA helicases in general. The mitochondrial DNA (mtDNA) 2The abbreviations used are:mtDNAmitochondrial DNAd-mtDNADrosophila mtDNASF4superfamily 4T7 gp4bacteriophage T7 gene 4 proteinPBSphosphate-buffered salineNTPasenucleoside-triphosphate hydrolase. helicase, also known as Twinkle, was identified as one of the causative genes for autosomal dominant progressive external ophthalmoplegia (1Spelbrink 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 (691) Google Scholar). Mutations of the mtDNA helicase have been reported in patients with multiple mtDNA deletions or depletions (1Spelbrink 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 (691) Google Scholar, 2Sarzi E. Goffart S. Serre V. Chretien D. Slama A. Munnich A. Spelbrink J.N. Rotig A. Ann. Neurol. 2007; 62: 579-587Crossref PubMed Scopus (150) Google Scholar, 3Hakonen A.H. Isohanni P. Paetau A. Herva R. Suomalainen A. Lonnqvist T. Brain. 2007; 130: 3032-3040Crossref PubMed Scopus (173) Google Scholar, 4Copeland W.C. Annu. Rev. Med. 2007; 59: 131-146Crossref Scopus (229) Google Scholar, 5Spinazzola A. Zeviani M. Gene (Amst.). 2005; 354: 162-168Crossref PubMed Scopus (97) Google Scholar, 6Kaguni L.S. Annu. Rev. Biochem. 2004; 73: 293-320Crossref PubMed Scopus (331) Google Scholar, 7Suomalainen A. Majander A. Wallin M. Setala K. Kontula K. Leinonen H. Salmi T. Paetau A. Haltia M. Valanne L. Lonnqvist J. Peltonen L. Somer H. Neurology. 1997; 48: 1244-1253Crossref PubMed Scopus (134) Google Scholar, 8Suomalainen A. Majander A. Haltia M. Somer H. Lonnqvist J. Savontaus M.L. Peltonen L. J. Clin. Investig. 1992; 90: 61-66Crossref PubMed Scopus (214) Google Scholar, 9Zeviani M. Servidei S. Gellera C. Bertini E. DiMauro S. DiDonato S. Nature. 1989; 339: 309-311Crossref PubMed Scopus (550) Google Scholar). The helicase domain of the enzyme, located in the carboxyl-terminal region, shares high homology with helicases from superfamily 4 (SF4), which includes bacteriophage T7 gene 4 protein (T7 gp4) and Escherichia coli DnaB protein. These enzymes catalyze DNA helix unwinding, translocating 5′-3′ on single-stranded DNA by utilizing the energy of nucleotide hydrolysis (10Singleton M.R. Dillingham M.S. Wigley D.B. Annu. Rev. Biochem. 2007; 76: 23-50Crossref PubMed Scopus (958) Google Scholar, 11Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (462) Google Scholar). Consistent with other ring-shaped SF4 enzymes, the mtDNA helicase forms a hexamer (1Spelbrink 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 (691) Google Scholar, 12Ziebarth T.D. Farr C.L. Kaguni L.S. J. Mol. Biol. 2007; 367: 1382-1391Crossref PubMed Scopus (33) Google Scholar, 13Matsushima Y. Kaguni L.S. J. Biol. Chem. 2007; 282: 9436-9444Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Farge G. Holmlund T. Khvorostova J. Rofougaran R. Hofer A. Falkenberg M. Nucleic Acids Res. 2008; 36: 393-403Crossref PubMed Scopus (53) Google Scholar). mitochondrial DNA Drosophila mtDNA superfamily 4 bacteriophage T7 gene 4 protein phosphate-buffered saline nucleoside-triphosphate hydrolase. The SF4 helicases share five conserved sequence motifs, H1, H1a, H2, H3, and H4. H1 and H2 are equivalent to the Walker A and Walker B motifs found in all AAA+ ATPases (10Singleton M.R. Dillingham M.S. Wigley D.B. Annu. Rev. Biochem. 2007; 76: 23-50Crossref PubMed Scopus (958) Google Scholar, 11Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (462) Google Scholar). The H4 motif contributes to DNA binding, whereas the remaining four play a role in NTP binding and hydrolysis. Additionally, individual amino acids, termed the arginine finger and base stack, have been shown to serve specific roles (15Donmez I. Patel S.S. Nucleic Acids Res. 2006; 34: 4216-4224Crossref PubMed Scopus (76) Google Scholar, 16Crampton D.J. Mukherjee S. Richardson C.C. Mol. Cell. 2006; 21: 165-174Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 17Crampton D.J. Guo S. Johnson D.E. