mtDNA Helicase Research Articles

<i>TWNK</i> Gene Associated Perrault Syndrome Patient with Neurological Features

Perrault syndrome 5 is a rare autosomal recessive disorder that is characterized by the association of sensorineural hearing loss and ovarian dysgenesis in females with diversity of neurologic deficits due to variants of twinkle mtDNA helicase (<i>TWNK</i>) gene. Since neurologic deficits develop gradually, patient is often misdiagnosed with other neurological disease during early age. Herein, we report a case of genetically diagnosed Perrault syndrome 5.

Whole-exome sequencing in 415,422 individuals identifies rare variants associated with mitochondrial DNA copy number.

SummaryInter-individual variation in the number of copies of the mitochondrial genome, called mitochondrial DNA copy number (mtDNA-CN), reflects mitochondrial function and has been associated with various aging-related diseases. We examined 415,422 exomes of self-reported White ancestry individuals from the UK Biobank and tested the impact of rare variants, at the level of single variants and through aggregate variant-set tests, on mtDNA-CN. A survey across nine variant sets tested enrichment of putatively causal variants and identified 14 genes at experiment-wide significance and three genes at marginal significance. These included associations at known mtDNA depletion syndrome genes (mtDNA helicase TWNK, p = 1.1 × 10−30; mitochondrial transcription factor TFAM, p = 4.3 × 10−15; mtDNA maintenance exonuclease MGME1, p = 2.0 × 10−6) and the V617F dominant gain-of-function mutation in the tyrosine kinase JAK2 (p = 2.7 × 10−17), associated with myeloproliferative disease. Novel genes included the ATP-dependent protease CLPX (p = 8.4 × 10−9), involved in mitochondrial proteome quality, and the mitochondrial adenylate kinase AK2 (p = 4.7 × 10−8), involved in hematopoiesis. The most significant association was a missense variant in SAMHD1 (p = 4.2 × 10−28), found on a rare, 1.2-Mb shared ancestral haplotype on chromosome 20. SAMHD1 encodes a cytoplasmic host restriction factor involved in viral defense response and the mitochondrial nucleotide salvage pathway, and is associated with Aicardi-Goutières syndrome 5, a childhood encephalopathy and chronic inflammatory response disorder. Rare variants were enriched in Mendelian mtDNA depletion syndrome loci, and these variants implicated core processes in mtDNA replication, nucleoid structure formation, and maintenance. These data indicate that strong-effect mutations from the nuclear genome contribute to the genetic architecture of mtDNA-CN.

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila 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 Drosophila melanogaster (Dm) 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 Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

Analysis of Mitochondrial SSB-DNA Complexes and Their Effects on DNA Polymerase γ Activity by Electron Microscopy and Enzymatic Assays.

The mitochondrial single-stranded DNA-binding protein (mtSSB) regulates the function of the mitochondrial DNA (mtDNA) replisome. In vitro, mtSSB stimulates the activity of enzymatic components of the replisome, namely mtDNA helicase and DNA polymerase gamma (Pol γ). We have demonstrated that the stimulatory properties of mtSSB result from its ability to organize the single-stranded DNA template in a specific manner. Here we present methods employing electron microscopy and enzymatic assays to characterize and classify the mtSSB-DNA complexes and their effects on the activity of Pol γ.

Stimulation of Variant Forms of the Mitochondrial DNA Helicase Twinkle by the Mitochondrial Single-Stranded DNA-Binding Protein.

Defects in mitochondrial DNA (mtDNA) maintenance may lead to disturbances in mitochondrial homeostasis and energy production in eukaryotic cells, causing diseases. During mtDNA replication, the mitochondrial single-stranded DNA-binding protein (mtSSB) stabilizes and protects the exposed single-stranded mtDNA from nucleolysis; perhaps more importantly, it appears to coordinate the actions of both the replicative mtDNA helicase Twinkle and DNA polymerase gamma at the replication fork. Here, we describe a helicase stimulation protocol to test in vitro the functional interaction between mtSSB and variant forms of Twinkle. We show for the first time that the C-terminal tail of Twinkle is important for such an interaction, and that it negatively regulates helicase unwinding activity in a salt-dependent manner.

