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Pol γ primer extension with structured oligo templates. A) Illustrative denaturing PAGE gel slices, at time = 0 or 10 minutes, showing Pol γ catalytic subunit pausing to form intermediate (left) or Pol γ heterotrimer catalyzing only full-length primer extension (right) during primer extension across the OriL template oligo. Full time course gel is shown in Supp. Figure S1. B) Primer extension time course for Pol γ catalytic subunit alone. Substrate template strand is an OriL mimic capable of adopting a stem-loop fold. Formation of reaction intermediates (red) and full-length product (orange) are shown. Reactions were carried out in 100 mM NaCl (closed symbols) and 200 mM NaCl (open symbols). In low salt, p140 (30 nM) catalyzed primer extension to form intermediate species at an initial rate of 0.51 nM/s and full-length product at 0.12 nM/s. At high salt, p140 catalyzed extension to intermediate length products at 0.04 nM/s. C) Primer extension time courses as described in (B) for the Pol γ heterotrimer with an OriL substrate template. The Pol γ complex catalyzed primer extension to produce full length products in low and high salt at 0.09 nM/s and 0.12 nM/s, respectively. D) Schematic representation of tRNA-coding oligo substrates, with Pol γ shown as a green box. The mtDNA gene encoding tRNA-Pro is templated on the light strand and coded by the heavy strand. E) and F) are reaction progress curves for Pol γ-catalyzed primer extension using either the catalytic subunit alone (E) or the heterotrimer (F) and the tRNA-Pro oligos. Synthesis of the mtDNA light strand is denoted by open symbols, while heavy strand synthesis is denoted by closed symbols. Red circles represent formation of intermediate species and orange squares represent formation of full-length product. When synthesizing L-stand (E), p140 alone (5 nM) catalyzed
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Faithful replication of the mitochondrial genome is carried out by a set of key nuclear-encoded proteins. DNA polymerase γ is a core component of the mtDNA replisome and the only replicative DNA polymerase localized to mitochondria. The asynchronous mechanism of mtDNA replication predicts that the replication machinery encounters double stranded (d...
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... products of primer extension reactions were resolved by urea-PAGE, and DNA synthesis both up to and past the structured region were determined by quantifying DNA substrates, reaction intermediates, and products by fluorescence image scanning. The catalytic subunit p140 was able to extend the DNA primer at both high and low salt concentrations ( Figure S1 lanes 1-23 and Figure 1A-C). Consistent with previous results, primer extension by p140 occurred at a higher rate in the low salt conditions ( Figure 1B). ...
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... products of primer extension reactions were resolved by urea-PAGE, and DNA synthesis both up to and past the structured region were determined by quantifying DNA substrates, reaction intermediates, and products by fluorescence image scanning. The catalytic subunit p140 was able to extend the DNA primer at both high and low salt concentrations ( Figure S1 lanes 1-23 and Figure 1A-C). Consistent with previous results, primer extension by p140 occurred at a higher rate in the low salt conditions ( Figure 1B). ...
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... catalytic subunit p140 was able to extend the DNA primer at both high and low salt concentrations ( Figure S1 lanes 1-23 and Figure 1A-C). Consistent with previous results, primer extension by p140 occurred at a higher rate in the low salt conditions ( Figure 1B). The initial rate of product formation was 0.10 nM/s at low salt and more than an order of magnitude slower at high salt. ...
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... inspection of the bands in the urea-PAGE gels indicate accumulation of reaction intermediates 4-6 nucleotides (nt) longer than the original primer, suggesting DNA synthesis proceeds immediately up to the beginning of the stem-loop structure prior to the enzyme stalling. In contrast, the Pol γ complex (p55+p140) catalyzes DNA synthesis across the stem-loop template to form full length products under both low and high salt conditions ( Figure S1 lanes 24-46 and Figure 1C). Thus, addition of the p55 accessory subunit fully alleviates Pol γ stalling on the stem-loop substrate in vitro. ...
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... inspection of the bands in the urea-PAGE gels indicate accumulation of reaction intermediates 4-6 nucleotides (nt) longer than the original primer, suggesting DNA synthesis proceeds immediately up to the beginning of the stem-loop structure prior to the enzyme stalling. In contrast, the Pol γ complex (p55+p140) catalyzes DNA synthesis across the stem-loop template to form full length products under both low and high salt conditions ( Figure S1 lanes 24-46 and Figure 1C). Thus, addition of the p55 accessory subunit fully alleviates Pol γ stalling on the stem-loop substrate in vitro. ...
