Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes

Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifunctional roles from DNA replication to damage tolerance and repair. This article focuses on the archaeo-eukaryotic primase (AEP) superfamily, drawing on recently characterized examples from all domains of life to highlight the functionally diverse pathways in which these enzymes are employed. The broad origins, functionalities and enzymatic capabilities of AEPs emphasizes their previous functional misannotation and supports the necessity for a reclassification of these enzymes under a category called primase-polymerases within the wider functional grouping of polymerases. Importantly, the repositioning of AEPs in this way better recognizes their broader roles in DNA metabolism and encourages the discovery of additional functions for these enzymes, aside from those highlighted here

DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein

[EN] Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB¿DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during `in situ¿ DNA synthesis. We show that HmtSSB binds to preformed ss-DNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.We are grateful to Prof. M. Salas laboratory (CBMSO-CSIC) for generously providing the Phi29 DNA polymerase and to Juan P. García Villaluenga (UCM) for useful discussions. Spanish Ministry of Economy and Competitiveness [MAT2015-71806-R to J.R.A-G, FIS2010-17440, FIS2015-67765-R to F.J.C., BFU2012-31825, BFU2015-63714-R to B.I.]; Spanish Ministry of Education, Culture and Sport [FPU13/02934 to J.J., FPU13/02826 to E.B-H.]; National Institutes of Health [GM45925 to L.S.K.]; University of Tampere (to G.L.C.); Programa de Financiacion Universidad Complutense de Madrid-Santander Universidades [CT45/15-CT46/15 to F.C.]. Funding for open access charge: Spanish Ministry of Economy and Competitiveness [BFU2015-63714-R].Morin, J.; Cerrón, F.; Jarillo, J.; Beltran-Heredia, E.; Ciesielski, G.; Arias-Gonzalez, JR.; Kaguni, L.... (2017). DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein. Nucleic Acids Research. 45(12):7237-7248. https://doi.org/10.1093/nar/gkx395S723772484512Shereda, R. D., Kozlov, A. G., Lohman, T. M., Cox, M. M., & Keck, J. L. (2008). 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Modeling Pathogenic Mutations of Human Twinkle in Drosophila Suggests an Apoptosis Role in Response to Mitochondrial Defects

The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase γ by Organization of the Template DNA

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pols γ with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ on these ssDNA templates is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and they are characterized by a similar organization of the template DNA for both Pols γ. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity and the variation among systems

Disruption of Mitochondrial DNA Replication in Drosophila Increases Mitochondrial Fast Axonal Transport In Vivo

Mutations in mitochondrial DNA polymerase (pol γ) cause several progressive human diseases including Parkinson's disease, Alper's syndrome, and progressive external ophthalmoplegia. At the cellular level, disruption of pol γ leads to depletion of mtDNA, disrupts the mitochondrial respiratory chain, and increases susceptibility to oxidative stress. Although recent studies have intensified focus on the role of mtDNA in neuronal diseases, the changes that take place in mitochondrial biogenesis and mitochondrial axonal transport when mtDNA replication is disrupted are unknown. Using high-speed confocal microscopy, electron microscopy and biochemical approaches, we report that mutations in pol γ deplete mtDNA levels and lead to an increase in mitochondrial density in Drosophila proximal nerves and muscles, without a noticeable increase in mitochondrial fragmentation. Furthermore, there is a rise in flux of bidirectional mitochondrial axonal transport, albeit with slower kinesin-based anterograde transport. In contrast, flux of synaptic vesicle precursors was modestly decreased in pol γ−α mutants. Our data indicate that disruption of mtDNA replication does not hinder mitochondrial biogenesis, increases mitochondrial axonal transport, and raises the question of whether high levels of circulating mtDNA-deficient mitochondria are beneficial or deleterious in mtDNA diseases

C-Terminal Extension of the Yeast Mitochondrial DNA Polymerase Determines the Balance between Synthesis and Degradation

