Physiological and biochemical defects in carboxyl-terminal mutants of mitochondrial DNA helicase

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.

FIGURE 1.

Sequence alignment and location of the deletion and amino acid substitution mutations in the carboxyl-terminal region of Drosophila mtDNA helicase. Schematic diagram of the sequence organization of the d-mtDNA helicase; five amino acid sequence motifs common to ring helicases are indicated in gray, and the bacteriophage T7 gp4 linker region is indicated in black. The deletion mutations in d-mtDNA helicase used in this study are designated below the schematic diagram along with the sequence alignments of the regions containing altered amino acids. Dm, mitochondrial DNA helicase of the fly; Ag, mosquito; Xl, frog; Hs, humans; DnaB, Thermus aquaticus DnaB helicase; T7, bacteriophage T7 gp4. Altered and conserved amino acids are indicated in gray. The positions of the deletion mutants and alanine substitutions are shown above the alignment as asterisks and A, respectively. The positions of the putative arginine finger and base stack are indicated below the alignment.

FIGURE 2.

Overexpression of Drosophila mtDNA helicase and deletion mutants in Schneider cells. A, Schneider cells containing no plasmid (control), carrying pMt/Hy (vector), pMt/WT/Hy (WT), pMt/Δ(607-613)/Hy (Δ(607-613)), pMt/Δ(597-613)/Hy (Δ(597-613)), pMt/Δ(586-613)/Hy (Δ(586-613)), or pMt/Δ(572-613)/Hy (Δ(572-613)), were cultured for 14 days in the presence of 0.2 mm CuSO₄. 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, effect of overexpression of the deletion mutants on mtDNA copy number. Upper panel, total mtDNA (10 μg) was extracted from Schneider cells (control) or Schneider cells carrying pMt/Hy (vector), pMt/WT/Hy (WT), pMt/Δ(607-613)/Hy (Δ(607-613)), pMt/Δ(597-613)/Hy (Δ(597-613)), pMt/Δ(586-613)/Hy (Δ(586-613)), or pMt/Δ(572-613)/Hy (Δ(572-613)) that were cultured for 14 days in the presence of 0.2 mm CuSO_4. 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 (designated as mtDNA). Lower panel, relative mtDNA copy number was quantitated as described under “Experimental Procedures.”

FIGURE 3.

Intramolecular cross-linking of Drosophila mtDNA helicase and its variants. Schneider cells carrying overexpressing constructs were treated with 1% formaldehyde for 5 min, and the reaction was quenched with glycine. Cell extracts were fractionated by 6% SDS-PAGE, transferred to nitrocellulose filters, and probed with affinity-purified rabbit antiserum against d-mtDNA helicase as above. A, lane 1, Schneider cells containing no plasmid (control); lane 2, wild type (WT); lane 3, Δ(607-613); lane 4, Δ(597-613). B, lane 1, Schneider cells containing no plasmid (control); lanes 2-10, wild type and mutant helicase as indicated above the lanes.

FIGURE 4.

Expression 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.”

FIGURE 5.

ATPase activities of human mtDNA helicase and its variants. ATP hydrolysis was measured in the presence of the indicated amounts of wild type mtDNA helicase and mutant forms. Open circles, wild type helicase; closed circles, R609A; open triangles, F621A; closed triangles, F628A.

FIGURE 6.

Homology 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 (22)). The figure was made using Pymol.