Role of the herpes simplex virus helicase-primase complex during adeno-associated virus DNA replication

A subset of DNA replication proteins of herpes simplex virus (HSV) comprising the single-strand DNA-binding protein, ICP8 (UL29), and the helicase-primase complex (UL5, UL8, and UL52 proteins) has previously been shown to be sufficient for the replication of adeno-associated virus (AAV). We recently demonstrated complex formation between ICP8, AAV Rep78, and the single-stranded DNA AAV genome, both in vitro and in the nuclear HSV replication domains of coinfected cells. In this study the functional role(s) of HSV helicase and primase during AAV DNA replication were analyzed. To differentiate between their necessity as structural components of the HSV replication complex or as active enzymes, point mutations within the helicase and primase catalytic domains were analyzed. In two complementary approaches the remaining HSV helper functions were either provided by infection with HSV mutants or by plasmid transfection. We show here that upon cotransfection of the minimal four HSV proteins (i.e., the four proteins constituting the minimal requirements for basal AAV replication), UL52 primase catalytic activity was not required for AAV DNA replication. In contrast, UL5 helicase activity was necessary for fully efficient replication. Confocal microscopy confirmed that all mutants retained the ability to support formation of ICP8-positive nuclear replication foci, to which AAV Rep78 colocalized in a manner strictly dependent on the presence of AAV single-stranded DNA (ssDNA). The data indicate that recruitment of AAV Rep78 and ssDNA to nuclear replication sites by the four HSV helper proteins is maintained in the absence of catalytic primase or helicase activities and suggest an involvement of the HSV UL5 helicase activity during AAV DNA replication.     

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.     

Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis

Initiation and synthesis of RNA primers in the lagging strand of the replication fork in Escherichia coli requires the replicative DnaB helicase and the DNA primase, the DnaG gene product. In addition, the physical interaction between these two replication enzymes appears to play a role in the initiation of chromosomal DNA replication. In vitro, DnaB helicase stimulates primase to synthesize primers on single-stranded (ss) oligonucleotide templates. Earlier studies hypothesized that multiple primase molecules interact with each DnaB hexamer and single-stranded DNA. We have examined this hypothesis and determined the exact stoichiometry of primase to DnaB hexamer. We have also demonstrated that ssDNA binding activity of the DnaB helicase is necessary for directing the primase to the initiator trinucleotide and synthesis of 11-20-nucleotide long primers. Although, association of these two enzymes determines the extent and rate of synthesis of the RNA primers in vitro, direct evidence of the formation of primase-DnaB complex has remained elusive in E. coli due to the transient nature of their interaction. Therefore, we stabilized this complex using a chemical cross-linker and carried out a stoichiometric analysis of this complex by gel filtration. This allowed us to demonstrate that the primase-helicase complex of E. coli is comprised of three molecules of primase bound to one DnaB hexamer. Fluorescence anisotropy studies of the interaction of DnaB with primase, labeled with the fluorescent probe Ru(bipy)3, and Scatchard analysis further supported this conclusion. The addition of DnaC protein, leading to the formation of the DnaB-DnaC complex, to the simple priming system resulted in the synthesis of shorter primers. Therefore, interactions of the DnaB-primase complex with other replication factors might be critical for determining the physiological length of the RNA primers in vivo and the overall kinetics of primer synthesis.     

Stimulation of mouse DNA primase-catalyzed oligoribonucleotide synthesis by mouse DNA helicase B.

Many prokaryotic and viral DNA helicases involved in DNA replication stimulate their cognate DNA primase activity. To assess the stimulation of DNA primase activity by mammalian DNA helicases, we analyzed the synthesis of oligoribonucleotides by mouse DNA polymerase alpha-primase complex on single-stranded circular M13 DNA in the presence of mouse DNA helicase B. DNA helicase B was purified by sequential chromatography through eight columns. When the purified DNA helicase B was applied to a Mono Q column, the stimulatory activity for DNA primase-catalyzed oligoribonucleotide synthesis and DNA helicase and DNA-dependent ATPase activities of DNA helicase B were co-eluted from the column. The synthesis of oligoribonucleotides 5-10 nt in length was markedly stimulated by DNA helicase B. The synthesis of longer species of oligoribonucleotides, which were synthesized at a low level in the absence of DNA helicase B, was inhibited by DNA helicase B. The stimulatory effect of DNA helicase B was marked at low template concentrations and little or no effect was observed at high concentrations. The mouse single-stranded DNA binding protein, replication protein A (RP-A), inhibited the primase activity of the DNA polymerase alpha-primase complex and DNA helicase B partially reversed the inhibition caused by RP-A.     

