Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase

BACKGROUND: DNA primases catalyse the synthesis of the short RNA primers that are required for DNA replication by DNA polymerases. Primases comprise three functional domains: a zinc-binding domain that is responsible for template recognition, a polymerase domain, and a domain that interacts with the replicative helicase, DnaB. RESULTS: We present the crystal structure of the zinc-binding domain of DNA primase from Bacillus stearothermophilus, determined at 1.7 A resolution. This is the first high-resolution structural information about any DNA primase. A model is discussed for the interaction of this domain with the single-stranded DNA template. CONCLUSIONS: The structure of the DNA primase zinc-binding domain confirms that the protein belongs to the zinc ribbon subfamily. Structural comparison with other nucleic acid binding proteins suggests that the beta sheet of primase is likely to be the DNA-binding surface, with conserved residues on this surface being involved in the binding and recognition of DNA.     

Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template

The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.     

Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.     

Staphylococcus aureus primase has higher initiation specificity, interacts with single-stranded DNA stronger, but is less stimulated by its helicase than Escherichia coli primase

The study of primases from model organisms such as Escherichia coli , phage T7 and phage T4 has demonstrated the essential nature of primase function, which is to generate de novo RNA polymers to prime DNA polymerase. However, little is known about the function of primases from other eubacteria. Their overall low primary sequence homology may result in functional differences. To help understand which primase functions were conserved, primase and its replication partner helicase from the pathogenic Gram-positive bacteria Staphylococcus aureus were compared in detail with that of E. coli primase and helicase. The conserved properties were to primer initiation and elongation and included slow kinetics, low fidelity and poor sugar specificity. The significant differences included S. aureus primase having sixfold higher kinetic affinity for its template than E. coli primase under equivalent conditions. This naturally higher activity was balanced by its fourfold lower stimulation by its replication fork helicase compared with E. coli primase. The most significant difference between the two primases was that S. aureus helicase stimulation did not broaden the S. aureus primase initiation specificity, which has important biological implications.     

Properties of an unusual DNA primase from an archaeal plasmid

Primases are specialized DNA-dependent RNA polymerases that synthesize a short oligoribonucleotide complementary to single-stranded template DNA. In the context of cellular DNA replication, primases are indispensable since DNA polymerases are not able to start DNA polymerization de novo.

The primase activity of the replication protein from the archaeal plasmid pRN1 synthesizes a rather unusual mixed primer consisting of a single ribonucleotide at the 5′ end followed by seven deoxynucleotides. Ribonucleotides and deoxynucleotides are strictly required at the respective positions within the primer. Furthermore, in contrast to other archaeo-eukaryotic primases, the primase activity is highly sequence-specific and requires the trinucleotide motif GTG in the template. Primer synthesis starts outside of the recognition motif, immediately 5′ to the recognition motif. The fidelity of the primase synthesis is high, as non-complementary bases are not incorporated into the primer.

     

Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis

DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.     

Staphylococcus aureus helicase but not Escherichia coli helicase stimulates S. aureus primase activity and maintains initiation specificity

Bacterial primases are essential for DNA replication due to their role in polymerizing the formation of short RNA primers repeatedly on the lagging-strand template and at least once on the leading-strand template. The ability of recombinant Staphylococcus aureus DnaG primase to utilize different single-stranded DNA templates was tested using oligonucleotides of the sequence 5'-CAGA (CA)5 XYZ (CA)3-3', where XYZ represented the variable trinucleotide. These experiments demonstrated that S. aureus primase synthesized RNA primers predominately on templates containing 5'-d(CTA)-3' or TTA and to a much lesser degree on GTA-containing templates, in contrast to results seen with the Escherichia coli DnaG primase recognition sequence 5'-d(CTG)-3'. Primer synthesis was initiated complementarily to the middle nucleotide of the recognition sequence, while the third nucleotide, an adenosine, was required to support primer synthesis but was not copied into the RNA primer. The replicative helicases from both S. aureus and E. coli were tested for their ability to stimulate either S. aureus or E. coli primase. Results showed that each bacterial helicase could only stimulate the cognate bacterial primase. In addition, S. aureus helicase stimulated the production of full-length primers, whereas E. coli helicase increased the synthesis of only short RNA polymers. These studies identified important differences between E. coli and S. aureus related to DNA replication and suggest that each bacterial primase and helicase may have adapted unique properties optimized for replication.     

Essential lysine residues in the RNA polymerase domain of the gene 4 primase-helicase of bacteriophage T7.

