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.     

Primer initiation and extension by T7 DNA primase

T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a two-fold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.     

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.     

Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6

The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.     

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.     

Interaction of bacteriophage T7 gene 4 primase with its template recognition site

The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5'-GGGTC-3', 5'-TGGTC-3', 5'-GTGTC-3', or 5'-TTGTC-3'). Although it is not copied into the oligoribonucleotides, the cytosine at the 3'-position is essential for synthesis and template binding. Two nucleotides flanking the 3'-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5'-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 microM. The tight DNA binding (<1 microM) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.     

Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase

DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.     

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.     

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.     

Class-specific restrictions define primase interactions with DNA template and replicative helicase

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the β4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase–helicase interaction has co-evolved within each species.     

Heterohexamer of 56- and 63-kDa Gene 4 Helicase-Primase of Bacteriophage T7 in DNA Replication

Bacteriophage T7 expresses two forms of gene 4 protein (gp4). The 63-kDa full-length gp4 contains both the helicase and primase domains. T7 phage also express a 56-kDa truncated gp4 lacking the zinc binding domain of the primase; the protein has helicase activity but no DNA-dependent primase activity. Although T7 phage grow better when both forms are present, the role of the 56-kDa gp4 is unknown. The two molecular weight forms oligomerize by virtue of the helicase domain to form heterohexamers. The 56-kDa gp4 and any mixture of 56- and 63-kDa gp4 show higher helicase activity in DNA unwinding and strand-displacement DNA synthesis than that observed for the 63-kDa gp4. However, single-molecule measurements show that heterohexamers have helicase activity similar to the 63-kDa gp4 hexamers. In oligomerization assays the 56-kDa gp4 and any mixture of the 56- and 63-kDa gp4 oligomerize to form more hexamers than does the 63-kDa gp4. The zinc binding domain of the 63-kDa gp4 interferes with hexamer formation, an inhibition that is relieved by the insertion of the 56-kDa species. Compared with the 63-kDa gp4, heterohexamers synthesize a reduced amount of oligoribonucleotides, mediated predominately by the 63-kDa subunits via a cis mode. During coordinated DNA synthesis 7% of the tetraribonucleotides synthesized are used as primers by both heterohexamers and hexamers of the 63-kDa gp4. Overall, an equimolar mixture of the two forms of gp4 shows the highest rate of DNA synthesis during coordinated DNA synthesis.     

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.     

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.     

The linker region between the helicase and primase domains of the gene 4 protein of bacteriophage T7. Role in helicase conformation and activity

The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for template-directed oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246-271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala257, Pro259, and Asp263) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala257 and Asp263 to be essential whereas Pro259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala257 or Asp263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg2+, all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.     

Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.     

A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis

The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.     

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Synthesis of the leading DNA strand requires the coordinated activity of DNA polymerase and DNA helicase, whereas synthesis of the lagging strand involves interactions of these proteins with DNA primase. We present the first structural model of a bacteriophage T7 DNA helicase-DNA polymerase complex using a combination of small angle x-ray scattering, single-molecule, and biochemical methods. We propose that the protein-protein interface stabilizing the leading strand synthesis involves two distinct interactions: a stable binding of the helicase to the palm domain of the polymerase and an electrostatic binding of the carboxyl-terminal tail of the helicase to a basic patch on the polymerase. DNA primase facilitates binding of DNA helicase to ssDNA and contributes to formation of the DNA helicase-DNA polymerase complex by stabilizing DNA helicase.     

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.     

Requirements for primer synthesis by bacteriophage T7 63-kDa gene 4 protein. Roles of template sequence and T7 56-kDa gene 4 protein.

Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication. The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively. Purified 56-kDa protein has helicase activity, but no primase activity. In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein. The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4. Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities. The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence. A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers. dTTP hydrolysis is essential for oligoribonucleotide synthesis. Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold. The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers. The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5'. T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers.     

Role of bacteriophage T7 DNA primase in the initiation of DNA strand synthesis.

Bacteriophage T7 DNA primase (gene-4 protein, 66,000 daltons) enables T7 DNA polymerase to initiate the synthesis of DNA chains on single-stranded templates. An initial step in the process of chain initiation is the formation of an oligoribonucleotide primer by T7 primase. The enzyme, in the presence of natural SS DNA, Mg++ (or Mn++), ATP and CTP (or a mixture of all 4 rNTPs), catalyzes the synthesis of di-, tri-, and tetraribonucleotides all starting at the 5' terminus with pppA. In a subsequent step requiring both T7 DNA polymerase and primase, the short oligoribonucleotides (predominantly pppA-C-C-AOH) are extended by covalent addition of deoxyribonucleotides. With the aid of primase, T7 DNA polymerase can also utilize efficiently a variety of synthetic tri-, tetra-, or pentanucleotides as chain initiators. T7 primase apparently plays an active role in primer extension by stabilizing the short primer segments in a duplex state on the template DNA.