Mitochondrial DNA Helicase Research Articles

Resolving Phenotypic Variability in Mitochondrial Diseases: Preliminary Findings of a Proteomic Approach

The introduction of new sequencing approaches into clinical practice has radically changed the diagnostic approach to mitochondrial diseases, significantly improving the molecular definition rate in this group of neurogenetic disorders. At the same time, there have been no equal successes in the area of in-depth understanding of disease mechanisms and few innovative therapeutic approaches have been proposed recently. In this regard, the identification of the molecular basis of phenotypic variability in primary mitochondrial disorders represents a key aspect for deciphering disease mechanisms with important therapeutic implications. In this study, we present data from proteomic investigations in two subjects affected by mitochondrial disease characterized by a different clinical severity and associated with the same variant in the TWNK gene, encoding the mitochondrial DNA and RNA helicase with a specific role in the mtDNA replisome. Heterozygous pathogenic variants in this gene are associated with progressive external ophthalmoplegia and ptosis, usually with adult onset. The overall results suggest an imbalance in glucose metabolism and ROS production/regulation, with possible consequences on the phenotypic manifestations of the enrolled subjects. Although the data will need to be validated in a large cohort, proteomic investigations have proven to be a valid approach for a deep understanding of these neurometabolic disorders.

Cochlear implantation in patients with Charcot-Marie-Tooth disease: two cases with a review of the literature.

To report two cases of bilateral cochlear implantation (CI) in Charcot-Marie-Tooth disease (CMT) patients with novel mutations. Furthermore, we conducted a detailed literature review on the profile and outcomes of CI in this uncommon clinical circumstance. Case 1 involved a 25-year-old woman who was referred for sudden hearing loss (HL) in her left ear and had a 7-year history of HL in her right ear. She was diagnosed with CMT type 1 with a thymidine phosphorylase gene mutation. CI was performed on her left side because her hearing gradually worsened to deafness in both ears. At 3months post-operation, her speech discrimination score without lip-reading improved from 0 to 100%. She underwent a second CI on her right ear 6months after her first CI. Two years from her first operation, the speech discrimination score was 100%. Case 2 received her first CI on her right ear at the age of nine for her bilateral HL. She was diagnosed with CMT type 2 with a Twinkle mitochondrial DNA helicase gene mutation. Preoperatively, the speech discrimination score in both ear-aided conditions was 70%. At the 7-year post-operation follow-up, the speech discrimination score was 76%. A second CI was performed due to decreasing hearing ability in her left ear. The speech discrimination score showed 100% at 7months after the second CI. CI is an effective hearing rehabilitation option for CMT patients with severe-to-profound SNHL. Neuro-otologists should consider CI as a treatment option, even though hearing loss in CMT is associated with auditory neuropathy spectrum disease (ANSD).

Method for the structural analysis of Twinkle mitochondrial DNA helicase by cryo-EM.

The mitochondrial replisome replicates the 16.6kb mitochondria DNA (mtDNA). The proper functioning of this multicomponent protein complex is vital for the integrity of the mitochondrial genome. One of the critical protein components of the mitochondrial replisome is the Twinkle helicase, a member of the Superfamily 4 (SF4) helicases. Decades of research has uncovered common themes among SF4 helicases including self-assembly, ATP-dependent translocation, and formation of protein-protein complexes. Some of the molecular details of these processes are still unknown for the mitochondria SF4 helicase, Twinkle. Here, we describe a protocol for expression, purification, and single-particle cryo-electron microscopy of the Twinkle helicase clinical variant, W315L, which resulted in the first high-resolution structure of Twinkle helicase. The methods described here serve as an adaptable protocol to support future high-resolution studies of Twinkle helicase or other SF4 helicases.

Clinical, Histological, and Genetic Features of 25 Patients with Autosomal Dominant Progressive External Ophthalmoplegia (ad-PEO)/PEO-Plus Due to TWNK Mutations.