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4373-4378Crossref PubMed Scopus (46) Google Scholar, 18Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar, 19Sawaya M.R. Guo S. Tabor S. Richardson C.C. Ellenberger T. Cell. 1999; 99: 167-177Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 20Wang G. Klein M.G. Tokonzaba E. Zhang Y. Holden L.G. Chen X.S. Nat. Struct. Mol. Biol. 2008; 15: 94-100Crossref PubMed Scopus (60) Google Scholar, 21Bailey S. Eliason W.K. Steitz T.A. Science. 2007; 318: 459-463Crossref PubMed Scopus (170) Google Scholar, 22Bailey S. Eliason W.K. Steitz T.A. Nucleic Acids Res. 2007; 35: 4728-4736Crossref PubMed Scopus (41) Google Scholar). The arginine finger of one subunit interacts with the phosphate of the nucleotide bound to a neighboring subunit, stabilizing the transition state of the reaction (10Singleton M.R. Dillingham M.S. Wigley D.B. Annu. Rev. Biochem. 2007; 76: 23-50Crossref PubMed Scopus (958) Google Scholar, 11Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (462) Google Scholar), whereas the amino acid functioning as the base stack contacts the base of the nucleotide bound on the same subunit. Furthermore, the extreme carboxyl-terminal region of many SF4 helicases has been shown to be required for interaction with other replication factors. Carboxyl-terminal residues of T7 gp4 interact directly with T7 DNA polymerase and thioredoxin, and the last three carboxyl-terminal residues in the RSF1010 plasmid-encoded RepA helicase are required for replication in vivo (23Hamdan S.M. Johnson D.E. Tanner N.A. Lee J.B. Qimron U. Tabor S. van Oijen A.M. Richardson C.C. Mol. Cell. 2007; 27: 539-549Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 24Lee S.J. Marintcheva B. Hamdan S.M. Richardson C.C. J. Biol. Chem. 2006; 281: 25841-25849Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 25Hamdan S.M. Marintcheva B. Cook T. Lee S.J. Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5096-5101Crossref PubMed Scopus (77) Google Scholar, 26Ziegelin G. Niedenzu T. Lurz R. Saenger W. Lanka E. Nucleic Acids Res. 2003; 31: 5917-5929Crossref PubMed Scopus (23) Google Scholar, 27Kong D. Richardson C.C. J. Biol. Chem. 1998; 273: 6556-6564Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 28Notarnicola S.M. Mulcahy H.L. Lee J. Richardson C.C. J. Biol. Chem. 1997; 272: 18425-18433Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). We have shown that the Drosophila (d-) mtDNA helicase is essential for mtDNA maintenance in vivo, and overexpression of protein variants carrying mutations in the H1 or H2 motifs or those with amino acid substitutions equivalent to human autosomal dominant progressive external ophthalmoplegia mutations results in the depletion of mtDNA in cultured cells (13Matsushima Y. Kaguni L.S. J. Biol. Chem. 2007; 282: 9436-9444Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Recently, similar results were obtained with human cultured cells (29Wanrooij S. Goffart S. Pohjoismaki J.L. Yasukawa T. Spelbrink J.N. Nucleic Acids Res. 2007; 35: 3238-3251Crossref PubMed Scopus (113) Google Scholar). Here, we extend our study by investigating the importance of the carboxyl-terminal region. We evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in the carboxyl-terminal region and characterize biochemically several recombinant human helicases carrying amino acid substitutions that