Mechanism of primer synthesis by the herpes simplex virus 1 helicase-primase

We utilized templates of defined sequence to investigate the mechanism of primer synthesis by herpes simplex virus 1 helicase-primase. Under steady-state conditions, the rate of primer synthesis and the size distribution of products remained constant with time, suggesting that the rate-limiting step(s) of primer synthesis occur(s) during primer initiation (at or before the formation of the pppNpN dinucleotide). Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis. Due to these effects on primer initiation, varying the NTP concentration could affect start site selection on templates containing multiple G-pyr-pyr initiation sites. Increasing the NTP concentration also increased the processivity of primase. However, even at very high concentrations of NTPs, elongation of the dinucleotide into longer products remained relatively inefficient. Primase did not readily elongate preexisting primers under conditions where free template was present in large excess of enzyme. However, if template concentrations were lowered such that primase synthesized primers on all or most of the template present in the reaction, then primase would elongate previously synthesized primers.     

Contribution of the 3'- to 5'-exonuclease activity of herpes simplex virus type 1 DNA polymerase to the fidelity of DNA synthesis

Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase catalytic subunit (pol) is less faithful than for most replicative DNA polymerases, despite the presence of an associated 3'- to 5'-exonuclease (exo) activity. To determine the aspects of fidelity affected by the exo activity, nucleotide incorporation and mismatch extension frequency for purified wild-type and an exo-deficient mutant (D368A) pol were compared using primer/templates that varied at only a single position. For both enzymes, nucleotide discrimination during incorporation occurred predominantly at the level of K(m) for nucleotide and was the major contributor to fidelity. The contribution of the exo activity to reducing the efficiency of formation of half of all possible mispairs was 6-fold or less, and 30-fold when averaged for the formation of all possible mispairs. In steady-state reactions, mismatches imposed a significant kinetic barrier to extension independent of exo activity. However, during processive DNA synthesis in the presence of only three nucleotides, misincorporation and mismatch extension were efficient for both exo-deficient and wild-type pol catalytic subunits, although slower kinetics of mismatch extension by the exo-deficient pol were observed. The UL42 processivity factor decreased the extent of misincorporation by both the wild-type and the exo-deficient pol to similar levels, but mismatch extension by the wild-type pol.UL42 complex was much less efficient than by the mutant pol.UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol.UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by the kinetic barrier to extending a mismatch. The kinetic barrier also increases the probability that a mismatched primer terminus will be transferred to the exo site where it can be excised by the associated exo activity and subsequently extended with correct nucleotide.     

Interaction of herpes primase with the sugar of a NTP

We analyzed the interaction of nucleoside triphosphates (NTPs) containing modified sugars to develop a better understanding of how DNA primase from herpes simplex virus I catalyzes primer synthesis. During the NTP binding reaction, primase tolerated a large number of modifications to the sugar ring. Altering the 2' and 3' carbons and even converting the furanose sugar into an acyclic sugar did not prevent binding. Whether or not the base on the NTP could form a correct base pair with the template base being replicated also had minimal effect on the binding reaction, indicating that primase does not use this process to discriminate between right and wrong NTPs. Rather, the key feature that primase recognizes to bind a NTP is the 5'-gamma-phosphate since converting a NTP into a NDP greatly compromised binding. During the polymerization reaction, primase tolerated substantial modification of the 2'-carbon, including the presence of either an ara or ribo hydroxyl, two hydrogens, or two fluorines. However, polymerization absolutely required that the NTP contain a 3'-hydroxyl and an intact sugar ring. Modifications at the 2'-carbon of the nucleotide at the primer 3'-terminus significantly impaired further polymerization events. Compared to a ribonucleotide, incorporation of a 2'-deoxyribo- or 2',2'-difluoro-2'-deoxyribonucleotide resulted in strong chain termination, while incorporation of an aranucleotide resulted in very strong chain termination. The implications of these data with respect to the mechanism of primase and the relationship between human and herpes primase are discussed.     

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.     

Human DNA primase: anion inhibition, manganese stimulation, and their effects on in vitro start-site selection.

We examined the effects of Mn(2+) on eukaryotic DNA primase both in the presence and absence of 5 mM Mg(2+). In the absence of Mg(2+), Mn(2+)-supported primase activity to a level 4-fold greater than that obtained with Mg(2+) alone, and adding low levels of Mn(2+) (100 microM) to assays containing 5 mM Mg(2+) greatly stimulated primase. Increased activity was primarily due to more efficient utilization of NTPs, as reflected in a lower K(M) for NTPs. Under conditions of saturating NTPs, Mn(2+) had minimal effects on both the rate of initiation (i.e., dinucleotide synthesis) and processivity. The effects of Mn(2+) involve multiple metal binding sites on primase and may involve both the catalytic p49 subunit as well as the p58 subunit. Physiological levels of salt can inhibit primase activity due to the presence of an anion binding site and low levels of Mn(2+) significantly decreased this salt sensitivity. The implications of these results with respect to the biological role of primase are discussed.     

Herpes simplex virus 1 primase employs watson-crick hydrogen bonding to identify cognate nucleoside triphosphates

We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-d-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethylbenzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed.     