At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C- and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5'-NNGTC-3' via a zinc motif and catalyzes the template-directed synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5'-ACCC-3' at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.     

Insight into the Human DNA Primase Interaction with Template-Primer

DNA replication in almost all organisms depends on the activity of DNA primase, a DNA-dependent RNA polymerase that synthesizes short RNA primers of defined size for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for the activity. The mode of interaction of primase subunits with substrates during the various steps of primer synthesis that results in the counting of primer length is not clear. Here we show that the C-terminal domain of the large subunit (p58_C) plays a major role in template-primer binding and also defines the elements of the DNA template and the RNA primer that interact with p58_C. The specific mode of interaction with a template-primer involving the terminal 5′-triphosphate of RNA and the 3′-overhang of DNA results in a stable complex between p58_C and the DNA/RNA duplex. Our results explain how p58_C participates in RNA synthesis and primer length counting and also indicate that the binding site for initiating NTP is located on p58_C. These findings provide notable insight into the mechanism of primase function and are applicable for DNA primases from other species.     

The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7

DNA primases catalyze the synthesis of oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Prokaryotic primases consist of a zinc-binding domain (ZBD) necessary for recognition of a specific template sequence and a catalytic RNA polymerase domain. Interactions of both domains with the DNA template and ribonucleotides are required for primer synthesis. Five tryptophan residues are dispersed in the primase of bacteriophage T7: Trp-42 in the ZBD and Trp-69, -97, -147, and -255 in the RNA polymerase domain. Previous studies showed that replacement of Trp-42 with alanine in the ZBD decreases primer synthesis, whereas substitution of non-aromatic residues for Trp-69 impairs both primer synthesis and delivery. However, the roles of tryptophan at position 97, 147, or 255 remain elusive. To investigate the essential roles of these residues, we replaced each tryptophan with the structurally similar tyrosine and examined the effect of this subtle alteration on primer synthesis. The substitution at position 42, 97, or 147 reduced primer synthesis, whereas substitution at position 69 or 255 did not. The functions of the tryptophans were further examined at each step of primer synthesis. Alteration of residue 42 disturbed the conformation of the ZBD and resulted in partial loss of the zinc ion, impairing binding to the ssDNA template. Replacement of Trp-97 with tyrosine reduced the binding affinity to NTP and the catalysis step. The replacement of Trp-147 with tyrosine also impaired the catalytic step. Therefore, Trp-42 is important in maintaining the conformation of the ZBD for template binding; Trp-97 contributes to NTP binding and the catalysis step; and Trp-147 maintains the catalysis step.     

Crosstalk between primase subunits can act to regulate primer synthesis in trans

The coordination of primase function within the replisome is an essential but poorly understood feature of lagging strand synthesis. By using crystallography and small-angle X-ray scattering (SAXS), we show that functional elements of bacterial primase transition between two dominant conformations: an extended form that uncouples a regulatory domain from its associated RNA polymerase core and a compact state that sequesters the regulatory region from the site of primer synthesis. FRET studies and priming assays reveal that the regulatory domain of one primase subunit productively associates with nucleic acid that is bound to the polymerase domain of a second protomer in trans. This intersubunit interaction allows primase to select initiation sites on template DNA and implicates the regulatory domain as a "molecular brake" that restricts primer length. Our data suggest that the replisome may cooperatively use multiple primases and this conformational switch to control initiation frequency, processivity, and ultimately, Okazaki fragment synthesis.     

Functional interplay of DnaE polymerase,DnaG primase and DnaC helicase within a ternary complex,and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis

Bacillus subtilis has two replicative DNA polymerases. PolC is a processive high-fidelity replicative polymerase, while the error-prone DnaE_Bs extends RNA primers before hand-off to PolC at the lagging strand. We show that DnaE_Bs interacts with the replicative helicase DnaC and primase DnaG in a ternary complex. We characterize their activities and analyse the functional significance of their interactions using primase, helicase and primer extension assays, and a ‘stripped down’ reconstituted coupled assay to investigate the coordinated displacement of the parental duplex DNA at a replication fork, synthesis of RNA primers along the lagging strand and hand-off to DnaE_Bs. The DnaG–DnaE_Bs hand-off takes place after de novo polymerization of only two ribonucleotides by DnaG, and does not require other replication proteins. Furthermore, the fidelity of DnaE_Bs is improved by DnaC and DnaG, likely via allosteric effects induced by direct protein–protein interactions that lower the efficiency of nucleotide mis-incorporations and/or the efficiency of extension of mis-aligned primers in the catalytic site of DnaE_Bs. We conclude that de novo RNA primer synthesis by DnaG and initial primer extension by DnaE_Bs are carried out by a lagging strand–specific subcomplex comprising DnaG, DnaE_Bs and DnaC, which stimulates chromosomal replication with enhanced fidelity.     