Mitochondrial spongiotic brain disease: astrocytic stress and harmful rapamycin and ketosis effect

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a group of severe, tissue-specific diseases of childhood with unknown pathogenesis. Brain-specific MDS manifests as devastating spongiotic encephalopathy with no curative therapy. Here, we report cell type-specific stress responses and effects of rapamycin treatment and ketogenic diet (KD) in mice with spongiotic encephalopathy mimicking human MDS, as these interventions were reported to improve some mitochondrial disease signs or symptoms. These mice with astrocyte-specific knockout of Twnk gene encoding replicative mtDNA helicase Twinkle (TwKOastro) show wide-spread cell-autonomous astrocyte activation and mitochondrial integrated stress response (ISRmt) induction with major metabolic remodeling of the brain. Mice with neuronal-specific TwKO show no ISRmt Both KD and rapamycin lead to rapid deterioration and weight loss of TwKOastro and premature trial termination. Although rapamycin had no robust effects on TwKOastro brain pathology, KD exacerbated spongiosis, gliosis, and ISRmt Our evidence emphasizes that mitochondrial disease treatments and stress responses are tissue- and disease specific. Furthermore, rapamycin and KD are deleterious in MDS-linked spongiotic encephalopathy, pointing to a crucial role of diet and metabolism for mitochondrial disease progression.

Roles of the mitochondrial replisome in mitochondrial DNA deletion formation.

Mitochondrial DNA (mtDNA) deletions are a common cause of human mitochondrial diseases. Mutations in the genes encoding components of the mitochondrial replisome, such as DNA polymerase gamma (Pol γ) and the mtDNA helicase Twinkle, have been associated with the accumulation of such deletions and the development of pathological conditions in humans. Recently, we demonstrated that changes in the level of wild-type Twinkle promote mtDNA deletions, which implies that not only mutations in, but also dysregulation of the stoichiometry between the replisome components is potentially pathogenic. The mechanism(s) by which alterations to the replisome function generate mtDNA deletions is(are) currently under debate. It is commonly accepted that stalling of the replication fork at sites likely to form secondary structures precedes the deletion formation. The secondary structural elements can be bypassed by the replication-slippage mechanism. Otherwise, stalling of the replication fork can generate single- and double-strand breaks, which can be repaired through recombination leading to the elimination of segments between the recombination sites. Here, we discuss aberrances of the replisome in the context of the two debated outcomes, and suggest new mechanistic explanations based on replication restart and template switching that could account for all the deletion types reported for patients.

Mitochondrial RNA granules are critically dependent on mtDNA replication factors Twinkle and mtSSB.

Newly synthesized mitochondrial RNA is concentrated in structures juxtaposed to nucleoids, called RNA granules, that have been implicated in mitochondrial RNA processing and ribosome biogenesis. Here we show that two classical mtDNA replication factors, the mtDNA helicase Twinkle and single-stranded DNA-binding protein mtSSB, contribute to RNA metabolism in mitochondria and to RNA granule biology. Twinkle colocalizes with both mitochondrial RNA granules and nucleoids, and it can serve as bait to greatly enrich established RNA granule proteins, such as G-rich sequence factor 1, GRSF1. Likewise, mtSSB also is not restricted to the nucleoids, and repression of either mtSSB or Twinkle alters mtRNA metabolism. Short-term Twinkle depletion greatly diminishes RNA granules but does not inhibit RNA synthesis or processing. Either mtSSB or GRSF1 depletion results in RNA processing defects, accumulation of mtRNA breakdown products as well as increased levels of dsRNA and RNA:DNA hybrids. In particular, the processing and degradation defects become more pronounced with both proteins depleted. These findings suggest that Twinkle is essential for RNA organization in granules, and that mtSSB is involved in the recently proposed GRSF1-mtRNA degradosome pathway, a route suggested to be particularly aimed at degradation of G-quadruplex prone long non-coding mtRNAs.