are shown to cause dominant negative phenotypes in vivo. Generation and Induction of Stable Cell Lines—Drosophila Schneider S2 cells were cultured at 25 °C in Drosophila Schneider Medium (Invitrogen) supplemented with 10% fetal bovine serum. Cells were subcultured to 5 × 106 cells/ml every third day. Cells were transfected using Effectene (Qiagen). Hygromycin-resistant cells were selected with 200 μg/ml hygromycin. Cells were passed at least five times in hygromycin-containing medium and then cultured in standard medium. The cell lines were grown to a density of 3 × 106/ml and then treated with 0.2 mm CuSO4 to induce high level expression from the metallothionein promoter. Immunoblotting—Total cellular protein (20 μg/lane) was fractionated by SDS-PAGE in 9% gels and transferred to nitrocellulose filters. Filters were preincubated for 1 h with 5% skim milk in phosphate-buffered saline (PBS) followed by incubation for 1 h with d-mtDNA helicase antibody (1:20 ml in PBS containing 0.1% Tween 20). Filters were washed 4 times with PBS containing 0.1% Tween 20, incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Bio-Rad), and washed with PBS containing 0.1% Tween 20. Protein bands were visualized using ECL Western blotting reagents (Amersham Biosciences). Rabbit polyclonal antibody against the Drosophila ATPase β subunit was provided by Rafael Garesse (Universidad Autonoma de Madrid-Consejo Superior de Investigaciones Científicas, Madrid). Protein Cross-linking Analysis—1 × 108 cells that were induced with 0.2 mm CuSO4 for 7 days were washed twice with PBS and resuspended in 200 μl cross-linking buffer (PBS containing 1% formaldehyde). After incubation for 5 min at room temperature, 200 μl of quenching solution (PBS containing 250 mm glycine, 10 mm EDTA, and 4% SDS) was added, and the samples were mixed thoroughly. 20 μl of the protein solution was fractionated by 6% SDS-PAGE and transferred to nitrocellulose filters. Immunoblot analysis was performed as described above. Southern Blotting—Genomic DNA was purified from Drosophila Schneider S2 cells by standard methods. DNA (10 μg/lane) was cleaved with XhoI, fractionated on a 0.7% agarose/Tris-buffered EDTA gel, and transferred to Hybond-N+ nylon membrane (Amersham Biosciences). Hybridization was performed as described previously (31Matsushima Y. Adan C. Garesse R. Kaguni L.S. J. Biol. Chem. 2005; 280: 16815-16820Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 32Matsushima Y. Garesse R. Kaguni L.S. J. Biol. Chem. 2004; 279: 26900-26905Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Filters were washed 3 times for 10 min at room temperature with 2× SSC containing 0.1% SDS, once for 30 min at 65 °C with 0.2× SSC containing 0.1% SDS, and then analyzed with a PhosphorImager. Blots were probed with radiolabeled DNAs for the mitochondrial gene Cytb and the nuclear histone gene cluster. The ratio of the signals for these two genes was used to determine the relative copy number of mtDNA. The Southern blot experiments shown in Figs. 2 and 4 were performed twice with each of the two independent cell lines carrying each plasmid construct, including the control (no plasmid), vector only, wild type, and each of the mutant d-mtDNA helicases. The data presented represent one such experiment, and quantitation is provided for the duplicate experiments from one of the two cell lines. All of the comparable data for each construct vary by less than 15%.FIGURE 4Expression in Schneider cells of Drosophila mtDNA helicases carrying mutations in the carboxyl-terminal region. Schneider cells containing no plasmid (control) or carrying pMt/WT/Hy (WT), pMt/K576A/Hy (K576A), pMt/R576A/Hy (R576A), pMt/Y577A/Hy (Y577A), pMt/D580A/Hy (D580A), pMt/E587A/Hy (E587A), pMt/F588A/Hy (F588A), pMt/K590A/Hy (K590A), or pMt/Y595A/Hy (Y595A) were cultured for 14 days in the absence or presence of 0.2 mm CuSO4. A, protein extracts (20 μg) were fractionated by 9% SDS-PAGE, transferred to nitrocellulose filters, and probed with affinity-purified rabbit antiserum against d-mtDNA helicase or antiserum against d-ATPase β as indicated. B, total mtDNA (10 μg) was extracted from Schneider cells (control) or Schneider cells carrying pMt/K576A/Hy (K576A), pMt/R576A/Hy (R576A), pMt/Y577A/Hy (Y577A), pMt/D580A/Hy (D580A), pMt/E587A/Hy (E587A), pMt/F588A/Hy (F588A), pMt/K590A/Hy (K590A), or pMt/Y595A/Hy (Y595A) were cultured for 14 days in the presence of 0.2 mm CuSO4. DNA was digested with XhoI, fractionated in a 0.7% agarose/Tris-buffered EDTA gel, and then blotted to a nylon membrane. The membrane was hybridized with a radiolabeled probe for the histone gene cluster (his genes) and then stripped and re-hybridized with radiolabeled probe for CytB (mtDNA). Lower panel, relative mtDNA copy number was quantitated as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) Preparation of an Inducible Plasmid Expressing d-mtDNA Helicase Variants—The construction of the plasmid pMt/WT/Hy was performed as described previously (13Matsushima Y. Kaguni L.S. J. Biol. Chem. 2007; 282: 9436-9444Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The expression vectors carrying mutant d-mtDNA helicases were prepared by QuikChange mutagenesis or PCR with Pfu DNA polymerase. A typical PCR was carried out in a 50-μl reaction mixture with 50 ng of pMt/WT/Hy and 2 units of Pfu DNA polymerase. A specific primer pair was used for each mutant as follows: Δ(607-613), 5′-gggctcgagAATCTTAAGAAATGGAAAATGAGACGCGCCGGTTTAATC-3′ and 5′-gcgcactagtTCACCGTTTTCGCTTGGCATTTTG-3′; Δ(597-613), 5′-CTTGAGCTACTCAtaaCAGATTCAAAATG-3′ and 5′-CATTTTGAATCTGttaTGAGTAGCTCAAG-3′; Δ(586-613), 5′-gcgcactagtTCACCGTTTTCGCTTGGCATTTTG-3′ and 5′-GcgcactagtTCATGGCATGATGCCCAGGTCGC-3′; Δ(572-613), 5′-gcgcactagtTCACCGTTTTCGCTTGGCATTTTG-3′ and 5′-GcgcactagtTCATTGGAGATACTTCTTGCCCC-3′; K574A, 5′-CTCCAAATTGCTgcGAATCGTTATTCCG-3′ and 5′-CGGAATAACGATTCgcAGCAATTTGGAG-3′; R576A, 5′-CAAATTGCTAAGAATgcTTATTCCGGCGAC-3′ and 5′-GTCGCCGGAATAAgcATTCTTAGCAATTTG-3′; Y577A, 5′-GCTAAGAATCGTgcTTCCGGCGACCTG-3′ and 5′-CAGGTCGCCGGAAgcACGATTCTTAGC-3′; D580A, 5′-CGTTATTCCGGCGcCCTGGGCATCATG-3′ and 5′-CATGATGCCCAGGgCGCCGGAATAACG-3′; E587A, 5′-TCATGCCACTGGcATTCGACAAAGA-3′ and 5′-TCTTTGTCGAATgCCAGTGGCATGA-3′; F588A, 5′-CATGCCACTGGAAgcCGACAAAGACGG-3′ and 5′-CCGTCTTTGTCGgcTTCCAGTGGCATG-3′; K590A, 5′-CTGGAATTCGACgcAGACGGCTTGAG-3′ and 5′-CTCAAGCCGTCTgcGTCGAATTCCAG-3′; Y595A, 5′-GACGGCTTGAGCgcCTCAACCCAGAT-3′ and 5′-ATCTGGGTTGAGgcGCTCAAGCCGTC-3′. Protein Overexpression and Purification of the Human mtDNA Helicase and Its Variants—Amino-terminal hexahistidine-tagged human mtDNA helicase and mutants, R609A, F621A, and F628A were produced by overexpression in Spodoptera frugiperda cells and purified to homogeneity as described by Ziebarth et al. (12Ziebarth T.D. Farr C.L. Kaguni L.S. J. Mol. Biol. 2007; 367: 1382-1391Crossref PubMed Scopus (33) Google Scholar). ATPase Assay—Reaction mixtures (20 μl) contained 20 mm Tris-HCl, pH 7.5, 4 mm MgCl2, 0.1 mg/ml bovine serum albumin, 10% glycerol, 0.5 mm ATP, 10 mm dithiothreitol, 4 μCi of [α-32P]dATP, 100 μm DNase I-activated calf thymus DNA, and amino-terminal His-tagged full-length mtDNA helicase, or the R609A, F621A, or F628A mutants. Incubation was for 15 min at 37 °C. The reactions were stopped by placing the reaction tubes on