Autosomal dominant mutations in the TWNK gene, which encodes a mitochondrial DNA helicase, cause adult-onset progressive external ophthalmoplegia (PEO) and PEO-plus presentations. In this retrospective observational study, we describe clinical and complementary data from 25 PEO patients with mutations in TWNK recruited from the Hospital 12 de Octubre Mitochondrial Disorders Laboratory Database. The mean ages of onset and diagnosis were 43 and 63 years, respectively. Family history was positive in 22 patients. Ptosis and PEO (92% and 80%) were the most common findings. Weakness was present in 48%, affecting proximal limbs, neck, and bulbar muscles. Exercise intolerance was present in 28%. Less frequent manifestations were cardiac (24%) and respiratory (4%) involvement, neuropathy (8%), ataxia (4%), and parkinsonism (4%). Only 28% had mild hyperCKemia. All 19 available muscle biopsies showed signs of mitochondrial dysfunction. Ten different TWNK mutations were identified, with c.1361T>G (p.Val454Gly) and c.1070G>C (p.Arg357Pro) being the most common. Before definitive genetic confirmation, 56% of patients were misdiagnosed (36% with myasthenia, 20% with oculopharyngeal muscle dystrophy). Accurate differential diagnosis and early confirmation with appropriately chosen complementary studies allow genetic counseling and the avoidance of unnecessary treatments. Thus, mitochondrial myopathies must be considered in PEO/PEO-plus presentations, and particularly, TWNK is an important cause when positive family history is present.

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.

Tau Pathology Associated With Parkinsonism and Mutation of Mitochondrial DNA Helicase Gene TWNK.

The TWNK gene encodes Twinkle, the mitochondrial DNA helicase that cooperates with the mitochondrial DNA polymerase (POLG) to maintain mitochondrial DNA integrity. Heterozygous TWNK mutations cause autosomal dominant progressive external ophthalmoplegia (PEO). Some patients with TWNK -linked PEO additionally develop late-onset neurodegenerative parkinsonism.1 However, little is known about the neuropathology of TWNK -linked parkinsonism. In this study, we describe neuropathologic findings in a patient with PEO and parkinsonism and a heterozygous TWNK mutation. The authors thank Dr. Guy Wilms (University Hospitals Leuven) for performing MRI, Dr. Valérie Race (University Hospitals Leuven) for genetic analysis, Dr. Peter Davies (Albert Einstein College of Medicine, New York, USA) for the gift of the PHF1 and MC1 antibodies, and the J. David Gladstone Institutes (San Francisco, USA) for the gift of the acK274-tau antibody (MAB359). The authors thank Dr. Guy Wilms (University Hospitals Leuven) for performing MRI, Dr. Valérie Race (University Hospitals Leuven) for genetic analysis, Dr. Peter Davies (Albert Einstein College of Medicine, New York, USA) for the gift of the PHF1 and MC1 antibodies, and the J. David Gladstone Institutes (San Francisco, USA) for the gift of the acK274-tau antibody (MAB359).

Defective mitochondrial function by mutation in THICK ALEURONE 1 encoding a mitochondrion-targeted single-stranded DNA-binding protein leads to increased aleurone cell layers and improved nutrition in rice

Cereal endosperm comprises an outer aleurone and an inner starchy endosperm. Although these two tissues have the same developmental origin, they differ in morphology, cell fate, and storage product accumulation, with the mechanism largely unknown. Here, we report the identification and characterization of rice thick aleurone 1 (ta1) mutant that shows an increased number of aleurone cell layers and increased contents of nutritional factors including proteins, lipids, vitamins, dietary fibers, and micronutrients. We identified that the TA1 gene, which is expressed in embryo, aleurone, and subaleurone in caryopses, encodes a mitochondrion-targeted protein with single-stranded DNA-binding activity named OsmtSSB1. Cytological analyses revealed that the increased aleurone cell layers in ta1 originate from a developmental switch of subaleurone toward aleurone instead of starchy endosperm in the wild type. We found that TA1/OsmtSSB1 interacts with mitochondrial DNA recombinase RECA3 and DNA helicase TWINKLE, and downregulation of RECA3 or TWINKLE also leads to ta1-like phenotypes. We further showed that mutation in TA1/OsmtSSB1 causes elevated illegitimate recombinations in the mitochondrial genome, altered mitochondrial morphology, and compromised energy supply, suggesting that the OsmtSSB1-mediated mitochondrial function plays a critical role in subaleurone cell-fate determination in rice.

