10 research outputs found
The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities
The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg2+ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome
The mitochondrial DNA helicase TWINKLE can assemble on a closed circular template and support initiation of DNA synthesis
Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase γ. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo
Molecular mechanisms of mitochondrial DNA replication
Mitochondria are the energy producing organelles of eukaryotic cells. The
organelle has its own genome, the mitochondrial DNA (mtDNA) that encodes
13 subunits of the respiratory chain (RC) complexes, two rRNAs and 22
tRNAs. Nuclear genes encode the majority of the RC subunits and all the
factors required for transcription and replication of the mtDNA.
Mutations in mtDNA replication factors are associated with human diseases
affecting mitochondrial genome stability and maintenance. The human mtDNA
replication system has been reconstituted in vitro and involves the
combined actions of the DNA polymerase gamma holoenzyme (POLgamma), the
TWINKLE helicase and the single-stranded DNA binding protein mtSSB. The
general aim of this thesis has been to further investigate the molecular
mechanisms of mtDNA replication, with a major focus on the mitochondrial
hexameric helicase TWINKLE, as well as the accessory B subunit of
POLgamma.
A biochemical characterization of POLgammaB demonstrated that the protein
blocks the exonuclease activity of the catalytic POLgammaA subunit. In
addition, the dsDNA-binding activity of POLgammaB was required for the
TWINKLE-dependant stimulation of the POLgamma holoenzyme.
TWINKLE displays sequence similarity to the bacteriophage T7 gene 4
protein (gp4) which contains the DNA helicase and primase activities
needed at the bacteriophage replication fork. The C-terminal domain of
TWINKLE is indeed an active helicase, but there have been no reports of
primase activity. The functional role of the TWINKLE N-terminus was
therefore investigated in this work. The N-terminal domain was found to
contribute to ssDNA-binding and helicase activities of TWINKLE, and was
ultimately required for full replisome activity. A structural model of
TWINKLE was constructed based on homology modeling with T7 gp4. This
model displayed a conserved region with significant electropositive
potential, which in structurally related primases has been suggested to
interact with ssDNA.
Mutations in both POLgamma and TWINKLE can cause autosomal dominant
progressive external ophtalmoplegia (adPEO). To investigate the molecular
mechanisms behind this disorder, we performed a detailed biochemical
analysis on eleven different adPEO-causing TWINKLE mutations, seven in
the linker-region and four in the N-terminal domain. Distinct molecular
phenotypes were observed, with individual consequences for
multimerization, ATPase activity, helicase activity and ability to
support mtDNA synthesis in vitro. The different molecular phenotypes
could be interpreted using our structural model of TWINKLE. Two of the
mutations in the linker region affected multimerization, whereas the
N-terminal mutations showed a striking reduction in ATPase activity and
were thus proposed to impair the interplay between ssDNA-binding and ATP
hydrolysis, an essential element of the catalytic cycle of related
hexameric helicases
The N-terminal domain of TWINKLE contributes
to single-stranded DNA binding and DNA helicase activitie
Structure-function defects of the twinkle amino-terminal region in progressive external ophthalmoplegia
AbstractTWINKLE is a DNA helicase needed for mitochondrial DNA replication. In lower eukaryotes the protein also harbors a primase activity, which is lost from TWINKLE encoded by mammalian cells. Mutations in TWINKLE underlie autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with multiple deletions in the mtDNA. Four different adPEO-causing mutations (W315L, K319T, R334Q, and P335L) are located in the N-terminal domain of TWINKLE. The mutations cause a dramatic decrease in ATPase activity, which is partially overcome in the presence of single-stranded DNA. The mutated proteins have defects in DNA helicase activity and cannot support normal levels of DNA replication. To explain the phenotypes, we use a molecular model of TWINKLE based on sequence similarities with the phage T7 gene 4 protein. The four adPEO-causing mutations are located in a region required to bind single-stranded DNA. These mutations may therefore impair an essential element of the catalytic cycle in hexameric helicases, i.e. the interplay between single-stranded DNA binding and ATP hydrolysis