Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase.
The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.
Matsushima
Yuichi
Y
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing MI 48824-1319, USA.
Kaguni
Laurie S
LS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D015139:Blotting, Southern; D017124:Conserved Sequence; D009033:Culicidae / Q000201:enzymology / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics*; D004261:DNA Replication; D004272:DNA, Mitochondrial / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004175:Diptera / Q000201:enzymology / Q000235:genetics; D004330:Drosophila / Q000201:enzymology / Q000235:genetics; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics; D014644:Genetic Variation; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D004272:DNA, Mitochondrial; D004279:DNA, Viral; D029721:Drosophila Proteins; D004265:DNA Helicases