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.
Highlights
The superfamily 4 (SF4) helicases share five conserved sequence motifs, H1, H1a, H2, H3, and H4
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 [10, 11], whereas the amino acid functioning as the base stack contacts the base of the nucleotide bound on the same subunit
We have shown that the Drosophila (d-) mitochondrial DNA (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 [13]
Summary
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. 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. Preparation of an Inducible Plasmid Expressing d-mtDNA Helicase Variants—The construction of the plasmid pMt/ WT/Hy was performed as described previously [13]. The expression vectors carrying mutant d-mtDNA helicases were prepared by QuikChange mutagenesis or PCR with Pfu DNA polymerase. ATPase Assay—Reaction mixtures (20 l) 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 Ci of [␣-32P]dATP, 100 M DNase I-activated calf thymus DNA, and amino-terminal His-tagged full-length mtDNA helicase, or the R609A, F621A, or F628A mutants. The homology model was constructed using Xfit software [34]
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