Functional and structural characterization of the human mitochondrial helicase

Sammanfattning: Mitochondria are the energy producing organelles of the eukaryotic cell. The human mitochondrial DNA (mtDNA) is a double stranded circular molecule of about 16 kb, usually present at 1000-10 000 copies per cell. The genome encodes 13 polypeptides involved in respiration, two ribosomal RNA s and a set of 22 transfer RNA s. Nuclear genes encode all the factors required for the transcription and replication of the mtDNA genome. Mutations in mtDNA replication factors are associated with human diseases affecting mitochondrial genome stability and maintenance. The general aim of this thesis has been to investigate the molecular mechanisms of human mtDNA replication with a focus on the recently identified TWINKLE protein, a mitochondrial DNA helicase with 5' to 3' directionality and distinct substrate requirements. TWINKLE displays sequence similarity to the bacteriophage T7 gene 4 protein, which contains the DNA helicase and primase activities needed at the bacteriophage DNA replication fork. TWINKLE alone is unable to unwind longer stretches of double-stranded DNA (dsDNA), but forms together with the mitochondrial DNA polymerase g (POL g ) a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. Addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. Mutations in both POL g and TWINKLE can cause autosomal dominant progressive external ophthalmoplegia (adPEO) with multiple deletions of mitochondrial DNA. Detailed analysis of seven different adPEO-causing mutations in the linker region of TWINKLE reveals different molecular phenotypes, with distinct consequences for multimerization, ATPase activity, DNA helicase activity, and ability to support mtDNA synthesis in vitro. A structural model of TWINKLE that is based on the extensive primary sequence similarities with the T7 gene 4 protein is used to explain these distinct molecular phenotypes. In this model, the TWINKLE linker region consists of two helical regions connected by a short turn. The N-terminal helical region makes intra-molecular contacts within the primase domain, whereas the second helical region interacts with the helicase domain of the neighboring monomer. The model suggests that mutations carried out in the first helical region may rather result in miss-folding or destabilization of the monomer itself, whereas mutations in the second helical region of the linker will weaken TWINKLE dimerization, which in turn affects hexamerization. Biochemical analyses of adPEO causing amino acid changes in the linker region support this interpretation. Four different adPEO-causing mutations in the N-terminal region of TWINKLE do not affect protein hexamerization, but display severely reduced ATPase activities. The decrease in ATPase activity can be partially overcome by the addition of ssDNA. The structural model of TWINKLE contains a conserved, positively charged surface region that has been implied in binding to ssDNA. All four adPEO-causing amino acid substitutions are located to this region and three of the mutants also display reduced binding to ssDNA in vitro.