Developmental arrest in Drosophila melanogaster caused by mitochondrial DNA replication defects cannot be rescued by the alternative oxidase

The xenotopic expression of the alternative oxidase AOX from the tunicate Ciona intestinalis in diverse models of human disease partially alleviates the phenotypic effects of mitochondrial respiratory chain defects. AOX is a non-proton pumping, mitochondrial inner membrane-bound, single-subunit enzyme that can bypass electron transport through the cytochrome segment, providing an additional site for ubiquinone reoxidation and oxygen reduction upon respiratory chain overload. We set out to investigate whether AOX expression in Drosophila could counteract the effects of mitochondrial DNA (mtDNA) replication defects caused by disturbances in the mtDNA helicase or DNA polymerase γ. We observed that the developmental arrest imposed by either the expression of mutant forms of these enzymes or their knockdown was not rescued by AOX. Considering also the inability of AOX to ameliorate the phenotype of tko25t, a fly mutant with mitochondrial translation deficiency, we infer that this alternative enzyme may not be applicable to cases of mitochondrial gene expression defects. Finding the limitations of AOX applicability will help establish the parameters for the future putative use of this enzyme in gene therapies for human mitochondrial diseases.

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. We found that elevation of mtDNA helicase levels increases the quantity of replication intermediates and alleviates pausing at the replication slow zones. Though we did not observe a concomitant alteration in mtDNA copy number, we observed deletions specific to the segment of repeated elements in the immediate vicinity of the origin of replication, and an accumulation of species characteristic of replication fork stalling. We also found elevated levels of RNA that are retained in the replication intermediates. Together, our results suggest that upregulation of mtDNA helicase promotes the process of mtDNA replication but also results in genome destabilization.

Loss of mtDNA activates astrocytes and leads to spongiotic encephalopathy

Mitochondrial dysfunction manifests as different neurological diseases, but the mechanisms underlying the clinical variability remain poorly understood. To clarify whether different brain cells have differential sensitivity to mitochondrial dysfunction, we induced mitochondrial DNA (mtDNA) depletion in either neurons or astrocytes of mice, by inactivating Twinkle (TwKO), the replicative mtDNA helicase. Here we show that astrocytes, the most abundant cerebral cell type, are chronically activated upon mtDNA loss, leading to early-onset spongiotic degeneration of brain parenchyma, microgliosis and secondary neurodegeneration. Neuronal mtDNA loss does not, however, cause symptoms until 8 months of age. Findings in astrocyte-TwKO mimic neuropathology of Alpers syndrome, infantile-onset mitochondrial spongiotic encephalopathy caused by mtDNA maintenance defects. Our evidence indicates that (1) astrocytes are dependent on mtDNA integrity; (2) mitochondrial metabolism contributes to their activation; (3) chronic astrocyte activation has devastating consequences, underlying spongiotic encephalopathy; and that (4) astrocytes are a potential target for interventions.

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. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research.

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys(68), Cys(71), Cys(102), and Cys(105)) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent Kd of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.

Structure, function and evolution of the mitochondrial replicative DNA helicase (570.1)

Mitochondrial DNA (mtDNA) helicase is an essential part of the mtDNA replisome. The mtDNA helicase shows high amino acid sequence homology with bacteriophage T7 gene 4 protein (T7gp4), which has both DNA primase and DNA helicase activities in its N‐terminal domain (NTD) and C‐terminal domain (CTD), respectively. Although the NTD of mtDNA helicase in metazoa does not have detectable primase activity, retention of extensive amino acid sequence homology throughout most eukaryotic lineages suggests other important roles, such as in protein stability and/ or protein‐protein interactions. Here, we investigate the structural and biochemical properties of the NTD of Drosophila mtDNA helicase by constructing two N‐terminal truncated variants: a full‐length NTD (including both the zinc binding domain (ZBD) and the RNA polymerase domain (RPD)), and a ZBD only construct. We show that NTD and ZBD constructs of Drosophila mtDNA helicase contain an iron‐sulfur cluster, most likely a 2Fe‐2S, as determined by mass spectrometry, and UV‐visible and atomic absorption spectroscopy. Mutagenesis studies show that the four conserved cysteine residues in the ZBD responsible for binding zinc in T7gp4, bind the iron‐sulfur cluster. We extend our biochemical studies in vivo through several approaches to identify partner proteins of the Drosophila mtDNA helicase that may elucidate the functional role of its iron‐sulfur cluster.Grant Funding Source: NIH GM45295