Initiation of bidirectional replication at the chromosomal origin is directed by the interaction between helicase and primase.

Several protein-protein interactions have been shown to be critical for proper replication fork function in Escherichia coli. These include interactions between the polymerase and the helicase, the helicase and the primase, and the primase and the polymerase. We have studied the influence of these interactions on proper initiation at oriC by using mutant primases defective in their interaction with the helicase and DNA polymerase III holoenzyme lacking the tau subunit so that it will not interact with the helicase. We show here that accurate initiation of bidirectional DNA replication from oriC is dependent on proper placement of the primers for leading strand synthesis and is thus governed primarily by the interaction between the helicase and primase.     

An in Trans Interaction at the Interface of the Helicase and Primase Domains of the Hexameric Gene 4 Protein of Bacteriophage T7 Modulates Their Activities

DNA helicase and primase are essential for DNA replication. The helicase unwinds the DNA to provide single-stranded templates for DNA polymerase. The primase catalyzes the synthesis of oligoribonucleotides for the initiation of lagging strand synthesis. The two activities reside in a single polypeptide encoded by gene 4 of bacteriophage T7. Their coexistence within the same polypeptide facilitates their coordination during DNA replication. One surface of helix E within the helicase domain is positioned to interact with the primase domain and the linker connecting the two domains within the functional hexamer. The interaction occurs in trans such that helix E interacts with the primase domain and the linker of the adjacent subunit. Most alterations of residues on the surface of helix E (Arg⁴⁰⁴, Lys⁴⁰⁸, Tyr⁴¹¹, and Gly⁴¹⁵) eliminate the ability of the altered proteins to complement growth of T7 phage lacking gene 4. Both Tyr⁴¹¹ and Gly⁴¹⁵ are important in oligomerization of the protein. Alterations G415V and K408A simultaneously influence helicase and primase activities in opposite manners that mimic events observed during coordinated DNA synthesis. The results suggest that Asp²⁶³ located in the linker of one subunit can interact with Tyr⁴¹¹, Lys⁴⁰⁸, or Arg⁴⁰⁴ in helix E of the adjacent subunit depending on the oligomerization state. Thus the switch in contacts between Asp²⁶³ and its three interacting residues in helix E of the adjacent subunit results in conformational changes that modulate helicase and primase activity.At the replication fork DNA helicase unwinds the duplex DNA to expose single-stranded DNA for use as templates for the leading and lagging strand DNA polymerases (1). The 5′ to 3′ polymerization of nucleotides by the leading strand DNA polymerase proceeds in a continuous manner, whereas synthesis on the lagging strand occurs in a discontinuous manner, generating Okazaki fragments. The synthesis of each Okazaki fragment is initiated by the extension of an oligoribonucleotide that serves as a primer for the lagging strand DNA polymerase. These oligoribonucleotides are synthesized in a template-directed manner by DNA primase. For the two polymerases to communicate with each other, the lagging strand folds back on itself such that the lagging strand DNA polymerase becomes part of the replisome. This association of the two polymerases enables both strands to be synthesized in the same overall direction, and synthesis of both strands proceeds at identical rates. The folding of the lagging strand creates a replication loop of lagging strand DNA that contains the nascent Okazaki fragment and the ssDNA³ extruded behind the helicase. Single-stranded DNA-binding protein binds to the exposed single-stranded DNA to remove secondary structure, but it also interacts with the other proteins of the replisome to assist in the coordination of DNA synthesis (2).Among the several protein interactions within the replisome, the interaction of the helicase with the primase is one of the most critical (2–6). The association of the primase with the helicase places it in position to catalyze primer synthesis on the single-stranded DNA extruded by the moving helicase. In addition, the higher affinity of the helicase for single-stranded DNA serves to stabilize the primase on the lagging strand. Perhaps the most important is the ability of the primase to communicate with the helicase. During the rate-limiting step of primer synthesis, leading strand synthesis would be expected to outpace lagging strand synthesis. The association of primase with