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... tDNA encoding tRNA proline (tRNA-Pro) is the first gene encountered by Pol γ during synthesis of the nascent H-strand in the Strand Displacement Model of mtDNA replication (10,25). tRNA-Pro is templated on the mtDNA L-strand and coded on the H-strand ( Figure 1D). Therefore, we hypothesized that any replication stalling would exhibit a strand bias, wherein synthesis of the L-strand may be impacted by the tRNAPro gene but not synthesis of the H-strand. ...
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... reactions utilizing only p140 exhibited brief stalling when extending the L-strand at tDNA-Pro as judged by the accumulation of replication intermediates ( Figure 1E). Initial rates for formation of the intermediate length product were 4-fold lower than the rate of generating the full-length product ( Table 1). ...
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... of full length tDNA-Pro H-strand was twice as efficient as L-strand synthesis, and p140 exhibited initial rates for full length product formation of 0.17 nM/s and 0.08 nM/s, respectively (Table 1). This increased apparent efficiency is likely due to the absence of detectable stalling by p140 when replicating the H-strand sequence ( Figure 1E). ...
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... to the results with the OriLbased substrate, the addition of accessory subunit p55 ablates the accumulation of any intermediate length species during either Hstrand or L-strand synthesis on the tDNAbased substrate ( Figure 1F). Synthesis of the L-strand catalyzed by p55+p140 occurs at a slower initial rate than synthesis of the Hstrand (Table 1). ...
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The small, multicopy mitochondrial genome (mtDNA) is essential for efficient energy production, as alterations in its coding information or a decrease in its copy number disrupt mitochondrial ATP synthesis. However, the mitochondrial replication machinery encounters numerous challenges that may limit its ability to duplicate this imp...
Citations
... They suggest that mitochondrial G4 are likely to be a source of genomic instability by perturbing the normal progression of the mitochondrial replication machinery ( 68 ). Sullivan et al. also identified a G4 sequence at the heavy-strand promoter (HSP1) that has the potential to cause significant stalling of mtDNA replication ( 69 ). G4 ligands can perturb mitochondrial genome replication, transcription processivity, and respiratory function in normal cells ( 70 ). ...
Current methods of processing archaeological samples combined with advances in sequencing methods lead to disclosure of a large part of H. neanderthalensis and Denisovans genetic information. It is hardly surprising that the genome variability between modern humans, Denisovans and H. neanderthalensis is relatively limited. Genomic studies may provide insight on the metabolism of extinct human species or lineages. Detailed analysis of G-quadruplex sequences in H. neanderthalensis and Denisovans mitochondrial DNA showed us interesting features. Relatively similar patterns in mitochondrial DNA are found compared to modern humans, with one notable exception for H. neanderthalensis. An interesting difference between H. neanderthalensis and H. sapiens corresponds to a motif found in the D-loop region of mtDNA, which is responsible for mitochondrial DNA replication. This area is directly responsible for the number of mitochondria and consequently for the efficient energy metabolism of cell. H. neanderthalensis harbor a long uninterrupted run of guanines in this region, which may cause problems for replication, in contrast with H. sapiens, for which this run is generally shorter and interrupted. One may propose that the predominant H. sapiens motif provided a selective advantage for modern humans regarding mtDNA replication and function.
... In this scenario, PolG2 participates in maintenance of the Dloop by stabilizing the forked DNA structure and perhaps protecting the 5 and 3 ends of 7S DNA from degradation by MGME1 or PolG exonuclease (43)(44)(45)(46). Similarly, Nucleic Acids Research, 2023 15 if PolG2 bound at the 5 -end of the D-loop can extract the PolG2 dimer from an approaching replicati v e Pol ␥ holoenzyme, then the remaining PolG subunit becomes incapable of strand displacement DNA synthesis ( 47 ). In any e v ent, our structural and biochemical data imply that formation of holoenzyme heterotrimers and PolG2 hexamers are mutually e xclusi v e and competiti v e, which suggests a vital role for PolG2 as a biological switch between mtDNA replication and maintenance functions. ...
