Summary DNA2, a helicase/nuclease family member, plays versatile roles in processing DNA intermediates during DNA replication and repair. Yeast Dna2 (yDna2) is essential in RNA primer removal during nuclear DNA replication and is important in repairing UV damage, base damage, and double-strand breaks. Our data demonstrate that, surprisingly, human DNA2 (hDNA2) does not localize to nuclei, as it lacks a nuclear localization signal equivalent to that present in yDna2. Instead, hDNA2 migrates to the mitochondria, interacts with mitochondrial DNA polymerase γ, and significantly stimulates polymerase activity. We further demonstrate that hDNA2 and flap endonuclease 1 synergistically process intermediate 5’ flap structures occurring in DNA replication and long-patch base excision repair (LP-BER) in mitochondria. Depletion of hDNA2 from a mitochondrial extract reduces its efficiency in RNA primer removal and LP-BER. Taken together, our studies illustrate an evolutionarily diversified role of hDNA2 in mitochondrial DNA replication and repair in a mammalian system.

Summary Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that the mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.

Mitochondrial DNA polymerase ␥ (pol ␥) is responsible for replication and repair of mtDNA and is mutated in individuals with genetic disorders such as chronic external ophthalmoplegia and Alpers syndrome. pol ␥ is also an adventitious target for toxic side effects of several antiviral compounds, and mutation of its proofreading exonuclease leads to accelerated aging in mouse models. We have used a variety of physical and functional approaches to study the interaction of the human pol ␥ catalytic subunit with both the wild-type accessory factor, pol ␥B, and a deletion derivative that is unable to dimerize and consequently is impaired in its ability to stimulate processive DNA synthesis. Our studies clearly showed that the functional human holoenzyme contains two subunits of the processivity factor and one catalytic subunit, thereby forming a heterotrimer. The structure of pol ␥ seems to be variable, ranging from a single catalytic subunit in yeast to a heterodimer in Drosophila and a heterotrimer in mammals.Mitochondria contain a single DNA polymerase, pol 4 ␥, responsible for replication and repair of mtDNA (reviewed in Ref. 1). Human pol ␥ is isolated from mitochondria as a complex containing two subunits, a catalytic subunit, pol ␥A, of 139 kDa and an accessory subunit, pol ␥B, of 53 kDa (2-5). The catalytic subunit is a family A DNA polymerase with separate polymerase and 3Ј-5Ј exonuclease domains. Two recent developments have stressed the importance of pol ␥. Mutations in the catalytic subunit of human pol ␥ cause mitochondrial disorders (6, 7), and a transgenic mouse engineered to express an error-prone form of DNA pol ␥ lacking the 3Ј-5Ј proofreading ability accumulates errors in mtDNA and undergoes accelerated aging (8, 9).The processivity and substrate binding properties of pol ␥A are enhanced by complex formation with the accessory subunit (2, 4, 5). Most interestingly, the presence of the accessory subunit has been shown to decrease the fidelity of DNA synthesis by the catalytic subunit because it increases the ability of the enzyme to extend a mismatched primer (10). Initial characterizations of pol ␥ suggested that the enzyme forms a heterodimer containing one copy of each subunit. However, when we solved the crystal structure of mouse pol ␥B, it became apparent that this accessory factor is itself a homodimer with remarkable structural similarity to prokaryotic tRNA synthetases (Protein Data Bank code 1G5H (11)). We considered it to be very unlikely that this dimerization is a crystal packing artifact, especially because we were able to show that wild-type pol ␥B sedimented more rapidly than a deletion derivative lacking a major portion of the dimerization interface (11).The incorporation of pol ␥B in the pol ␥ holoenzyme appears to be a relatively recent event in evolutionary terms. The protein has not been reported in yeast, where pol ␥ was first cloned as a product of the mip1 gene (12), and efforts to find evidence for it have been unsuccessful (13). Drosophila pol ␥ differs from the enzyme in v...

SUMMARY MTERF4 is the first MTERF family member shown to bind RNA, and plays an essential role as a regulator of ribosomal biogenesis in mammalian mitochondria. It forms a complex with the rRNA methyltransferase NSUN4 and recruits it to the large ribosomal subunit. In this paper, we characterize the interaction between both proteins, demonstrate that MTERF4 strongly stimulates the specificity of NSUN4 during in vitro methylation experiments and present the 2.0 Å resolution crystal structure of the MTERF4:NSUN4 protein complex, lacking 48 residues of the MTERF4 C-terminal acidic tail, bound to S-adenosyl-L-methionine, revealing the nature of the interaction between both proteins and the structural conservation of the most divergent of the human MTERF family members. Moreover, the structure suggests a model for RNA binding by the MTERF4:NSUN4 complex, providing insight into the mechanism by which an MTERF family member facilitates rRNA methylation.