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.     

Crystal structure of the C-terminal domain of human DNA primase large subunit: Implications for the mechanism of the primase—polymerase α switch

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes'' large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.Key words: DNA primase, prim1, prim2, replication, 4Fe-4S cluster, crystal structure, DNA polymerase α     

Flexible tethering of primase and DNA Pol α in the eukaryotic primosome

The Pol α/primase complex or primosome is the primase/polymerase complex that initiates nucleic acid synthesis during eukaryotic replication. Within the primosome, the primase synthesizes short RNA primers that undergo limited extension by Pol α. The resulting RNA–DNA primers are utilized by Pol δ and Pol ε for processive elongation on the lagging and leading strands, respectively. Despite its importance, the mechanism of RNA–DNA primer synthesis remains poorly understood. Here, we describe a structural model of the yeast primosome based on electron microscopy and functional studies. The 3D architecture of the primosome reveals an asymmetric, dumbbell-shaped particle. The catalytic centers of primase and Pol α reside in separate lobes of high relative mobility. The flexible tethering of the primosome lobes increases the efficiency of primer transfer between primase and Pol α. The physical organization of the primosome suggests that a concerted mechanism of primer hand-off between primase and Pol α would involve coordinated movements of the primosome lobes. The first three-dimensional map of the eukaryotic primosome at 25 Å resolution provides an essential structural template for understanding initiation of eukaryotic replication.     

Mechanisms of primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli

In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates. These ‘primers’ are synthesized by various enzymatic mechanisms in Escherichia coli. Studies on primer RNA synthesis on single-stranded DNA templates containing specific ‘priming signals’ revealed the presence of two distinct modes, ie immobile and mobile priming. The former includes primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a σ⁷⁰ subunit. Priming is initiated at a specific site in immobile priming. Novel immobile priming signals were identified from various plasmid replicaons, some of which function in initiation of the leading strand synthesis. The latter, on the other hand, involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication. Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes primer RNAs at multiple sites. Two distinct primosomes. DnaA-dependent primosome supports normal chromosomal identified, which are differentially utilized for E coli chromosomal replication. Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNaseH activity. In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates primer RNA synthesis and replication fork progression.     

Crystal Structure of the Human Pol α B Subunit in Complex with the C-terminal Domain of the Catalytic Subunit

In eukaryotic DNA replication, short RNA-DNA hybrid primers synthesized by primase-DNA polymerase α (Prim-Pol α) are needed to start DNA replication by the replicative DNA polymerases, Pol δ and Pol ϵ. The C terminus of the Pol α catalytic subunit (p180_C) in complex with the B subunit (p70) regulates the RNA priming and DNA polymerizing activities of Prim-Pol α. It tethers Pol α and primase, facilitating RNA primer handover from primase to Pol α. To understand these regulatory mechanisms and to reveal the details of human Pol α organization, we determined the crystal structure of p70 in complex with p180_C. The structured portion of p70 includes a phosphodiesterase (PDE) domain and an oligonucleotide/oligosaccharide binding (OB) domain. The N-terminal domain and the linker connecting it to the PDE domain are disordered in the reported crystal structure. The p180_C adopts an elongated asymmetric saddle shape, with a three-helix bundle in the middle and zinc-binding modules (Zn1 and Zn2) on each side. The extensive p180_C-p70 interactions involve 20 hydrogen bonds and a number of hydrophobic interactions resulting in an extended buried surface of 4080 Ų. Importantly, in the structure of the p180_C-p70 complex with full-length p70, the residues from the N-terminal to the OB domain contribute to interactions with p180_C. The comparative structural analysis revealed both the conserved features and the differences between the human and yeast Pol α complexes.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. III. A polymerase-primase interaction governs primer size.

Studies with a rolling-circle DNA replication system reconstituted in vitro with a tailed form II DNA template, the DNA polymerase III holoenzyme (Pol III HE), the Escherichia coli single-stranded DNA binding protein, and the primosome, showed that within the context of a replication fork, the oligoribonucleotide primers that were formed were limited to a length in the range of 9 to 14 nucleotides, regardless of whether they were subsequently elongated by the lagging-strand DNA polymerase. This is in contrast to the 8-60-nucleotide-long primers synthesized by the primosome in the absence of DNA replication on a bacteriophage phi X174 DNA template, although when primer synthesis and DNA replication were catalyzed concurrently in this system, the extent of RNA polymerization decreased. As described in this report, we therefore examined the effect of the DNA Pol III HE on the length of primers synthesized by primase in vitro in the absence of DNA replication. When primer synthesis was catalyzed either: i) by the primosome on a phi X174 DNA template, ii) by primase on naked DNA with the aid of the DnaB protein (general priming), or iii) by primase alone at the bacteriophage G4 origin, the presence of the DNA Pol III HE in the reaction mixtures resulted in a universal reduction in the length of the heterogeneous RNA products to a uniform size of approximately 10 nucleotides. dNTPs were not required, and the addition of dGMP, an inhibitor of the 3'----5' exonuclease of the DNA Pol III HE, did not alter the effect; therefore, neither the 5'----3' DNA polymerase activity nor the 3'----5' exonuclease activity of the DNA Pol III HE was involved. E. coli DNA polymerase I, and the DNA polymerases of bacteriophages T4 and T7 could not substitute for the DNA Pol III HE. The Pol III core plays a crucial role in mediating this effect, although other subunits of the DNA Pol III HE are also required. These observations suggest that the association of primase with the DNA Pol III HE during primer synthesis regulates its catalytic activity and that this regulatory interaction occurs independently of, and prior to, formation of a preinitiation complex of the DNA Pol III HE on the primer terminus.     