The homodimeric PolG2 accessory subunit of the mitochondrial DNA polymerase gamma (Pol γ) enhances DNA binding and processive DNA synthesis by the PolG catalytic subunit. PolG2 also directly binds DNA, although the underlying molecular basis and functional significance are unknown. Here, data from Atomic Force Microscopy (AFM) and X-ray structures of PolG2-DNA complexes define dimeric and hexameric PolG2 DNA binding modes. Targeted disruption of PolG2 DNA-binding interfaces impairs processive DNA synthesis without diminishing Pol γ subunit affinities. In addition, a structure-specific DNA-binding role for PolG2 oligomers is supported by X-ray structures and AFM showing that oligomeric PolG2 localizes to DNA crossings and targets forked DNA structures resembling the mitochondrial D-loop. Overall, data indicate that PolG2 DNA binding has both PolG-dependent and -independent functions in mitochondrial DNA replication and maintenance, which provide new insight into molecular defects associated with PolG2 disruption in mitochondrial disease.
... It has been demonstrated that re-combinant mitochondrial DN A pol ymerase ␥ (POL G) can pause DNA synthesis upon encountering G4 structures on the DNA template ( 11 , 45 ). Howe v er, the degree of pausing may vary depending on the specific G4 structure being tested ( 11 ). DN A pol ymerase stop assays using a previousl y tested mtDN A G4 template (Supplementary Figure S3A) ( 11 ) that has an anti-parallel topology (Supplementary Figure S3D and Supplementary Table S2) confirmed that G4 structures are an obstacle for POLG (Supplementary Figure S3B lanes 1 and 2). ...
Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.
... Pol␥ also exhibits DNA strand displacement activity, in which the holoenzyme displaces downstream DNA encountered during synthesis. This activity is relevant for replication through stable secondary structures (8)(9)(10), maintenance of the D-loop DNA structure at the origin of replication of the heavy strand (11), and removing RNA/DNA primers in coordination with primer processing factors (nucleases, helicases and/or mtSSBs) (12)(13)(14). As in the case of other replicative DNA polymerases (DNApols) (15)(16)(17), strand displacement DNA synthesis is usually limited to a few nucleotides (13,(18)(19)(20)(21). ...
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. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.
... The effect of SSB on strand displacement DNA replication has also been examined using single-molecule tools [176,177] (Figure 6B). It was previously demonstrated that strand displacement DNA synthesis, such as by Polγ, accomplishes replication by utilizing stable secondary structures [178,179], ensuring that the D-loop DNA structure is maintained at the origin of heavy strands [180], and removing primers through the coordination of primer processing factors [181][182][183]. However, the efficiency of Polγ is limited to a few nucleotides [182,[184][185][186][187], in accordance with other DNA polymerases involved in strand displacement synthesis [176,188,189]. ...
Single-stranded DNA-binding proteins (SSBs) play vital roles in DNA metabolism. Proteins of the SSB family exclusively and transiently bind to ssDNA, preventing the DNA double helix from re-annealing and maintaining genome integrity. In the meantime, they interact and coordinate with various proteins vital for DNA replication, recombination, and repair. Although SSB is essential for DNA metabolism, proteins of the SSB family have been long described as accessory players, primarily due to their unclear dynamics and mechanistic interaction with DNA and its partners. Recently-developed single-molecule tools, together with biochemical ensemble techniques and structural methods, have enhanced our understanding of the different coordination roles that SSB plays during DNA metabolism. In this review, we discuss how single-molecule assays, such as optical tweezers, magnetic tweezers, Förster resonance energy transfer, and their combinations, have advanced our understanding of the binding dynamics of SSBs to ssDNA and their interaction with other proteins partners. We highlight the central coordination role that the SSB protein plays by directly modulating other proteins’ activities, rather than as an accessory player. Many possible modes of SSB interaction with protein partners are discussed, which together provide a bigger picture of the interaction network shaped by SSB.
... Strand displacement DNA synthesis by Polis relevant for replication through stable secondary structures (17)(18)(19), maintenance of the D-loop DNA structure at the origin of replication of the heavy strand (20), and removing RNA/DNA primers in coordination with primer processing factors (21)(22)(23). Similarly to other DNApols (13,15,24), the efficiency of strand displacement activity of Pol is limited to a few nucleotides (16,22,(25)(26)(27). Biochemical studies have shown that at a nick Pol is prone to enter the idling state, characterized by repeated addition and excision of a nucleotide, which is driven by the intramolecular and reversible transfer of the primer between the pol and the exo active sites (11,16). ...
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. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ~55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ~30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ~25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ~3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.
