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

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• Mitochondrial DNA helicase (Twinkle) is crucial for maintaining mitochondrial DNA (mtDNA).
• The carboxyl-terminal region of Twinkle, homologous to superfamily 4 helicases, is implicated in NTP hydrolysis and DNA unwinding.
• Limited genetic and biochemical data exist regarding the specific roles of these carboxyl-terminal residues.

Purpose of the Study:

• To investigate the function of the carboxyl-terminal residues of mitochondrial DNA helicase (Twinkle) in vivo.
• To identify critical amino acids within the carboxyl terminus essential for helicase activity.
• To understand the role of these residues in nucleotide hydrolysis and DNA unwinding.

Main Methods:

• Overexpression of Twinkle variants with carboxyl-terminal deletions and alanine substitutions in Drosophila cell culture.
• Analysis of dominant-negative phenotypes resulting from specific amino acid substitutions.
• Biochemical assays of human recombinant proteins to assess nucleotide hydrolysis defects.

Main Results:

• Identified critical residues between amino acids 572 and 596 essential for Twinkle's in vivo function.
• Demonstrated that mutations K574A, R576A, Y577A, F588A, and F595A cause dose-dependent dominant-negative effects.
• Showed that human recombinant proteins analogous to these mutants exhibit impaired nucleotide hydrolysis.
• Highlighted the functional analogy of Arg-576 and Phe-588 to conserved motifs in other superfamily 4 helicases.

Conclusions:

• The carboxyl-terminal region of Twinkle contains residues critical for its essential function in mtDNA maintenance.
• Specific amino acid substitutions in this region disrupt nucleotide hydrolysis and DNA unwinding.
• Findings provide insights into the structure-function relationship of superfamily 4 helicases and may have broader implications for understanding DNA helicase mechanisms.