Distinct Roles of the Mitochondrial DNA Helicase Twinkle in Drosophila Neurons and Muscles

Mutations in the human gene TWNK, which encodes the mitochondrial DNA (mtDNA) helicase Twinkle, frequently affect the neurolocomotor system. Patients may present accumulation of multiple large-scale mtDNA deletions in muscle fibers, cerebral cortex, basal ganglia, and cerebellum throughout lifetime, as observed in cases of autosomal dominant progressive external ophthalmoplegia (adPEO). Specially in high energy demanding tissues such as muscles and neurons, mtDNA deletions and/or depletion can affect mitochondrial function, since this genome plays a central role in ATP production by means of oxidative phosphorylation (OXPHOS). Maintaining this genome intact and at the appropriate level requires an array of proteins involved in replication, transcription, repair, and recombination. Although Twinkle may participate in recombinational repair, its roles in the replication process is best understood. We have been using Drosophila melanogaster as a model to understand the role of Twinkle in mtDNA maintenance in a tissue-specific pattern, with focus on the central nervous system and the musculature. We have shown that defective versions of Twinkle that are ubiquitously expressed cause lethality at the larval stage, but directing the expression only to neurons or to muscles does not impact development. Furthermore, our results indicate that neuron-specific, but not muscle-specific, expression of ATPase-deficient versions of Twinkle leads to a dramatic shortening of adult lifespan, which is accompanied by severe locomotor problems. The lack of phenotype upon Twinkle dysfunction in muscles, however, is unexpected considering the high OXPHOS activity and high mtDNA copy number of the Drosophila flight muscles. We also overexpressed Twinkle mutants in Drosophila S2 cells in culture to get a first insight into how mtDNA maintenance may be disturbed. We have previously shown that overexpressing wild-type (WT) Twinkle leads to a decrease in intermediates at two important replication pausing regions in the mitochondrial genome. Interestingly, our preliminary findings indicate that the overexpression of an ATPase-deficient Twinkle mutant causes severe accumulation of replication intermediates at the same pausing sites. Because in whole adult fly samples the vast majority of mitochondria (and therefore mtDNA) come from the flight musculature, it is safe to assume that the mode of mtDNA replication previously described for adult individuals is in fact the mode for muscle mtDNA. In this case, it is intriguing that mtDNA maintenance in S2 cells and in adult flight muscles is so similar, and yet a severely defective Twinkle enzyme causes a strong mtDNA depletion in the former and no detectable phenotype in the latter. Since there is no available effective treatment for adPEO and other mtDNA diseases, determining if, how and when Twinkle participates in DNA maintenance and repair inside mitochondria may provide insights into how to regulate Twinkle's functions to improve conditions of mitochondrial dysfunction.

Human Mitochondrial Helicase Twinkle Catalyzes Toehold Mediated DNA Strand Displacement

DNA nanotechnology often utilizes ‘toeholds,’ which are small single-stranded DNA overhangs, in kinetically controlled complex biochemical circuits for designing nano-devices. We have previously reported that human mitochondrial DNA helicase Twinkle possesses an unexpected DNA annealing activity and can catalyze helicase-coupled homologous DNA strand exchange reactions. Here, we demonstrate that Twinkle can catalyze toehold mediated DNA strand displacement (TMSD) on various DNA substrates. In contrast to the homologous DNA strand exchange, which is driven by Twinkle’s active helicase activity, the observed increase in the rate of Twinkle-catalyzed TMSD is independent of the helicase activity. The Twinkle catalyzed TMSD can be kinetically modulated by changing the length of external or internal toehold domains, therefore providing additional tunability and control over reaction outcomes. Furthermore, we show that the Twinkle catalyzed strand displacement discriminates single base changes, and thus, can be utilized for developing diagnostic probes for the detection of single nucleotide polymorphisms.

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.

Using Atomic Force Microscopy to Study the Real Time Dynamics of DNA Unwinding by Mitochondrial Twinkle Helicase.

Understanding the structure and dynamics of DNA-protein interactions during DNA replication is crucial for elucidating the origins of disorders arising from its dysfunction. In this study, we employed Atomic Force Microscopy as a single-molecule imaging tool to examine the mitochondrial DNA helicase Twinkle and its interactions with DNA. We used imaging in air and time-lapse imaging in liquids to observe the DNA binding and unwinding activities of Twinkle hexamers at the single-molecule level. These procedures helped us visualize Twinkle loading onto and unloading from the DNA in the open-ring conformation. Using traditional methods, it has been shown that Twinkle is capable of unwinding dsDNA up to 20-55 bps. We found that the addition of mitochondrial single-stranded DNA binding protein (mtSSB) facilitates a 5-fold increase in the DNA unwinding rate for the Twinkle helicase. The protocols developed in this study provide new platforms to examine DNA replication and to explore the mechanism driving DNA deletion and human diseases. Graphic abstract: Mitochondrial Twinkle Helicase Dynamics.

