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Reconstitution of a minimal mtDNA replisome in vitro.

Journal article
Authors Jenny A Korhonen
Xuan Hoi Pham
Mina Pellegrini
Maria Falkenberg
Published in The EMBO journal
Volume 23
Issue 12
Pages 2423-9
ISSN 0261-4189
Publication year 2004
Published at
Pages 2423-9
Language en
Keywords Base Sequence, DNA Replication, DNA, Mitochondrial, biosynthesis, genetics, Molecular Sequence Data, Templates, Genetic
Subject categories Chemistry

Abstract

We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLgamma) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLgamma and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The 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. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLgamma cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLgamma thus explain why mutations in these two proteins cause an identical syndrome.

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