Primase Domain Research Articles

Initial Primer Synthesis of a DNA Primase Monitored by Real-Time NMR Spectroscopy.

Primases are crucial enzymes for DNA replication, as they synthesize a short primer required for initiating DNA replication. We herein present time-resolved nuclear magnetic resonance (NMR) spectroscopy in solution and in the solid state to study the initial dinucleotide formation reaction of archaeal pRN1 primase. Our findings show that the helix-bundle domain (HBD) of pRN1 primase prepares the two substrates and then hands them over to the catalytic domain to initiate the reaction. By using nucleotide triphosphate analogues, the reaction is substantially slowed down, allowing us to study the initial dinucleotide formation in real time. We show that the sedimented protein-DNA complex remains active in the solid-state NMR rotor and that time-resolved 31P-detected cross-polarization experiments allow monitoring the kinetics of dinucleotide formation. The kinetics in the sedimented protein sample are comparable to those determined by solution-state NMR. Protein conformational changes during primer synthesis are observed in time-resolved 1H-detected experiments at fast magic-angle spinning frequencies (100 kHz). A significant number of spectral changes cluster in the HBD pointing to the importance of the HBD for positioning the nucleotides and the dinucleotide.

Structural insight into the assembly and working mechanism of helicase-primase D5 from Mpox virus.

The Mpox pandemic, caused by the Mpox virus (or monkeypox virus, MPXV), has gained global attention. The D5 protein, a putative helicase-primase found in MPXV, plays a vital role in viral replication and genome uncoating. Here we determined multiple cryo-EM structures of full-length hexameric D5 in diverse states. These states were captured during ATP hydrolysis while moving along the single-stranded DNA (ssDNA) track. Through comprehensive structural analysis combined with the helicase activity system, we revealed that when the primase domain is truncated or the interaction between the primase and helicase domains is disrupted, the double-stranded DNA (dsDNA) unwinds into ssDNA, suggesting a critical regulatory role of the primase domain. Two transition states bound with ssDNA substrate during unwinding reveals that two ATP molecules were consumed to drive DNA moving forward two nucleotides. Collectively, our findings shed light on the molecular mechanism that links ATP hydrolysis to the DNA unwinding in poxviruses.

The plant organellar primase-helicase directs template recognition and primosome assembly via its zinc finger domain

BackgroundThe mechanisms and regulation for DNA replication in plant organelles are largely unknown, as few proteins involved in replisome assembly have been biochemically studied. A primase-helicase dubbed Twinkle (T7 gp4-like protein with intramitochondrial nucleoid localization) unwinds double-stranded DNA in metazoan mitochondria and plant organelles. Twinkle in plants is a bifunctional enzyme with an active primase module. This contrast with animal Twinkle in which the primase module is inactive. The organellar primase-helicase of Arabidopsis thaliana (AtTwinkle) harbors a primase module (AtPrimase) that consists of an RNA polymerase domain (RPD) and a Zn + + finger domain (ZFD).ResultsHerein, we investigate the mechanisms by which AtTwinkle recognizes its templating sequence and how primer synthesis and coupling to the organellar DNA polymerases occurs. Biochemical data show that the ZFD of the AtPrimase module is responsible for template recognition, and this recognition is achieved by residues N163, R166, and K168. The role of the ZFD in template recognition was also corroborated by swapping the RPDs of bacteriophage T7 primase and AtPrimase with their respective ZFDs. A chimeric primase harboring the ZFD of T7 primase and the RPD of AtPrimase synthesizes ribonucleotides from the T7 primase recognition sequence and conversely, a chimeric primase harboring the ZFD of AtPrimase and the RPD of T7 primase synthesizes ribonucleotides from the AtPrimase recognition sequence. A chimera harboring the RPDs of bacteriophage T7 and the ZBD of AtTwinkle efficiently synthesizes primers for the plant organellar DNA polymerase.ConclusionsWe conclude that the ZFD is responsible for recognizing a single-stranded sequence and for primer hand-off into the organellar DNA polymerases active site. The primase activity of plant Twinkle is consistent with phylogeny-based reconstructions that concluded that Twinkle´s last eukaryotic common ancestor (LECA) was an enzyme with primase and helicase activities. In plants, the primase domain is active, whereas the primase activity was lost in metazoans. Our data supports the notion that AtTwinkle synthesizes primers at the lagging-strand of the organellar replication fork.