RecJ-like protein from Pyrococcus furiosus has 3′–5′ exonuclease activity on RNA: implications for proofreading of 3′-mismatched RNA primers in DNA replication

Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3′-mismatched RNA primer because it cannot remove the 3′-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3′-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3′–5′ direction, and the kinetic parameters (K_m and K_cat) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3′-mismatched RNA primers. Replication protein A, the single-stranded DNA–binding protein, stimulates the removal of 3′-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3′-mismatched RNA primer after the 3′-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3′-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo.     

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.     

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.

     

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.     

Crystal structure of the C-terminal domain of human DNA primase large subunit

DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes’ large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.     

Arg304 of human DNA primase is a key contributor to catalysis and NTP binding: primase and the family X polymerases share significant sequence homology.

Comparison of the amino acid sequences of eucaryotic DNA primase and the family X polymerases indicates that primase shares significant sequence homology with this family. With the use of DNA polymerase beta (pol beta) as a paradigm for family X polymerases, these homologies include both the catalytic core domain/subunit of each enzyme (31 kDa domain of pol beta and p49 subunit of primase) as well as the accessory domain/subunit (8 kDa domain of pol beta and p58 subunit of primase). To further explore these homologies as well as provide insights into the mechanism of primase, we generated three mutants (R304K, R304Q, and R304A) of the p49 subunit at an arginine that is highly conserved between primase and the eukaryotic family X polymerases. These mutations significantly decreased the rate of primer synthesis, due primarily to a decreased rate of initiation, and the extent of impairment correlated with the severity of the mutation (A > Q > K). R304 also contributes to efficient utilization of the NTP that will become the 5'-terminus of the new primer, and these effects are at least partially mediated through interactions with the phosphates of this NTP. The implications of these results with respect to the structure and biological role of primase, as well as its relationship to the family X polymerases, are discussed.     

The C-terminal Domain of the DNA Polymerase Catalytic Subunit Regulates the Primase and Polymerase Activities of the Human DNA Polymerase α-Primase Complex

The initiation of DNA synthesis during replication of the human genome is accomplished primarily by the DNA polymerase α-primase complex, which makes the RNA-DNA primers accessible to processive DNA pols. The structural information needed to understand the mechanism of regulation of this complex biochemical reaction is incomplete. The presence of two enzymes in one complex poses the question of how these two enzymes cooperate during priming of DNA synthesis. Yeast two-hybrid and direct pulldown assays revealed that the N-terminal domain of the large subunit of primase (p58N) directly interacts with the C-terminal domain of the catalytic subunit of polα (p180C). We found that a complex of the C-terminal domain of the catalytic subunit of polα with the second subunit (p180C-p70) stimulated primase activity, whereas the whole catalytically active heterodimer of polα (p180ΔN-p70) inhibited RNA synthesis by primase. Conversely, the polα catalytic domain without the C-terminal part (p180ΔN-core) possessed a much higher propensity to extend the RNA primer than the two-subunit polα (p180ΔN-p70), suggesting that p180C and/or p70 are involved in the negative regulation of DNA pol activity. We conclude that the interaction between p180C, p70, and p58 regulates the proper primase and polymerase function. The composition of the template DNA is another important factor determining the activity of the complex. We have found that polα activity strongly depends on the sequence of the template and that homopyrimidine runs create a strong barrier for DNA synthesis by polα.     

Archaeal primase: bridging the gap between RNA and DNA polymerases

In the evolution of life, DNA replication is a fundamental process, by which species transfer their genetic information to their offspring. DNA polymerases, including bacterial and eukaryotic replicases, are incapable of de novo DNA synthesis. DNA primases are required for this function, which is sine qua non to DNA replication. In Escherichia coli, the DNA primase (DnaG) exists as a monomer and synthesizes a short RNA primer. In Eukarya, however, the primase activity resides within the DNA polymerase alpha-primase complex (Pol alpha-pri) on the p48 subunit, which synthesizes the short RNA segment of a hybrid RNA-DNA primer. To date, very little information is available regarding the priming of DNA replication in organisms in Archaea. Available sequenced genomes indicate that the archaeal DNA primase is a homolog of the eukaryotic p48 subunit. Here, we report investigations of a p48-like DNA primase from Pyrococcus furiosus, a hyperthermophilic euryarchaeote. P. furiosus p48-like protein (Pfup41), unlike hitherto-reported primases, does not catalyze by itself the synthesis of short RNA primers but preferentially utilizes deoxynucleotides to synthesize DNA fragments up to several kilobases in length. Pfup41 is the first DNA polymerase that does not require primers for the synthesis of long DNA strands.     