helicase provides a mechanism by which helicase movement can be coordinated with primer synthesis (7).The gene 4 protein of bacteriophage T7 is unique in that it contains both helicase and primase activities within the same polypeptide chain (see Fig. 1A). Although separate genes encode other replicative helicases and primases, they nonetheless require a physical association to function properly (2, 5). The helicase activity resides in the C-terminal 295 residues, and the primase activity resides in the N-terminal 245 residues (8). A linker of 26 residues separates the helicase and primase domains. The linker plays a critical role in the oligomerization of gene 4 protein (9). The primase and the helicase domains have been purified separately and shown to exhibit their activities independently (9–11). However, the presence of each domain has striking effects on the activity of the other (2).Open in a separate windowFIGURE 1.Elements involved in the interaction between helicase and primase in E. coli and bacteriophage T7. A, schematic presentation of helicase and primase together with the structural elements involved in their interaction. In E. coli the helicase and primase interact via contacts of the C-terminal p16 of the primase with the N-terminal p17 of the helicase. In bacteriophage T7 the two activities are found in a single polypeptide where the primase and helicase domains are covalently connected via a flexible linker. Helix E is located in the helicase domain. B, top view of the hexameric T7 helicase (right panel) (Protein Data Bank accession code 1E0J). C, side view of the heptameric gene 4 protein containing both the helicase and primase domains (right panel) (Protein Data Bank accession code 1Q57). In B and C, two adjacent subunits are shown in green and yellow, respectively. The linker region and residues Ala²²⁵–Gly²²⁶ in the primase domain of the green subunit are shown in blue and magenta, respectively. Helix E in the helicase domain of the adjacent yellow subunit is shown in red. Residues potentially involved in the in trans interaction at the interface are indicated (left panels). In the heptameric structure (C), Gly⁴¹⁵ in Helix E is potentially interacting with Ala²²⁵ and/or Gly²²⁶ from the primase domain of the adjacent subunit. Lys⁴⁰⁸ is close to Asp²⁶³ in the linker from the adjacent subunit. In the hexameric structure (B), because the primase domain and a portion of the linker region are missing in this structure, the counterpart of Gly⁴¹⁵ is not present. Another obvious difference in this structure compared with that shown in C is that Asp²⁶³ in the linker of the heptamer is oriented toward Lys⁴⁰⁸, whereas it is close to Tyr⁴¹¹ in the hexamer structure. Distances shown in B and C are in similar ranges regardless of locations of interfaces in both hexameric and heptameric gene 4 protein structures. Structures from the Protein Data Bank were analyzed using PyMOL (DeLano Scientific LLC).Like other members of the Family 4 helicases, the helicase domain of gene 4 protein functions as a hexamer (see Fig. 1B). Members of this family assemble on single-stranded DNA with the DNA passing through the central channel formed by the oligomerization (4, 12). The nucleotide-binding site of the helicase is located at the subunit interface located between two RecA-like subdomains that bind dTTP, the preferred nucleotide for T7 helicase (13–17). The location of the nucleotide-binding site at the subunit interface provides multiple interactions of residues with the bound dTTP (18). These interactions assist in oligomerization, in binding to DNA, and in coupling the hydrolysis of dTTP to mechanical movement of the helicase (19–23).The primase domain, residing in the N-terminal half of the gene 4 protein, is a member of the DnaG family of prokaryotic primases. Three structural features distinguish members of this family. An N-terminal zinc-binding domain plays a critical role in recognizing sites for primer synthesis in ssDNA. An RNA polymerase domain, linked to the zinc-binding domain by a flexible linker, contains the catalytic site where metal-dependent polymerization of nucleotides occurs. A C-terminal segment covalently attaches the primase to the helicase. In other primases of this family, this segment interacts with the cognate helicase. T7 primase, like the primases of phage T4 and Escherichia coli, recognizes a trinucleotide sequence (5). T7 primase recognizes the sequence 5′-GTC-3′, at which it catalyzes the template directed synthesis of a dinucleotide (pppAC); the 3′-cytosine is essential for recognition, although this “cryptic” nucleotide is not