ice. Aliquots (2 μl) were spotted onto Polygram polyethyleneimine cellulose paper with 0.5 μl of 50 mm ADP/ATP. The Polygram polyethyleneimine cellulose paper was developed in 1 m formic acid, 0.5 m LiCl. The position of both ADP and ATP bands was visualized by ultraviolet light. The corresponding bands were isolated, and the radioactivity was measured by liquid scintillation counting. Structural Modeling—Human mtDNA helicase shares significant sequence homology with the T7 gp4 protein within the helicase domain (1Spelbrink 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 (691) Google Scholar). Using the crystal structure of the T7 gp4 helicase (PDB code 1EOJ (18Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar)) as the template, we developed a homology model of the three-dimensional structure of human mtDNA helicase domain (residues 383-634) based on multiple sequence alignment by ClustalW among homologs from humans, mouse, and fly with the T7 gp4 helicase domain (residues 281-549) and in consideration of structural conservation among known crystal structures of DnaB-like helicases (33Niedenzu T. Roleke D. Bains G. Scherzinger E. Saenger W. J. Mol. Biol. 2001; 306: 479-487Crossref PubMed Scopus (87) Google Scholar). The homology model was constructed using Xfit software (34McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2022) Google Scholar). Overexpression of Carboxyl-terminal Deletion Mutants of Drosophila mtDNA Helicase in Schneider Cells—To examine the role of the carboxyl-terminal region of the d-mtDNA helicase, we created four variants of the enzyme lacking 7, 17, 28, or 42 amino acids at the carboxyl terminus (Fig. 1). The constructs were transfected into Schneider cells, and after a 14-day incubation in the presence of 0.2 mm CuSO4, an immunoblot analysis revealed a 10-20-fold increase in the levels of the wild type, Δ(607-613) mutant, and Δ(597-613) mutant relative to the endogenous d-mtDNA helicase level (Fig. 2A). The ATP synthase β subunit probed as a control showed no significant change in expression level. Cell lines expressing the Δ(586-613) and Δ(572-613) mutants showed increases of only 1.5- and 1.3-fold, respectively, relative to the endogenous d-mtDNA helicase level. As a result, we could not isolate high level expression cell lines for these two variants. The effects of overexpression of the d-mtDNA helicase deletion mutants on mtDNA maintenance were examined in cells expressing the mutants. To determine the mtDNA copy number, total cellular DNA was isolated, cleaved with XhoI, and analyzed by Southern blot (see “Experimental Procedures”). Blots were hybridized sequentially with probes for the nuclear histone gene cluster and the mitochondrial gene CytB. Relative mtDNA copy number was determined from the ratio of CytB hybridization to histone gene cluster hybridization. After 14 days of induction, the relative mtDNA copy number was increased 1.2-fold in cells overexpressing the wild type enzyme as compared with the control cells (Fig. 2B). In the case of the overexpression of the Δ(607-613) and Δ(597-613) mutants, the relative mtDNA copy number was increased to 105-120% that of the control cells, values that are comparable with the wild type-overexpressing cells. In contrast, in the cells overexpressing the Δ(586-613) and Δ(572-613) mutants, the relative mtDNA copy number was decreased to 57-65% that of the control despite their very low levels of expression. These results indicate that the region between 572 and 596 is essential for helicase function. d-mtDNA Helicase Deletion Mutants Form Hexamers—To explain the dominant negative effects we observed in the