Molecular mechanisms of mitochondrial DNA replication

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Laboratory Medicine

Sammanfattning: 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.

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