copied into the product (24). The dinucleotide is then extended by the primase, provided the proper nucleotides, T and G, are present in the template. Consequently, the predominant T7 primase recognition sites are 5′-GGGTC-3′, 5′-TGGTC-3′, and 5′-GTGTC-3′ (25, 26). Thus T7 primase catalyzes the synthesis of the tetraribonucleotides pppACCC, pppACCA, and pppACAC. The lagging strand DNA polymerase then extends these functional tetranucleotides.The covalent linkage of primase and helicase in the gene 4 protein of bacteriophage T7 distinguishes it from most other replication systems where the association of the two proteins is dependent on a physical interaction of the two separate proteins. In bacteria such as E. coli, Bacillus stearothermophilus, and Staphylococcus aureus, this interaction is mediated through two structurally similar regions: the helicase-binding domain (p16 domain) located at the C terminus of the DnaG primase and the p17 domain of the DnaB helicase located at the N terminus of the protein (see Fig. 1A) (27–31). The association of DnaB with DnaG alters sequence recognition by DnaG and affects the length of primers synthesized (28, 31–33). Furthermore, cooperative binding of two or three DnaG monomers to the hexameric DnaB can halt translocation of DnaB on DNA (34). Such “association and dissociation” between the helicase and primase mediated by the p16 and p17 domains are believed to coordinate DNA synthesis by regulating the initiation of Okazaki fragment synthesis (6, 35, 36). Mutations in the p16 domain of DnaG can either affect the ability of the two proteins to form a complex, enhance the primase activity, or modulate the ATPase and/or helicase activities allosterically (31).The covalent association of primase and helicase in the bacteriophage T7 system clearly provides several of the advantages derived from the physical association of the two proteins in other systems. The primase is positioned correctly for primer synthesis, and DNA binding is achieved via the helicase. Furthermore, communication between the two domains of the gene 4 protein is dramatically revealed by the cessation of helicase movement during primer synthesis (7). However, the covalent association of the two activities precludes regulation by dissociation as in the other replication systems. The frequency of primase recognition sites in the phage genome is considerably more than that required for the initiation of Okazaki fragments. Consequently, primase activity in the T7 replication system must be highly regulated to ensure the translocation of helicase and the almost constant length of Okazaki fragments (2).T7 gene 4 protein is present in solution as a mixture of hexamers and heptamers (37), and the crystal structures of both oligomeric forms have been determined (see Fig. 1, B and C) (15, 38). In the heptameric structure an interaction of the helicase and primase domains occurs through helix E (see Fig. 1C). Located at the front of the helicase domain facing toward the primase domain, helix E is not only in proximity to the primase of the adjacent subunit but also in contact with the linker region connecting the two domains of the adjacent subunit. By this trans-packing interaction, the primase domain from one subunit is loosely stacked on the top of the helicase from the adjacent subunit (38) (see Fig. 1C). In the six-membered ring structure (15), the functional form of gene 4 protein, the primase domain is missing. However, the contact between helix E and the linker region from the adjacent subunit is present (see Fig. 1B). Some residues in the linker region have been identified previously as key factors involved in the conformational switch of helicase (39).How does the primase domain of gene 4 protein communicate with the helicase domain? Although the two domains cannot dissociate into solution, a transient dissociation of the two domains is possible as a result of the flexible linker through which they are connected. Alternatively, primase activity or helicase activity may be conveyed to the other domain as a result of conformational changes in the protein at the interface between the two domains. In either instance the linker region and the interface between the two domains are certain to be critical for this communication. Helix E, although quite distant from the catalytic sites of either the helicase or primase, contacts both the primase domain and the linker. In the present study we have examined the role of helix E in the function of gene 4 by genetically altering several residues and examining the function of the altered proteins in vivo and in vitro.     