Schneider cells overexpressing the d-mtDNA helicase deletion mutants, we examined the oligomeric state of the overexpressed proteins. To do so, we induced stable cell lines carrying either no construct or the wild type or mutant constructs for 7 days in the presence of 0.2 mm CuSO4. Cells were harvested and treated with formaldehyde to cross-link the proteins as described under “Experimental Procedures.” Immunoblot analysis after native gel electrophoresis of the wild type d-mtDNA helicase revealed monomers, dimers, and hexamers (Fig. 3A, lane 2), whereas the endogenous protein in cells carrying no construct was undetectable under these conditions (lane 1). Analysis of the deletion mutants (lanes 3 and 4) showed that the efficiency of hexamer formation with the Δ(607-613) and Δ(597-613) mutants is nearly equivalent to that of the overexpressed wild type protein. Because of the low level of expression of the Δ(586-613) mutants, we were unable to detect multimeric helicase forms using the formaldehyde cross-linking analysis. However, upon velocity sedimentation of these two mutants from mitochondrial extracts of Schneider cells, we found them to have sedimentation coefficients similar to endogenous mtDNA helicase (data not shown). Thus, we conclude that the dominant-negative phenotype and mtDNA depletion observed in Drosophila Schneider cells expressing these deletion mutants also results from the formation of functionally defective heterohexamers. Overexpression of Carboxyl-terminal Point Mutants of the d-mtDNA Helicase in Schneider Cells—In the carboxyl-terminal region between amino acids 572 and 596 of the d-mtDNA helicase, eight charged or aromatic amino acids, Lys-574, Arg-576, Tyr-577, Asp-580, Glu-587, Phe-588, Lys-590, and Tyr-595, are well conserved between the various mtDNA helicases aligned in Fig. 1. Arg-576 and Phe-588 of the d-mtDNA helicase are analogous to the arginine finger, Arg-522, and base stack, Tyr-535, of T7 gp4, respectively. To examine their physiological significance in the mitochondrial enzyme, we constructed metallothionein-inducible plasmids expressing eight d-mtDNA helicase variants carrying the alanine substitutions K574A, R576A, Y577A, D580A, E587A, F588A, K590A, and Y595A. The constructs were transfected into Schneider cells, and two independent stable cell lines were established for each of them. After 14 days of incubation in the absence or presence of 0.2 mm CuSO4, immunoblot analysis indicated increases in the levels of all of the variants in the range of 2.5-3.5- and 12-20-fold, respectively, relative to the control cell lines (Fig. 4A). At the same time, the ATP synthase β subunit showed no significant change in expression levels. Cross-linking analysis of the mutants as described above showed that all were capable of forming hexamers (Fig. 3B). Next, we evaluated their effects on mtDNA maintenance. In cells overexpressing the putative arginine finger mutant R576A, the relative mtDNA copy number was reduced to 70 and 9% of the control cells (Fig. 4B), correlating with the 3- and 20-fold overexpression of R576A in the absence and presence of induction, respectively. Similarly, overexpression of the putative base stack mutant F588A resulted in a 7-fold decrease in mtDNA copy number upon induction. These data are comparable with those we obtained using cells overexpressing the Walker A and Walker B active-site mutants (13Matsushima Y. Kaguni L.S. J. Biol. Chem. 2007; 282: 9436-9444Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In addition to the putative arginine finger and base stack mutants, we found 20- and 9-fold decreases in relative mtDNA copy number