The crystal structure of the bifunctional primase-helicase of bacteriophage T7

Within minutes after infecting Escherichia coli, bacteriophage T7 synthesizes many copies of its genomic DNA. The lynchpin of the T7 replication system is a bifunctional primase-helicase that unwinds duplex DNA at the replication fork while initiating the synthesis of Okazaki fragments on the lagging strand. We have determined a 3.45 A crystal structure of the T7 primase-helicase that shows an articulated arrangement of the primase and helicase sites. The crystallized primase-helicase is a heptamer with a crown-like shape, reflecting an intimate packing of helicase domains into a ring that is topped with loosely arrayed primase domains. This heptameric isoform can accommodate double-stranded DNA in its central channel, which nicely explains its recently described DNA remodeling activity. The double-jointed structure of the primase-helicase permits a free range of motion for the primase and helicase domains that suggests how the continuous unwinding of DNA at the replication fork can be periodically coupled to Okazaki fragment synthesis.     

Interactions of bacteriophage T4-coded primase (gp61) with the T4 replication helicase (gp41) and DNA in primosome formation.

One primase (gp61) and six helicase (gp41) subunits interact to form the bacteriophage T4-coded primosome at the DNA replication fork. In order to map some of the detailed interactions of the primase within the primosome, we have constructed and characterized variants of the gp61 primase that carry kinase tags at either the N or the C terminus of the polypeptide chain. These tagged gp61 constructs have been probed using several analytical methods. Proteolytic digestion and protein kinase protection experiments show that specific interactions with single-stranded DNA and the T4 helicase hexamer significantly protect both the N- and the C-terminal regions of the T4 primase polypeptide chain against modification by these procedures and that this protection becomes more pronounced when the primase is assembled within the complete ternary primosome complex. Additional discrete sites of both protection and apparent hypersensitivity along the gp61 polypeptide chain have also been mapped by proteolytic footprinting reactions for the binary helicase-primase complex and in the three component primosome. These studies provide a detailed map of a number of gp61 contact positions within the primosome and reveal interactions that may be important in the structure and function of this central component of the T4 DNA replication complex.     

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.     

A mutation in the human herpes simplex virus type 1 UL52 zinc finger motif results in defective primase activity but can recruit viral polymerase and support viral replication efficiently

Herpes simplex virus type 1 (HSV-1) encodes a heterotrimeric helicase/primase complex consisting of UL5, UL8, and UL52. UL5 contains conserved helicase motifs, while UL52 contains conserved primase motifs, including a zinc finger motif. Although HSV-1 and HSV-2 UL52s contain a leucine residue at position 986, most other herpesvirus primase homologues contain a phenylalanine at this position. We constructed an HSV-1 UL52 L986F mutation and found that it can complement a UL52 null virus more efficiently than the wild type (WT). We thus predicted that the UL5/8/52 complex containing the L986F mutation might possess increased primase activity; however, it exhibited only 25% of the WT level of primase activity. Interestingly, the mutant complex displayed elevated levels of DNA binding and single-stranded DNA-dependent ATPase and helicase activities. This result confirms a complex interdependence between the helicase and primase subunits. We previously showed that primase-defective mutants failed to recruit the polymerase catalytic subunit UL30 to prereplicative sites, suggesting that an active primase, or primer synthesis, is required for polymerase recruitment. Although L986F exhibits decreased primase activity, it can support efficient replication and recruit UL30 efficiently to replication compartments, indicating that a partially active primase is capable of recruiting polymerase. Extraction with detergents prior to fixation can extract nucleosolic proteins but not proteins bound to chromatin or the nuclear matrix. We showed that UL30 was extracted from replication compartments while UL42 remained bound, suggesting that UL30 may be tethered to the replication fork by protein-protein interactions.     