Key role of template sequence for primer synthesis by the herpes simplex virus 1 helicase-primase

We investigated the effects of ssDNA template sequence on both primer synthesis and NTP hydrolysis by herpes simplex virus 1 helicase-primase. Primer synthesis was found to be profoundly dependent upon template sequence. Although not absolutely required, an important sequence feature for significant production of longer primers (beyond four nucleotides in length) is a deoxyguanylate-pyrimidine-pyrimidine (3'-G-pyr-pyr-5') triplet in the template. The deoxyguanylate serves both to direct primase to initiate synthesis opposite the adjacent pyrimidine and to dramatically increase primer length. While primase synthesized significantly more long primers on those templates containing a G-pyr-pyr triplet, the enzyme still synthesized massive quantities of di- and trinucleotides on many templates containing this sequence. Varying the sequences around the G-pyr-pyr recognition sequence dramatically altered both the rate of primer synthesis and the fraction of primers longer than four nucleotides, indicating that primase must interact with both the G-pyr-pyr and flanking sequences in the template. In contrast to the large effects that varying the template sequence had on primase activity, ssDNA-dependent NTPase activity was essentially unaffected by changes in template sequence, including the presence or absence of the G-pyr-pyr trinucleotide. In addition to hydrolyzing NTPs the NTPase could also hydrolyze the 5'-terminal phosphate from newly synthesized primers.     

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.     

The E249K mutator mutant of DNA polymerase beta extends mispaired termini

The DNA polymerase beta mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol beta wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase beta is critical for mispair extension fidelity.     

Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back 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.

     

Interplay between primase and replication factor C in the hyperthermophilic archaeon Sulfolobus solfataricus

The heterodimeric primase from the hyperthermophilic archaeon Sulfolobus solfataricus synthesizes long RNA and DNA products in vitro. How primer synthesis by primase is coupled to primer extension by DNA polymerase in this organism is unclear. Here we show that the small subunit of the clamp loader replication factor C (RFC) of S. solfataricus interacted with both the catalytic and non-catalytic subunits of the primase by yeast two-hybrid and co-immunoprecipitation assays. Further, the primase-RFC interaction was also identified in the cell extract of S. solfataricus. Deletion analysis indicated that the small subunit of RFC interacted strongly with the N-terminal domain of the catalytic subunit of the primase. RFC stimulated dinucleotide formation but decreased the amount of primers synthesized by the primase. The inhibition of primer synthesis is consistent with the observation that RFC reduced the affinity of the primase for DNA templates. On the other hand, primase stimulated the ATPase activity of RFC. These findings suggest that the primase-RFC interaction modulates the activities of both enzymes and therefore may be involved in the regulation of primer synthesis and the transfer of primers to DNA polymerase in Archaea.     

Mechanism and evolution of DNA primases

DNA primase synthesizes short RNA primers that replicative polymerases further elongate in order to initiate the synthesis of all new DNA strands. Thus, primase owes its existence to the inability of DNA polymerases to initiate DNA synthesis starting with 2 dNTPs. Here, we discuss the evolutionary relationships between the different families of primases (viral, eubacterial, archael, and eukaryotic) and the catalytic mechanisms of these enzymes. This includes how they choose an initiation site, elongate the growing primer, and then only synthesize primers of defined length via an inherent ability to count. Finally, the low fidelity of primases along with the development of primase inhibitors is described.     

Fidelity of DNA polymerase epsilon holoenzyme from budding yeast Saccharomyces cerevisiae

DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain.     

Accuracy of DNA primase

The base-pairing fidelity of DNA primase from calf thymus was studied in vitro by using a misinsertion assay based on synthetic polydeoxynucleotide templates. With poly(dT) as template, GMP misinsertions occurred with a frequency of one error per 1600 correctly incorporated nucleotides, while UMP and CMP were inserted with a frequency of 1/300 and 1/500, respectively. Accuracy with poly(dC, dT) as template was 1/200 for the misinsertion of UMP, and 1/300 for the misinsertion of CMP. Thus, DNA primase is the least accurate polynucleotide-synthesizing enzyme known. The results are discussed in terms of an obvious necessity for a priming mechanism at the beginning of DNA synthesis.     

Cleavage of substrates with mismatched nucleotides by Flap endonuclease-1. Implications for mammalian Okazaki fragment processing.

Flap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5'-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase alpha/primase. Because the fidelity of DNA polymerase alpha is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5'-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5'-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5'-end of the fragment.     

Structures of DNA polymerase beta with active-site mismatches suggest a transient abasic site intermediate during misincorporation

We report the crystallographic structures of DNA polymerase beta with dG-dAMPCPP and dC-dAMPCPP mismatches in the active site. These premutagenic structures were obtained with a nonhydrolyzable incoming nucleotide analog, dAMPCPP, and Mn(2+). Substituting Mn(2+) for Mg(2+) significantly decreases the fidelity of DNA synthesis. The structures reveal that the enzyme is in a closed conformation like that observed with a matched Watson-Crick base pair. The incorrect dAMPCPP binds in a conformation identical to that observed with the correct nucleotide. To accommodate the incorrect nucleotide and closed protein conformation, the template strand in the vicinity of the active site has shifted upstream over 3 A, removing the coding base from the active site and generating an abasic templating pocket. The primer terminus rotates as its complementary template base is repositioned. This rotation moves O3' of the primer terminus away from the alpha-phosphate of the incoming nucleotide, thereby deterring misincorporation.