Mutation of TWNK Gene Is One of the Reasons of Runting and Stunting Syndrome Characterized by mtDNA Depletion in Sex-Linked Dwarf Chicken.

Runting and stunting syndrome (RSS), which is characterized by low body weight, generally occurs early in life and leads to considerable economic losses in the commercial broiler industry. Our previous study has suggested that RSS is associated with mitochondria dysfunction in sex-linked dwarf (SLD) chickens. However, the molecular mechanism of RSS remains unknown. Based on the molecular diagnostics of mitochondrial diseases, we identified a recessive mutation c. 409G > A (p. Ala137Thr) of Twinkle mitochondrial DNA helicase (TWNK) gene and mitochondrial DNA (mtDNA) depletion in RSS chickens’ livers from strain N301. Bioinformatics investigations supported the pathogenicity of the TWNK mutation that is located on the extended peptide linker of Twinkle primase domain and might further lead to mtDNA depletion in chicken. Furthermore, overexpression of wild-type TWNK increases mtDNA copy number, whereas overexpression of TWNK A137T causes mtDNA depletion in vitro. Additionally, the TWNK c. 409G > A mutation showed significant associations with body weight, daily gain, pectoralis weight, crureus weight, and abdominal fat weight. Taken together, we corroborated that the recessive TWNK c. 409G > A (p. Ala137Thr) mutation is associated with RSS characterized by mtDNA depletion in SLD chicken.

The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication

BackgroundDNA replication requires contributions from various proteins, such as DNA helicases; in mitochondria Twinkle is important for maintaining and replicating mitochondrial DNA. Twinkle helicases are predicted to also possess primase activity, as has been shown in plants; however this activity appears to have been lost in metazoans. Given this, the study of Twinkle in other organisms is required to better understand the evolution of this family and the roles it performs within mitochondria.ResultsHere we describe the characterization of a Twinkle homologue, Twm1, in the amoeba Dictyostelium discoideum, a model organism for mitochondrial genetics and disease. We show that Twm1 is important for mitochondrial function as it maintains mitochondrial DNA copy number in vivo. Twm1 is a helicase which unwinds DNA resembling open forks, although it can act upon substrates with a single 3′ overhang, albeit less efficiently. Furthermore, unlike human Twinkle, Twm1 has primase activity in vitro. Finally, using a novel in bacterio approach, we demonstrated that Twm1 promotes DNA replication.ConclusionsWe conclude that Twm1 is a replicative mitochondrial DNA helicase which is capable of priming DNA for replication. Our results also suggest that non-metazoan Twinkle could function in the initiation of mitochondrial DNA replication. While further work is required, this study has illuminated several alternative processes of mitochondrial DNA maintenance which might also be performed by the Twinkle family of helicases.

Linear mitochondrial DNA is rapidly degraded by components of the replication machinery

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5′-3′exonuclease MGME1, elimination of the 3′-5′exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.

Infantile onset spinocerebellar ataxia caused by compound heterozygosity for Twinkle mutations and modeling of Twinkle mutations causing recessive disease.

Mutations in nuclear genes required for the replication and maintenance of mitochondrial DNA cause progressive multisystemic neuromuscular disorders with overlapping phenotypes. Biallelic mutations in C10orf2, encoding the Twinkle mitochondrial DNA helicase, lead to infantile-onset cerebellar ataxia (IOSCA), as well as milder and more severe phenotypes. We present a 13-year-old girl with ataxia, severe hearing loss, optic atrophy, peripheral neuropathy, and hypergonadotropic hypogonadism. Whole-exome sequencing revealed that the patient is compound heterozygous for previously unreported variants in the C10orf2 gene: a paternally inherited frameshift variant (c.333delT; p.L112Sfs*3) and a maternally inherited missense variant (c.904C>T; p.R302W). The identification of novel C10orf2 mutations extends the spectrum of mutations in the Twinkle helicase causing recessive disease, in particular the intermediate IOSCA phenotype. Structural modeling suggests that the p.R302W mutation and many other recessively inherited Twinkle mutations impact the position or interactions of the linker region, which is critical for the oligomeric ring structure and activity of the helicase. This study emphasizes the utility of whole-exome sequencing for the genetic diagnosis of a complex multisystemic disorder.

Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA.

Purification and Comparative Assay of the Human Mitochondrial Replicative DNA Helicase.

The replicative mitochondrial DNA (mtDNA) helicase is essential for mtDNA replication and maintenance of the mitochondrial genome. Despite substantial advances that have been made in its characterization, there is still much to be understood about the functional roles of its domains and its interactions with the other components of the minimal mitochondrial DNA replisome. Critical to achieving this is the ability to isolate the enzyme in a stable, active form. In this chapter we describe a modified, streamlined purification strategy for recombinant forms of the enzyme. We also present assays to assess its helix unwinding activity and the stimulatory effects of the mitochondrial single-stranded DNA-binding protein (mtSSB). Finally, we describe a concentration/buffer exchange method that we have employed to achieve greater enzyme stability and appropriate conditions for biochemical and biophysical studies.

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.

Alpers disease mutations in human DNA polymerase gamma cause catalytic defects in mitochondrial DNA replication by distinct mechanisms.

The human mitochondrial DNA polymerase gamma (Pol-γ) is nuclearly encoded and is responsible for the replication and repair of the mitochondrial genome. Mutations S305R and P1073L in the POLG gene have been reported to be associated with early childhood Alpers syndrome. One patient harboring both mutations as compound heterozygous died at 2 years of age after disease onset at 9 months. Quantitative kinetic analysis on purified enzyme showed that the S305R mutation reduces the DNA binding affinity by 10-fold, and reduces the specificity constant (kcat/Km) for correct nucleotide incorporation by fourfold. It also causes a ∼threefold reduction in the excision rate to remove mismatched nucleotides. Compared to the wild-type Pol-γ, the S305R mutant showed no product formation in a reconstituted rolling circle replisome assay. Interestingly, the P1073L mutant exhibited wild-type activity in single turnover kinetics to quantify changes in kcat/Km, kcat, kexo, or processivity, and showed a twofold decrease in the net polymerization rate in the reconstituted replisome assay, while in yeast, P1073L caused a 60–70% mtDNA reduction in haploid cells. The heterozygous diploid yeast cells carrying S305R and P1073L mutations in trans showed ∼75% reduction of mtDNA content, relative to homozygous diploid cells with two wild-type alleles. Taken together, we show clearly in both the rolling circle and the humanized yeast system that the P1073L mutation caused significant defects in mtDNA replication, and our results suggest a role for P1073 in the functioning of the Pol-γ with the mitochondrial DNA helicase, and provide a rationale for understanding the physiological consequences of the S305R/P1073L compound heterozygote in humans.

Chimeric proteins constructed from bacteriophage T7 gp4 and a putative primase-helicase from Arabidopsis thaliana.

An open reading frame from Arabidopsis thaliana, which is highly homologous to the human mitochondrial DNA helicase TWINKLE, was previously cloned, expressed, and shown to have DNA primase and DNA helicase activity. The level of DNA primase activity of this Arabidopsis Twinkle homolog (ATH) was low, perhaps due to an incomplete zinc binding domain (ZBD). In this study, N-terminal truncations of ATH implicate residues 80-102 interact with the RNA polymerase domain (RPD). In addition, chimeric proteins, constructed using domains from ATH and the well-characterized T7 phage DNA primase-helicase gp4, were created to determine if the weak primase activity of ATH could be enhanced. Two chimeric proteins were constructed: ATHT7 contains the ZBD and RPD domains of ATH tethered to the helicase domain of T7, while T7ATH contains the ZBD and RPD domains of T7 tethered to the helicase domain of ATH. Both chimeric proteins were successfully expressed and purified in E. coli, and assayed for traditional primase and helicase activities. T7ATH was able to generate short oligoribonucleotide primers, but these primers could not be cooperatively extended by a DNA polymerase. Although T7ATH contains the ATH helicase domain, it exhibited few of the characteristics of a functional helicase. ATHT7 lacked primase activity altogether and also demonstrated only weak helicase activities. This work demonstrates the importance of interactions between structurally and functionally distinct domains, especially in recombinant, chimeric proteins.