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA.

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity.

Bacterial genomes contain an abundance of transposable insertion sequence (IS) elements that are essential for genome evolution and fitness. Among them, IS629 is present in most strains of enterohemorrhagic Escherichia coli O157 and accounts for many polymorphisms associated with gene inactivation and/or genomic deletions. The excision of IS629 from the genome is promoted by IS-excision enhancer (IEE) protein. Despite IEE has been identified in the most pathogenic serotypes of E. coli, its biochemical features that could explain its role in IS excision are not yet understood. We show that IEE is present in >30% of all available E. coli genome assemblies, and is highly conserved and very abundant within enterohemorrhagic, enteropathogenic and enterotoxigenic genomes. In vitro analysis of the recombinant protein from E. coli O157:H7 revealed the presence of a Mn2+-dependent error-prone DNA polymerase activity in its N-terminal archaeo-eukaryotic primase (AEP) domain able to promote dislocations of the primer and template strands. Importantly, IEE could efficiently perform in vitro an end-joining reaction of 3'-single-strand DNA overhangs with≥4 bp of homology requiring both the N-terminal AEP and C-terminal helicase domains. The proposed role for IEE in the novel IS excision mechanism is discussed.

Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA.

Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5'-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5'-triphosphate (ATP) at the subunit interface stabilizes the subunit-subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5'-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein-protein and protein-DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein-ssDNA force field and elucidate the molecular basis of replicative helicase translocation.

Residues located in the primase domain of the bacteriophage T7 primase-helicase are essential for loading the hexameric complex onto DNA

The T7 primase-helicase plays a pivotal role in the replication of T7 DNA. Using affinity isolation of peptide–nucleic acid crosslinks and mass spectrometry, we identify protein regions in the primase-helicase and T7 DNA polymerase that form contacts with the RNA primer and DNA template. The contacts between nucleic acids and the primase domain of the primase-helicase are centered in the RNA polymerase subdomain of the primase domain, in a cleft between the N-terminal subdomain and the topoisomerase-primase fold. We demonstrate that residues along a beta sheet in the N-terminal subdomain that contacts the RNA primer are essential for phage growth and primase activity in vitro. Surprisingly, we found mutations in the primase domain that had a dramatic effect on the helicase. Substitution of a residue conserved in other DnaG-like enzymes, R84A, abrogates both primase and helicase enzymatic activities of the T7 primase-helicase. Alterations in this residue also decrease binding of the primase-helicase to ssDNA. However, mass photometry measurements show that these mutations do not interfere with the ability of the protein to form the active hexamer.

DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain

The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB-DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB-DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I-binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46 DnaB Leu160 is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I-bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA-DnaB-DnaC complex, which could be relevant to the process of DnaB loading onto oriC.

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.

Arabidopsis thaliana organelles mimic the T7 phage DNA replisome with specific interactions between Twinkle protein and DNA polymerases Pol1A and Pol1B

BackgroundPlant chloroplasts and mitochondria utilize nuclear encoded proteins to replicate their DNA. These proteins are purposely built for replication in the organelle environment and are distinct from those involved in replication of the nuclear genome. These organelle-localized proteins have ancestral roots in bacterial and bacteriophage genes, supporting the endosymbiotic theory of their origin. We examined the interactions between three of these proteins from Arabidopsis thaliana: a DNA helicase-primase similar to bacteriophage T7 gp4 protein and animal mitochondrial Twinkle, and two DNA polymerases, Pol1A and Pol1B. We used a three-pronged approach to analyze the interactions, including Yeast-two-hybrid analysis, Direct Coupling Analysis (DCA), and thermophoresis.ResultsYeast-two-hybrid analysis reveals residues 120–295 of Twinkle as the minimal region that can still interact with Pol1A or Pol1B. This region is a part of the primase domain of the protein and slightly overlaps the zinc-finger and RNA polymerase subdomains located within. Additionally, we observed that Arabidopsis Twinkle interacts much more strongly with Pol1A versus Pol1B. Thermophoresis also confirms that the primase domain of Twinkle has higher binding affinity than any other region of the protein. Direct-Coupling-Analysis identified specific residues in Twinkle and the DNA polymerases critical to positive interaction between the two proteins.ConclusionsThe interaction of Twinkle with Pol1A or Pol1B mimics the minimal DNA replisomes of T7 phage and those present in mammalian mitochondria. However, while T7 and mammals absolutely require their homolog of Twinkle DNA helicase-primase, Arabidopsis Twinkle mutants are seemingly unaffected by this loss. This implies that while Arabidopsis mitochondria mimic minimal replisomes from T7 and mammalian mitochondria, there is an extra level of redundancy specific to loss of Twinkle function.