Structure and primase-mediated activation of a bacterial dodecameric replicative helicase

Replicative helicases are essential ATPases that unwind DNA to initiate chromosomal replication. While bacterial replicative DnaB helicases are hexameric, Helicobacter pylori DnaB (HpDnaB) was found to form double hexamers, similar to some archaeal and eukaryotic replicative helicases. Here we present a structural and functional analysis of HpDnaB protein during primosome formation. The crystal structure of the HpDnaB at 6.7 Å resolution reveals a dodecameric organization consisting of two hexamers assembled via their N-terminal rings in a stack-twisted mode. Using fluorescence anisotropy we show that HpDnaB dodecamer interacts with single-stranded DNA in the presence of ATP but has a low DNA unwinding activity. Multi-angle light scattering and small angle X-ray scattering demonstrate that interaction with the DnaG primase helicase-binding domain dissociates the helicase dodecamer into single ringed primosomes. Functional assays on the proteins and associated complexes indicate that these single ringed primosomes are the most active form of the helicase for ATP hydrolysis, DNA binding and unwinding. These findings shed light onto an activation mechanism of HpDnaB by the primase that might be relevant in other bacteria and possibly other organisms exploiting dodecameric helicases for DNA replication.     

Organization and evolution of bacterial and bacteriophage primase-helicase systems

Summary Amino acid sequences of primases and associated helicases involved in the DNA replication of eubacteria and bacteriophages T7, T3, T4, P4, and P22 were compared by computer-assisted methods. There are two types of such systems, the first one represented by distinct helicase and primase proteins (e.g., DnaB and DnaG proteins of Escherichia coli), and the second one by single polypeptides comprising both activities (gp4 of bacteriophages T7 and T3, and alpha protein of bacteriophage P4). Pronounced sequence similarity was revealed between approximately 250 amino acid residue N-terminal domains of stand-alone primases and the primase-helicase proteins of T7(T3) and P4. All these domains contain, close to their N-termini, a conserved Zn-finger pattern that may be implicated in template DNA recognition by the primases. In addition, they encompass five other conserved motifs some of which may be involved in substrate (NTP) binding. Significant similarity was also observed between the primase-associated helicases (DnaB, gp12 of P22 and gp41 of T4) and the C-terminal domain of T7(T3) gp4. On the other hand the C-terminal domain of P-alpha of P4 is related to another group of DNA and RNA helicases. Tentative phylogenetic trees generated for the primases and the associated helicases showed no grouping of the phage proteins, with the exception of the primase domains of bacteriophages T4 and P4. This may indicate a common origin for one-component primase-helicase systems. Two scenarios for the evolution of primase-helicase systems are discussed. The first one involves fusion of the primase and helicase components (T7 and T3) or fusion of the primase component with a different type of helicase domain (P4). The second possibility is the duplication of an ancestral gene encoding a gp4-like bifunctional protein followed by divergence of the copies, one of which retains the primase and the other the helicase domain.     

Insights into Eukaryotic Primer Synthesis from Structures of the p48 Subunit of Human DNA Primase

DNA replication in all organisms requires polymerases to synthesize copies of the genome. DNA polymerases are unable to function on a bare template and require a primer. Primases are crucial RNA polymerases that perform the initial de novo synthesis, generating the first 8–10 nucleotides of the primer. Although structures of archaeal and bacterial primases have provided insights into general priming mechanisms, these proteins are not well conserved with heterodimeric (p48/p58) primases in eukaryotes. Here, we present X-ray crystal structures of the catalytic engine of a eukaryotic primase, which is contained in the p48 subunit. The structures of p48 reveal that eukaryotic primases maintain the conserved catalytic prim fold domain, but with a unique subdomain not found in the archaeal and bacterial primases. Calorimetry experiments reveal that Mn^2 + but not Mg^2 + significantly enhances the binding of nucleotide to primase, which correlates with higher catalytic efficiency in vitro. The structure of p48 with bound UTP and Mn^2 + provides insights into the mechanism of nucleotide synthesis by primase. Substitution of conserved residues involved in either metal or nucleotide binding alter nucleotide binding affinities, and yeast strains containing the corresponding Pri1p substitutions are not viable. Our results reveal that two residues (S160 and H166) in direct contact with the nucleotide were previously unrecognized as critical to the human primase active site. Comparing p48 structures to those of similar polymerases in different states of action suggests changes that would be required to attain a catalytically competent conformation capable of initiating dinucleotide synthesis.     

The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes.     

枯草芽孢杆菌全长引物酶的溶液结构研究

在细菌DNA复制中,DnaG引物酶合成RNA引物,然后合成的引物通过DNA聚合酶进行延伸. DnaG引物酶由3个结构域组成,N端锌结合结构域（zinc-binding domain,ZBD）、RNA聚合酶结构域（RNA polymerase domain,RPD）和C端解旋酶结合结构域（helicase binding domain,HBD）. 在合成引物的过程中,引物酶的3个结构域协同作用,缺一不可. 尽管引物酶3个结构域的结构均已有研究报道,但到目前为止,引物酶的全长结构尚不清楚. 我们在上海光源利用小角X射线散射技术研究了枯草芽孢杆菌全长引物酶的溶液结构,首次构建了全长引物酶结构模型. 我们发现,枯草芽孢杆菌引物酶在溶液中处于伸展状态,且ZBD和HBD结构域相对于RPD结构域呈现出连续的构象变化. 本文研究表明DnaG引物酶中的结构域重排可能有助于其在DNA复制中发挥功能.