Physiological and biochemical defects in carboxyl-terminal mutants of mitochondrial DNA helicase.
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.
Matsushima
Yuichi
Y
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
Farr
Carol L
CL
Fan
Li
L
Kaguni
Laurie S
LS
D019943:Amino Acid Substitution; D000818:Animals; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D002460:Cell Line; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D006801:Humans; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D024101:Mitochondrial Proteins; D004265:DNA Helicases; C431576:TWNK protein, human