Modes of mitochondrial DNA replication and its regulation in Drosophila melanogaster (573.3)

Although replication of the mitochondrial DNA genome has been studied extensively in mammalian and yeast models, the mechanisms of mitochondrial DNA (mtDNA) replication in the widely‐used model organism Drosophila melanogaster remain largely unknown. We employ a comparative approach to analyze mtDNA replication intermediates using two‐dimensional neutral agarose gel electrophoresis (2DNAGE), and to visualize their architecture by transmission electron microscopy (TEM). Our studies target the nuclear‐encoded replisome proteins (DNA polymerase γ, mtDNA helicase and single‐stranded DNA‐binding protein), as well as transcription factors (mTTF and mTerf5), and their roles in the regulation of mtDNA replication through overexpression of both wild type proteins and biochemical variants in cultured insect cells and fly strains. Whereas 2DNAGE allows the analysis of minimally manipulated DNA preparations, TEM provides molecular details regarding the length of Okazaki fragments and on the presence of double‐stranded DNA and/ or RNA loops at the replication fork. In addition to the evaluation of the modes of replication in normal and perturbed cells, biotin labeling of the replisome proteins enables the determination of their positions on the replicating mtDNA molecule. Together, these methods can be used to identify novel structures and proteins involved in the progression of the replication process.Grant Funding Source: Supported by Academy of Finland and NIH GM45295

Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication

Mitochondrial DNA (mtDNA) is organized in discrete protein–DNA complexes, nucleoids, that are usually considered to be mitochondrial-inner-membrane associated. Here we addressed the association of replication factors with nucleoids and show that endogenous mtDNA helicase Twinkle and single-stranded DNA-binding protein, mtSSB, co-localize only with a subset of nucleoids. Using nucleotide analogs to identify replicating mtDNA in situ, the fraction of label-positive nucleoids that is Twinkle/mtSSB positive, is highest with the shortest labeling-pulse. In addition, the recruitment of mtSSB is shown to be Twinkle dependent. These proteins thus transiently associate with mtDNA in an ordered manner to facilitate replication. To understand the nature of mtDNA replication complexes, we examined nucleoid protein membrane association and show that endogenous Twinkle is firmly membrane associated even in the absence of mtDNA, whereas mtSSB and other nucleoid-associated proteins are found in both membrane-bound and soluble fractions. Likewise, a substantial amount of mtDNA is found as soluble or loosely membrane bound. We show that, by manipulation of Twinkle levels, mtDNA membrane association is partially dependent on Twinkle. Our results thus show that Twinkle recruits or is assembled with mtDNA at the inner membrane to form a replication platform and amount to the first clear demonstration that nucleoids are dynamic both in composition and concurrent activity.

The Overexpression of Twinkle Helicase Ameliorates the Progression of Cardiac Fibrosis and Heart Failure in Pressure Overload Model in Mice

Myocardial mitochondrial DNA (mtDNA) copy number decreases in heart failure. In post-myocardial infarction mice, increasing mtDNA copy number by overexpressing mitochondrial transcription factor attenuates mtDNA deficiency and ameliorates pathological remodeling thereby markedly improving survival. However, the functional significance of increased mtDNA copy number in hypertensive heart disease remains unknown. We addressed this question using transgenic mice that overexpress Twinkle helicase (Twinkle; Tg), the mtDNA helicase, and examined whether Twinkle overexpression protects the heart from left ventricular (LV) remodeling and failure after pressure overload created by transverse aortic constriction (TAC). Twinkle overexpression increased mtDNA copy number by 2.2±0.1-fold. Heart weight, LV diastolic volume and wall thickness were comparable between Tg and wild type littermates (WT) at 28 days after TAC operation. LV end-diastolic pressure increased in WT after TAC (8.6±2.8 mmHg), and this increase was attenuated in Tg (4.6±2.6 mmHg). Impaired LV fractional shortening after TAC operation was also suppressed in Tg, as measured by echocardiography (WT: 16.2±7.2% vs Tg: 20.7±6.2%). These LV functional improvements were accompanied by a decrease in interstitial fibrosis (WT: 10.6±1.1% vs Tg: 3.0±0.6%). In in vitro studies, overexpressing Twinkle using an adenovirus vector in cultured cardiac fibroblasts significantly suppressed mRNA of collagen 1a, collagen 3a and connective tissue growth factor, and angiotensin II-induced transforming growth factor β1 expression. The findings suggest that Twinkle overexpression prevents LV function deterioration. In conclusion, Twinkle overexpression increases mtDNA copy number and ameliorates the progression of LV fibrosis and heart failure in a mouse pressure overload model. Increasing mtDNA copy number by Twinkle overexpression could be a novel therapeutic strategy for hypertensive heart disease.

