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

Yuichi Matsushima, Carol L. Farr, Li Fan, Laurie S. Kaguni

Research output: Contribution to journalArticle

19 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)23964-23971
Number of pages8
JournalJournal of Biological Chemistry
Volume283
Issue number35
DOIs
Publication statusPublished - Aug 29 2008
Externally publishedYes

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DNA Helicases
Mitochondrial DNA
Substitution reactions
Amino Acids
Alanine
Defects
Hydrolysis
DnaB Helicases
DNA Primase
Essential Amino Acids
Amino Acid Substitution
Cell culture
Recombinant Proteins
Fingers
Drosophila
Arginine
Molecular Biology
Nucleotides
Cell Culture Techniques
Maintenance

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Physiological and biochemical defects in carboxyl-terminal mutants of mitochondrial DNA helicase. / Matsushima, Yuichi; Farr, Carol L.; Fan, Li; Kaguni, Laurie S.

In: Journal of Biological Chemistry, Vol. 283, No. 35, 29.08.2008, p. 23964-23971.

Research output: Contribution to journalArticle

Matsushima, Yuichi ; Farr, Carol L. ; Fan, Li ; Kaguni, Laurie S. / Physiological and biochemical defects in carboxyl-terminal mutants of mitochondrial DNA helicase. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 35. pp. 23964-23971.
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