... A clue to the mechanism underlying POLZ-dependent Del3895 formation could come from the fact that POLZ is thought to bind to difficult-to-replicate sites such as G4 structures already arising in the absence of exogenous stress [81]. One of the direct repeat regions (located in the NCR region) at the extremities of the 3895 bp deletion was shown to overlap with a G4 structure, causing stalling of POLG [82]. POLG is known to interact with POLZ [36], so POLG may contribute to the recruitment of POLZ to stalled replication forks to promote TLS [83,84]. ...
Mitochondrial DNA (mtDNA) damaged by reactive oxygen species (ROS) triggers so far poorly understood processes of mtDNA maintenance that are coordinated by a complex interplay among DNA repair, DNA degradation, and DNA replication. This study was designed to identify the proteins involved in mtDNA maintenance by applying a special long-range PCR, reflecting mtDNA integrity in the minor arc. A siRNA screening of literature-based candidates was performed under conditions of enforced oxidative phosphorylation revealing the functional group of polymerases and therein polymerase ζ (POLZ) as top hits. Thus, POLZ knockdown caused mtDNA accumulation, which required the activity of the base excision repair (BER) nuclease APE1, and was followed by compensatory mtDNA replication determined by the single-cell mitochondrial in situ hybridization protocol (mTRIP). Quenching reactive oxygen species (ROS) in mitochondria unveiled an additional, ROS-independent involvement of POLZ in the formation of a typical deletion in the minor arc region. Together with data demonstrating the localization of POLZ in mitochondria, we suggest that POLZ plays a significant role in mtDNA turnover, particularly under conditions of oxidative stress.
... G4s have been shown to perturb mtDNA replication, transcription, and respiratory function in non-cancerous cells [70]. In vitro, a specific G-quadruplex-forming sequence at HSP1 blocks DNA synthesis by DNA polymerase γ [71]. Additional effects of mitochondrial G4 are summarized in a recent review [72]. ...
Mitochondria have a plethora of functions in eukaryotic cells, including cell signaling, programmed cell death, protein cofactor synthesis, and various aspects of metabolism. The organelles carry their own genomic DNA, which encodes transfer and ribosomal RNAs and crucial protein subunits in the oxidative phosphorylation system. Mitochondria are vital for cellular and organismal functions, and alterations of mitochondrial DNA (mtDNA) have been linked to mitochondrial disorders and common human diseases. As such, how the cell maintains the integrity of the mitochondrial genome is an important area of study. Interactions of mitochondrial proteins with mtDNA damage are critically important for repairing, regulating, and signaling mtDNA damage. Mitochondrial transcription factor A (TFAM) is a key player in mtDNA transcription, packaging, and maintenance. Due to the extensive contact of TFAM with mtDNA, it is likely to encounter many types of mtDNA damage and secondary structures. This review summarizes recent research on the interaction of human TFAM with different forms of non-canonical DNA structures and discusses the implications on mtDNA repair and packaging.
... The mtDNA is replicated by a core set of proteins: polymerase γ, Twinkle, and the single-stranded DNA binding protein [10]. Polymerase γ efficiently replicates through many natural template barriers (e.g., double stranded DNA, structured genes, G-quadruplexes) [11]. ...
... A change of paradigm seems necessary to improve the effectivity of the treatment of chronic diseases. This should include mitochondria-based medicine [11]. ...
Mitochondria are of great relevance to health, and their dysregulation is associated with major chronic diseases. Research on mitochondria—156 brand new publications from 2019 and 2020—have contributed to this review. Mitochondria have been fundamental for the evolution of complex organisms. As important and semi-autonomous organelles in cells, they can adapt their function to the needs of the respective organ. They can program their function to energy supply (e.g., to keep heart muscle cells going, life-long) or to metabolism (e.g., to support hepatocytes and liver function). The capacity of mitochondria to re-program between different options is important for all cell types that are capable of changing between a resting state and cell proliferation, such as stem cells and immune cells. Major chronic diseases are characterized by mitochondrial dysregulation. This will be exemplified by cardiovascular diseases, metabolic syndrome, neurodegenerative diseases, immune system disorders, and cancer. New strategies for intervention in chronic diseases will be presented. The tumor microenvironment can be considered a battlefield between cancer and immune defense, competing for energy supply and metabolism. Cancer cachexia is considered as a final stage of cancer progression. Nevertheless, the review will present an example of complete remission of cachexia via immune cell transfer. These findings should encourage studies along the lines of mitochondria, energy supply, and metabolism.
The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polγ heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polγ at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polγ complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polγ aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.
