Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB.

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs.

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Mitochondrial Replicative DNA Helicase.

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the mtDNA helicase.

Physical and Functional Interaction of Mitochondrial Single-Stranded DNA-Binding Protein and the Catalytic Subunit of DNA Polymerase Gamma.

The maintenance of the mitochondrial genome depends on a suite of nucleus-encoded proteins, among which the catalytic subunit of the mitochondrial replicative DNA polymerase, Pol γα, plays a pivotal role. Mutations in the Pol γα-encoding gene, , are a major cause of human mitochondrial disorders. Here we present a study of direct and functional interactions of Pol γα with the mitochondrial single-stranded DNA-binding protein (mtSSB).

Sphingolipid lysosomal storage diseases: from bench to bedside.

Johann Ludwig Wilhelm Thudicum described sphingolipids (SLs) in the late nineteenth century, but it was only in the past fifty years that SL research surged in importance and applicability. Currently, sphingolipids and their metabolism are hotly debated topics in various biochemical fields. Similar to other macromolecular reactions, SL metabolism has important implications in health and disease in most cells.

A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1.

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD.

Iron-sulfur clusters in nucleic acid metabolism: Varying roles of ancient cofactors.

Despite their relative simplicity, iron-sulfur clusters have been omnipresent as cofactors in myriad cellular processes such as oxidative phosphorylation and other respiratory pathways. Recent research advances confirm the presence of different clusters in enzymes involved in nucleic acid metabolism. Iron-sulfur clusters can therefore be considered hallmarks of cellular metabolism.

Lethal Interaction of Nuclear and Mitochondrial Genotypes in .

, like most animal species, displays considerable genetic variation in both nuclear and mitochondrial DNA (mtDNA). Here we tested whether any of four natural mtDNA variants was able to modify the effect of the phenotypically mild, nuclear mutation, affecting mitochondrial protein synthesis. When combined with , the mtDNA from wild strain KSA2 produced pupal lethality, accompanied by the presence of melanotic nodules in L3 larvae.

Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis.

Genome replication induces the generation of large stretches of single-stranded DNA (ssDNA) intermediates that are rapidly protected by single-stranded DNA-binding (SSB) proteins. To date, the mechanism by which tightly bound SSBs are removed from ssDNA by the lagging strand DNA polymerase without compromising the advance of the replication fork remains unresolved. Here, we aimed to address this question by measuring, with optical tweezers, the real-time replication kinetics of the human mitochondrial and bacteriophage T7 DNA polymerases on free-ssDNA, in comparison with ssDNA covered with homologous and non-homologous SSBs under mechanical tension.

Mitochondrial Protein Synthesis and mtDNA Levels Coordinated through an Aminoacyl-tRNA Synthetase Subunit.

The aminoacylation of tRNAs by aminoacyl-tRNA synthetases (ARSs) is a central reaction in biology. Multiple regulatory pathways use the aminoacylation status of cytosolic tRNAs to monitor and regulate metabolism. The existence of equivalent regulatory networks within the mitochondria is unknown.

Structural rearrangements in the mitochondrial genome of Drosophila melanogaster induced by elevated levels of the replicative DNA helicase.

Pathological conditions impairing functions of mitochondria often lead to compensatory upregulation of the mitochondrial DNA (mtDNA) replisome machinery, and the replicative DNA helicase appears to be a key factor in regulating mtDNA copy number. Moreover, mtDNA helicase mutations have been associated with structural rearrangements of the mitochondrial genome. To evaluate the effects of elevated levels of the mtDNA helicase on the integrity and replication of the mitochondrial genome, we overexpressed the helicase in Drosophila melanogaster Schneider cells and analyzed the mtDNA by two-dimensional neutral agarose gel electrophoresis and electron microscopy.

Drosophila protease ClpXP specifically degrades DmLRPPRC1 controlling mitochondrial mRNA and translation.

ClpXP is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. ClpXP is composed of a proteolytic subunit, ClpP, and a chaperone-like subunit, ClpX. Although it has been proposed that ClpXP is required for the mitochondrial unfolded protein response, additional roles for ClpXP in mitochondrial biogenesis are unclear.

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

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.

Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database.

DNA polymerase gamma (POLG) is the replicative polymerase responsible for maintaining mitochondrial DNA (mtDNA). Disorders related to its functionality are a major cause of mitochondrial disease. The clinical spectrum of POLG syndromes includes Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), the ataxia neuropathy spectrum (ANS) and progressive external ophthalmoplegia (PEO).

Mitochondrial genotype modulates mtDNA copy number and organismal phenotype in Drosophila.

We evaluated the role of natural mitochondrial DNA (mtDNA) variation on mtDNA copy number, biochemical features and life history traits in Drosophila cybrid strains. We demonstrate the effects of both coding region and non-coding A+T region variation on mtDNA copy number, and demonstrate that copy number correlates with mitochondrial biochemistry and metabolically important traits such as development time. For example, high mtDNA copy number correlates with longer development times.

Iron-Sulfur Clusters in Mitochondrial Metabolism: Multifaceted Roles of a Simple Cofactor.

Iron-sulfur metabolism is essential for cellular function and is a key process in mitochondria. In this review, we focus on the structure and assembly of mitochondrial iron-sulfur clusters and their roles in various metabolic processes that occur in mitochondria. Iron-sulfur clusters are crucial in mitochondrial respiration, in which they are required for the assembly, stability, and function of respiratory complexes I, II, and III.

Animal Mitochondrial DNA Replication.

Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the relative simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein-the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although substantial integrative research is warranted to establish firmly these links.

Structure, function and evolution of the animal mitochondrial replicative DNA helicase.

The mitochondrial replicative DNA helicase is essential for animal mitochondrial DNA (mtDNA) maintenance. Deleterious mutations in the gene that encodes it cause mitochondrial dysfunction manifested in developmental delays, defects and arrest, limited life span, and a number of human pathogenic phenotypes that are recapitulated in animals across taxa. In fact, the replicative mtDNA helicase was discovered with the identification of human disease mutations in its nuclear gene, and based upon its deduced amino acid sequence homology with bacteriophage T7 gene 4 protein (T7 gp4), a bi-functional primase-helicase.

Biolayer Interferometry: A Novel Method to Elucidate Protein-Protein and Protein-DNA Interactions in the Mitochondrial DNA Replisome.

A lack of effective treatment for mitochondrial diseases prompts scientists to investigate the molecular processes that underlie their development. The major cause of mitochondrial diseases is dysfunction of the sole mitochondrial DNA polymerase, DNA polymerase γ (Pol γ). The development of treatment strategies will require a detailed characterization of the molecular properties of Pol γ.

Purification and Comparative Assay of Human Mitochondrial Single-Stranded DNA-Binding Protein.

The mitochondrial single-stranded DNA-binding protein (mtSSB) coordinates the function of replisome components at the mitochondrial replication fork. In recent years, it has been demonstrated that mtSSB stimulates the activities of DNA polymerase γ (Pol γ) and mitochondrial DNA (mtDNA) helicase in a concentration-dependent manner. Here we present a new approach to purify the human mtSSB and our standard assays to evaluate its biochemical properties, including a Gel Mobility Shift Assay (GMSA) to assess single-stranded DNA (ssDNA) binding activity, and an assay to assess SSB stimulation of Pol γ activity.