in cells overexpressing the K574A and Y595A variants, respectively, and cells overexpressing Y577A showed a 2-fold decrease relative to the control cells. In contrast, in cells overexpressing D580A, E587A, and K590A, the relative mtDNA copy number was increased to 114-135% that of the control upon induction, values that are comparable with those we obtained upon overexpression of the wild type d-mtDNA helicase. Correspondingly, whereas copper induction had no adverse effects on the growth of cells expressing the Y577A, D580A, E587A, and K590A mutants, cells expressing the K574A, R576A, F588A, and Y595A mutants grew very poorly. After 4 weeks of induction, the mtDNA levels in the latter mutants decreased to an undetectable level and produced a lethal phenotype between 4 to 6 weeks (data not shown). ATPase Activities of the Human mtDNA Helicase and Its Variants—To evaluate biochemically mutants producing dominant negative phenotypes in Drosophila cells, we produced recombinant human proteins carrying alanine substitutions at analogous positions: R609A, F621A, and F628A. Consistent with the dominant negative effects seen in vivo for the Drosophila Arg-576, Phe-588, and Tyr-595 helicase mutants, the human R609A variant lacks ATP hydrolysis activity, and the F621A and F628A mutants exhibit 10- and 4-fold decreases in activity, respectively (Fig. 5). These data indicate that in addition to an arginine finger at amino acid Arg-609 and a base stack at Phe-621, amino acid Phe-628 also serves a role in the nucleotide hydrolysis reaction. Structural modeling of the human mtDNA helicase using the crystal structure of the T7 gp4 helicase as the template ((PDB code 1EOJ (18Singleton M.R. Sawaya M.R. Ellenberger T. Wigley D.B. Cell. 2000; 101: 589-600Abstract Full Text Full Text PDF PubMed Scopus (425) Google Scholar); see “Experimental Procedures”) illustrates that the positions of these amino acids are consistent with their proposed roles in the human protein (Fig. 6).FIGURE 6Homology model of the helicase domain of human mtDNA helicase showing amino acid substitutions in the carboxyl terminus. Upper panel, two adjacent protomers within the hexameric ring are presented in gray ribbons. The five conserved helicase motifs in one protomer are colored in red (H1), yellow (H1a), green (H2), blue (H3), and cyan (H4). The carboxyl-terminal region in which amino acid substitutions were investigated is colored in purple in both. Middle panel, mutations within the carboxyl-terminal region. Amino acid substitutions residues are shown in stick form and are labeled by numbers and single letter code. Dominant negative residues (Lys-607, Arg-609, Phe-610, Phe-621, Phe-628) are shown red, and substituted residues without a phenotype are shown in blue (Asp-613, Glu-620, Lys-623) and yellow (Phe-424). Lower panel, corresponding residues in DnaB helicase (PDB code 2Q6T (22Bailey S. Eliason W.K. Steitz T.A. Nucleic Acids Res. 2007; 35: 4728-4736Crossref PubMed Scopus (41) Google Scholar)). The figure was made using Pymol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The ring-shaped helicases require the energy nucleotide hydrolysis for helicase movement and double-stranded DNA unwinding. Nucleotide binding and hydrolysis requires amino acids within the Walker A and B motifs, with additional contributions from a number of other signature sequences (10Singleton M.R. Dillingham M.S. Wigley D.B. Annu. Rev. Biochem. 2007; 76: 23-50Crossref PubMed Scopus (958) Google Scholar, 11Patel S.S. Picha K.M. Annu. Rev. Biochem. 2000; 69: 651-697Crossref PubMed Scopus (4" @default.
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