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.     

The Alarmone (p)ppGpp Regulates Primer Extension by Bacterial Primase

Primase is an essential component of the DNA replication machinery, responsible for synthesizing RNA primers that initiate leading and lagging strand DNA synthesis. Bacterial primase activity can be regulated by the starvation-inducible nucleotide (p)ppGpp. This regulation contributes to a timely inhibition of DNA replication upon amino acid starvation in the Gram-positive bacterium Bacillus subtilis. Here, we characterize the effect of (p)ppGpp on B. subtilis DnaG primase activity in vitro. Using a single-nucleotide resolution primase assay, we dissected the effect of ppGpp on the initiation, extension, and fidelity of B. subtilis primase. We found that ppGpp has a mild effect on initiation, but strongly inhibits primer extension and reduces primase processivity, promoting termination of primer extension. High (p)ppGpp concentration, together with low GTP concentration, additively inhibit primase activity. This explains the strong inhibition of replication elongation during starvation which induces high levels of (p)ppGpp and depletion of GTP in B. subtilis. Finally, we found that lowering GTP concentration results in mismatches in primer base pairing that allow priming readthrough, and that ppGpp reduces readthrough to protect priming fidelity. These results highlight the importance of (p)ppGpp in protecting replisome integrity and genome stability in fluctuating nucleotide concentrations upon onset of environmental stress.     

Crystal Structure of the Human Primase

DNA replication in bacteria and eukaryotes requires the activity of DNA primase, a DNA-dependent RNA polymerase that lays short RNA primers for DNA polymerases. Eukaryotic and archaeal primases are heterodimers consisting of small catalytic and large accessory subunits, both of which are necessary for RNA primer synthesis. Understanding of RNA synthesis priming in eukaryotes is currently limited due to the lack of crystal structures of the full-length primase and its complexes with substrates in initiation and elongation states. Here we report the crystal structure of the full-length human primase, revealing the precise overall organization of the enzyme, the relative positions of its functional domains, and the mode of its interaction with modeled DNA and RNA. The structure indicates that the dramatic conformational changes in primase are necessary to accomplish the initiation and then elongation of RNA synthesis. The presence of a long linker between the N- and C-terminal domains of p58 provides the structural basis for the bulk of enzyme''s conformational flexibility. Deletion of most of this linker affected the initiation and elongation steps of the primer synthesis.     

Structure of a DNA Polymerase α-Primase Domain That Docks on the SV40 Helicase and Activates the Viral Primosome

DNA polymerase α-primase (pol-prim) plays a central role in DNA replication in higher eukaryotes, initiating synthesis on both leading and lagging strand single-stranded DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68 (also termed B-subunit), that is thought to regulate the complex. Although significant knowledge about single-subunit primases of prokaryotes has accumulated, the functions and regulation of pol-prim remain poorly understood. In the SV40 replication model, the p68 subunit is required for primosome activity and binds directly to the hexameric viral helicase T antigen, suggesting a functional link between T antigen-p68 interaction and primosome activity. To explore this link, we first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the T antigen helicase domain. NMR spectroscopy was used to determine the solution structure of p68N and map its interface with the T antigen helicase domain. Structure-guided mutagenesis of p68 residues in the interface diminished T antigen-p68 interaction, confirming the interaction site. SV40 primosome activity of corresponding pol-prim mutants decreased in proportion to the reduction in p68N-T antigen affinity, confirming that p68-T antigen interaction is vital for primosome function. A model is presented for how this interaction regulates SV40 primosome activity, and the implications of our findings are discussed in regard to the molecular mechanisms of eukaryotic DNA replication initiation.     

The DNA primase of Sulfolobus solfataricus is activated by substrates containing a thymine-rich bubble and has a 3'-terminal nucleotidyl-transferase activity

DNA primases are responsible for the synthesis of the short RNA primers that are used by the replicative DNA polymerases to initiate DNA synthesis on the leading- and lagging-strand at the replication fork. In this study, we report the purification and biochemical characterization of a DNA primase (Sso DNA primase) from the thermoacidophilic crenarchaeon Sulfolobus solfataricus. The Sso DNA primase is a heterodimer composed of two subunits of 36 kDa (small subunit) and 38 kDa (large subunit), which show sequence similarity to the eukaryotic DNA primase p60 and p50 subunits, respectively. The two polypeptides were co-expressed in Escherichia coli and purified as a heterodimeric complex, with a Stokes radius of about 39.2 Å and a 1:1 stoichiometric ratio among its subunits. The Sso DNA primase utilizes poly-pyrimidine single-stranded DNA templates with low efficiency for de novo synthesis of RNA primers, whereas its synthetic function is specifically activated by thymine-containing synthetic bubble structures that mimic early replication intermediates. Interestingly, the Sso DNA primase complex is endowed with a terminal nucleotidyl-tranferase activity, being able to incorporate nucleotides at the 3′ end of synthetic oligonucleotides in a non-templated manner.     

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.