Saccharomyces cerevisiae mitochondrial DNA polymerase (Mip1) contains a C-terminal extension (CTE) of 279 amino acid residues. The CTE is required for mitochondrial DNA maintenance in yeast but is absent in higher eukaryotes. Here we use recombinant Mip1 C-terminal deletion mutants to investigate functional importance of the CTE. We show that partial removal of the CTE in Mip1Δ216 results in strong preference for exonucleolytic degradation rather than DNA polymerization. This disbalance in exonuclease and polymerase activities is prominent at suboptimal dNTP concentrations and in the absence of correctly pairing nucleotide. Mip1Δ216 also displays reduced ability to synthesize DNA through double-stranded regions. Full removal of the CTE in Mip1Δ279 results in complete loss of Mip1 polymerase activity, however the mutant retains its exonuclease activity. These results allow us to propose that CTE functions as a part of Mip1 polymerase domain that stabilizes the substrate primer end at the polymerase active site, and is therefore required for efficient mitochondrial DNA replication in vivo

POLG1 p.R722H mutation associated with multiple mtDNA deletions and a neurological phenotype

<p>Abstract</p> <p>Background</p> <p>The c.2447G>A (p.R722H) mutation in the gene <it>POLG1 </it>of the catalytic subunit of human mitochondrial polymerase gamma has been previously found in a few occasions but its pathogenicity has remained uncertain. We set out to ascertain its contribution to neuromuscular disease.</p> <p>Methods</p> <p>Probands from two families with probable mitochondrial disease were examined clinically, muscle and buccal epithelial DNA were analyzed for mtDNA deletions, and the <it>POLG1, POLG2, ANT1 </it>and <it>Twinkle </it>genes were sequenced.</p> <p>Results</p> <p>An adult proband presented with progressive external ophthalmoplegia, sensorineural hearing impairment, diabetes mellitus, dysphagia, a limb myopathy and dementia. Brain MRI showed central and cortical atrophy, and ¹⁸F-deoxyglucose PET revealed reduced glucose uptake. Histochemical analysis of muscle disclosed ragged red fibers and cytochrome c oxidase-negative fibers. Electron microscopy showed subsarcolemmal aggregates of morphologically normal mitochondria. Multiple mtDNA deletions were found in the muscle, and sequencing of the <it>POLG1 </it>gene revealed a homozygous c.2447G>A (p.R722H) mutation. His two siblings were also homozygous with respect to the p.R722H mutation and presented with dementia and sensorineural hearing impairment. In another family the p.R722H mutation was found as compound heterozygosity with the common p.W748S mutation in two siblings with mental retardation, ptosis, epilepsy and psychiatric symptoms. The estimated carrier frequency of the p.R722H mutation was 1:135 in the Finnish population. No mutations in <it>POLG2</it>, <it>ANT1 </it>and <it>Twinkle </it>genes were found. Analysis of the POLG1 sequence by homology modeling supported the notion that the p.R722H mutation is pathogenic.</p> <p>Conclusions</p> <p>The recessive c.2447G>A (p.R722H) mutation in the linker region of the <it>POLG1 </it>gene is pathogenic for multiple mtDNA deletions in muscle and is associated with a late-onset neurological phenotype as a homozygous state. The onset of the disease can be earlier in compound heterozygotes.</p

Association of the Chromosome Replication Initiator DnaA with the Escherichia coli Inner Membrane In Vivo: Quantity and Mode of Binding

DnaA initiates chromosome replication in most known bacteria and its activity is controlled so that this event occurs only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and the resulting ADP is replaced with ATP on the verge of the next initiation. Two putative recycling mechanisms depend on the binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation. While there is no doubt that DnaA interacts with artificial membranes in vitro, it is still controversial as to whether it binds the cytoplasmic membrane in vivo. In this work we looked for DnaA-membrane interaction in E. coli cells by employing cell fractionation with both native and fluorescent DnaA hybrids. We show that about 10% of cellular DnaA is reproducibly membrane-associated. This small fraction might be physiologically significant and represent the free DnaA available for initiation, rather than the vast majority bound to the datA reservoir. Using the combination of mCherry with a variety of DnaA fragments, we demonstrate that the membrane binding function is delocalized on the surface of the protein’s domain III, rather than confined to a particular sequence. We propose a new binding-bending mechanism to explain the membrane-induced nucleotide release from DnaA. This mechanism would be fundamental to the initiation of replication

Classification of Plant Associated Bacteria Using RIF, a Computationally Derived DNA Marker

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format