Towards Efficient Reconstitution of the Human Mitochondrial DNA Replication Complex

The minimal human mitochondrial DNA replisome, comprising mitochondrial DNA polymerase (Pol γ), mitochondrial DNA helicase (mtDNA helicase) and mitochondrial single-stranded binding protein (mtSSB) was reconstituted. Assembly of these replication factors on the minicircle template, resembling the replication fork in vivo, supported the rolling-circle DNA replication. Quantitative analysis of the product formed from the minicircle template with the radioactive label on the primer strand or on the incoming ribonucleotides yielded consistent results, which demonstrated the poor efficiency (less than 1% active fraction) of the reconstituted replisome described (Korhonen et al., 2003). We sought to screen conditions to optimize the assembly of the replisome. The optimized assembly of replisome improves the replication efficiency to ∼14%. The relative low efficiency may indicate a poor assembly of mtDNA helicase or that the mitochondrial replisome in vivo comprises unidentified essential components. Notably, we found that stimulation of processive DNA synthesis by mtSSB occurs over a narrow range of salt concentration. We obtained more quantitative reconstitution the human mitochondrial replisome using a small synthetic replication fork and measured the kinetics of coupled DNA unwinding and polymerization. Optimal reconstitution was achieved by preincubation of the DNA with the polymerase, helicase and two of the four deoxyribonucleotides. Following the addition of the remaining two nucleotides, 80% of the primers were extended with a half-life of 84 seconds.

Replicating animal mitochondrial DNA

The field of mitochondrial DNA (mtDNA) replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s) used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark) has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading- and lagging-strand synthesis (resembling bacterial genome replication) and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS). The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase γ, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase). Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

The N‐terminal Primase‐like Domain of the Drosophila Mitochondrial Replicative DNA Helicase Binds an Iron Sulfur Cluster: Structural and Functional Implications

The N‐terminal region of the metazoan mitochondrial replicative DNA helicase (mtDNA helicase) shares extensive sequence conservation with the primase domain of bacteriophage T7 primase‐helicase. Primase activity has not been demonstrated in metazoan mtDNA helicases, most likely because several Mg2+‐binding residues of the primase active site in T7 primase are not conserved in Metazoa, although several conserved primase sequence motifs are well conserved. Interestingly, insects have retained the four cysteines of conserved motif I that have been shown to bind zinc in the T7 enzyme. Therefore, to study the function of the N‐terminal domain of metazoan mtDNA helicase, we designed two N‐terminal constructs of the Drosophila melanogaster protein comprising residues N24‐A333 (NTD) and N24‐P123 (ZBD). Purified fractions of the full N‐terminal domain construct (NTD) contain an iron sulfur cluster, as determined by UV‐Visible spectroscopy, as well as by iron and sulfide determination. Based on T7 homology, the NTD contains the conserved zinc binding (ZBD) and RNA polymerase (RPD) domains. Purified fractions of the ZBD construct also contained an Fe‐S cluster, suggesting the zinc‐binding site has been converted to an Fe‐S binding site in the Dm mtDNA helicase. We will present data using differential scanning fluorimetry, fluorescence resonance energy transfer and differential scanning calorimetry regarding the involvement of the Fe‐S cluster in the structural stability and in nucleic acid ligand binding by the NTD form.