Crystal structure and biochemical studies of the bifunctional DNA primase-polymerase from phage NrS-1

A novel DNA polymerase found in the deep-sea vent phage NrS-1, was confirmed to have both DNA polymerase and primase activities. In this polymerase, the N-terminal residues 1–300 (referred to as N300) are the core region required for polymerizing DNA and catalyzing de novo DNA synthesis. Here, the crystal structure of N300 was solved at a resolution of 1.80 Å. The overall structure consists of a prim/pol domain and a helix bundle domain, which are separated by a 14-residue-long flexible tether (residues 177–190). Both the prim/pol domain of N300 and other primase–polymerases (prim-pol) encompass an analogous fold with conserved catalytic residues. Mutagenesis and enzymatic activity assays show that the acidic active-site residue E139 is required for both polymerase and primase activities. Functional assays confirm the essentiality of the helix bundle domain for primase activity. Furthermore, we identified a mutant (N300-Y261A) of the helix bundle domain, which probably plays an indispensable role in the primer initiation and recognition of template DNA.

Deciphering the Essential Interaction between Primase and Helicase in Mycobacterium tuberculosis

The helicase-primase interaction is an essential event in DNA replication and is mediated by highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation with a low sequence homology of the DnaB binding domain of the DnaG among the eubacteria, we determined the crystal structure of the helicase binding domain of DnaG from M. tuberculosis (MtDnaG-CTD) at 1.58 Å. The overall structure of MtDnaG-CTD consists two subdomains, the N-terminal globe region (GR) and the C-terminal helix hairpin region (HHR) connected with a small loop. On comparing with other DnaG-CTDs structures, the MtDnaG-CTD structure shows conservation of the folds primarily in the GR. Further, to insight into the crucial helicase-primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor binding studies show ten-fold decrease in binding affinity of MtDnaB-NTD with mutant Ile605Ala MtDnaG-CTD (KD-250 nM) compared to native MtDnaG-CTD (KD-2.5 μM). The loop, connecting both helixes of the HHR, was largely responsible for the stability of DnaB-DnaG complex. The MtDnaB-NTD shows the micromolar affinity with DnaG-CTDs from E. coli and H. pylori while the MtDnaB-NTD shows unstable binding with DnaG-CTD from V. cholerae. The interaction pattern of DnaG and DnaB seems to be unique for an organism. The conserved catalytic domains and highly variable interacting domains of both DnaG and DnaB suggest the species-specific evolution of the system.

Theoretical Chemistry Answers Bimolecular Signaling Debate in [4Fe4S] Proteins

In this issue of Chem, Migliore and co-workers performed a theoretical study on redox signaling between the [4Fe4S] clusters in the C-terminal domain of DNA primase and polymerase α. They found that redox signaling cannot be accomplished solely via DNA-mediated charge transport, which requires [4Fe4S]-to-DNA and DNA-to-[4Fe4S] charge transfer.

Structural basis for adPEO-causing mutations in the mitochondrial TWINKLE helicase.

TWINKLE is the helicase involved in replication and maintenance of mitochondrial DNA (mtDNA) in mammalian cells. Structurally, TWINKLE is closely related to the bacteriophage T7 gp4 protein and comprises a helicase and primase domain joined by a flexible linker region. Mutations in and around this linker region are responsible for autosomal dominant progressive external ophthalmoplegia (adPEO), a neuromuscular disorder associated with deletions in mtDNA. The underlying molecular basis of adPEO-causing mutations remains unclear, but defects in TWINKLE oligomerization are thought to play a major role. In this study, we have characterized these disease variants by single-particle electron microscopy and can link the diminished activities of the TWINKLE variants to altered oligomeric properties. Our results suggest that the mutations can be divided into those that (i) destroy the flexibility of the linker region, (ii) inhibit ring closure and (iii) change the number of subunits within a helicase ring. Furthermore, we demonstrate that wild-type TWINKLE undergoes large-scale conformational changes upon nucleoside triphosphate binding and that this ability is lost in the disease-causing variants. This represents a substantial advancement in the understanding of the molecular basis of adPEO and related pathologies and may aid in the development of future therapeutic strategies.

Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis.

The helicase-primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase-primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB-DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.

DnaC, the indispensable companion of DnaB helicase, controls the accessibility of DnaB helicase by primase

Former studies relying on hydrogen/deuterium exchange analysis suggest that DnaC bound to DnaB alters the conformation of the N-terminal domain (NTD) of DnaB to impair the ability of this DNA helicase to interact with primase. Supporting this idea, the work described herein based on biosensor experiments and enzyme-linked immunosorbent assays shows that the DnaB-DnaC complex binds poorly to primase in comparison with DnaB alone. Using a structural model of DnaB complexed with the C-terminal domain of primase, we found that Ile-85 is located at the interface in the NTD of DnaB that contacts primase. An alanine substitution for Ile-85 specifically interfered with this interaction and impeded DnaB function in DNA replication, but not its activity as a DNA helicase or its ability to bind to ssDNA. By comparison, substitutions of Asn for Ile-136 (I136N) and Thr for Ile-142 (I142T) in a subdomain previously named the helical hairpin in the NTD of DnaB altered the conformation of the helical hairpin and/or compromised its pairwise arrangement with the companion subdomain in each brace of protomers of the DnaB hexamer. In contrast with the I85A mutant, the latter were defective in DNA replication due to impaired binding to both ssDNA and primase. In view of these findings, we propose that DnaC controls the ability of DnaB to interact with primase by modifying the conformation of the NTD of DnaB.

The Polyphyletic Origins of Primase-Helicase Bifunctional Proteins.

We studied the evolutionary relationships of different primase-helicase bifunctional proteins, found mostly in viruses, virophages, plasmids, and organellar genomes, by phylogeny and correlation analysis. Our study suggests independent origins of primase-helicase bifunctional proteins resulting from multiple fusion events between genes encoding primase and helicase domains of different families. The correlation analysis further indicated strong functional dependencies of domains in the bifunctional proteins that are part of smaller genomes and plasmids. Bifunctional proteins found in some bacterial genomes exhibited weak coevolution probably suggesting that these are the non-functional remnants of the proteins acquired via horizontal transfer. We have put forward possible scenarios for the origin of primase-helicase bifunctional proteins in large eukaryotic DNA viruses and virophages.

Response to Comments on "The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport".

Baranovskiy et al and Pellegrini argue that, based on structural data, the path for charge transfer through the [4Fe4S] domain of primase is not feasible. Our manuscript presents electrochemical data directly showing charge transport through DNA to the [4Fe4S] cluster of a primase p58C construct and a reversible switch in the DNA-bound signal with oxidation/reduction, which is inhibited by mutation of three tyrosine residues. Although the dispositions of tyrosines differ in different constructs, all are within range for microsecond electron transfer.

Comment on "The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport".

O'Brien et al (Research Article, 24 February 2017, eaag1789) report that the iron-sulfur cluster of primase has a redox role in enzyme activity. Their analysis is based on a partially misfolded structure of the iron-sulfur cluster domain of primase. In the correctly folded structure, two of the three tyrosines putatively involved in electron transfer, Y345 and Y347, contact the RNA/DNA helix, providing an alternative explanation for the data of O'Brien et al.

Modulation of RNA primer formation by Mn(II)-substituted T7 DNA primase

Lagging strand DNA synthesis by DNA polymerase requires RNA primers produced by DNA primase. The N-terminal primase domain of the gene 4 protein of phage T7 comprises a zinc-binding domain that recognizes a specific DNA sequence and an RNA polymerase domain that catalyzes RNA polymerization. Based on its crystal structure, the RNA polymerase domain contains two Mg(II) ions. Mn(II) substitution leads to elevated RNA primer synthesis by T7 DNA primase. NMR analysis revealed that upon binding Mn(II), T7 DNA primase undergoes conformational changes near the metal cofactor binding site that are not observed when the enzyme binds Mg(II). A machine-learning algorithm called linear discriminant analysis (LDA) was trained by using the large collection of Mn(II) and Mg(II) binding sites available in the protein data bank (PDB). Application of the model to DNA primase revealed a preference in the enzyme’s second metal binding site for Mn(II) over Mg(II), suggesting that T7 DNA primase activity modulation when bound to Mn(II) is based on structural changes in the enzyme.