Leading and lagging strand synthesis at the replication fork of bacteriophage T7. Distinct properties of T7 gene 4 protein as a helicase and primase

Reactions at the replication fork of bacteriophage T7 have been reconstituted in vitro on a preformed replication fork. A minimum of three proteins is required to catalyze leading and lagging strand synthesis. The T7 gene 4 protein, which exists in two forms of molecular weight 56,000 and 63,000, provides helicase and primase activities. A tight complex of the T7 gene 5 protein and Escherichia coli thioredoxin provides DNA polymerase activity. Gene 4 protein and DNA polymerase catalyze processive leading strand synthesis. Gene 4 protein molecules serving as helicase remain bound to the template as leading strand synthesis proceeds greater than 40 kilobases. Primer synthesis for lagging strand synthesis is catalyzed by additional gene 4 protein molecules that undergo multiple association/dissociation steps to catalyze multiple rounds of primer synthesis. The smaller molecular weight form of gene 4 protein has been purified from an equimolar mixture of both forms. Removal of the large form results in the loss of primase activity but not of helicase activity. Submolar amounts of the large form present in a mixture of both forms are sufficient to restore high specific activity of primase characteristic of an equimolar mixture of both forms. These results suggest that the gene 4 primase is an oligomer which is composed of both molecular weight forms. The large form may be the distributive component of the primase which dissociates from the template after each round of primer synthesis.     

Crystal and solution structures of the helicase-binding domain of Escherichia coli primase

During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase. In Escherichia coli, this occurs by transient interaction of primase with the helicase. Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase is responsible for interaction with DnaB6. Determination of the 2.8-angstroms crystal structure of the C-terminal domain of primase revealed an asymmetric dimer. The monomers have an N-terminal helix bundle similar to the N-terminal domain of DnaB, followed by a long helix that connects to a C-terminal helix hairpin. The connecting helix is interrupted differently in the two monomers. Solution studies using NMR showed that an equilibrium exists between a monomeric species with an intact, extended but naked, connecting helix and a dimer in which this helix is interrupted in the same way as in one of the crystal conformers. The other conformer is not significantly populated in solution, and its presence in the crystal is due largely to crystal packing forces. It is proposed that the connecting helix contributes necessary structural flexibility in the primase-helicase complex at replication forks.     

Replication initiation at the Escherichia coli chromosomal origin

To initiate DNA replication, DnaA recognizes and binds to specific sequences within the Escherichia coli chromosomal origin (oriC), and then unwinds a region within oriC. Next, DnaA interacts with DnaB helicase in loading the DnaB-DnaC complex on each separated strand. Primer formation by primase (DnaG) induces the dissociation of DnaC from DnaB, which involves the hydrolysis of ATP bound to DnaC. Recent evidence indicates that DnaC acts as a checkpoint in the transition from initiation to the elongation stage of DNA replication. Freed from DnaC, DnaB helicase unwinds the parental duplex DNA while interacting the cellular replicase, DNA polymerase III holoenzyme, and primase as it intermittently forms primers that are extended by the replicase in duplicating the chromosome.     

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.     

Three-Dimensional Structure of N-Terminal Domain of DnaB Helicase and Helicase-Primase Interactions in Helicobacter pylori

Replication initiation is a crucial step in genome duplication and homohexameric DnaB helicase plays a central role in the replication initiation process by unwinding the duplex DNA and interacting with several other proteins during the process of replication. N-terminal domain of DnaB is critical for helicase activity and for DnaG primase interactions. We present here the crystal structure of the N-terminal domain (NTD) of H. pylori DnaB (HpDnaB) helicase at 2.2 Å resolution and compare the structural differences among helicases and correlate with the functional differences. The structural details of NTD suggest that the linker region between NTD and C-terminal helicase domain plays a vital role in accurate assembly of NTD dimers. The sequence analysis of the linker regions from several helicases reveals that they should form four helix bundles. We also report the characterization of H. pylori DnaG primase and study the helicase-primase interactions, where HpDnaG primase stimulates DNA unwinding activity of HpDnaB suggesting presence of helicase-primase cohort at the replication fork. The protein-protein interaction study of C-terminal domain of primase and different deletion constructs of helicase suggests that linker is essential for proper conformation of NTD to interact strongly with HpDnaG. The surface charge distribution on the primase binding surface of NTDs of various helicases suggests that DnaB-DnaG interaction and stability of the complex is most probably charge dependent. Structure of the linker and helicase-primase interactions indicate that HpDnaB differs greatly from E.coli DnaB despite both belong to gram negative bacteria.     

The Escherichia coli primosome can translocate actively in either direction along a DNA strand

The primosome is a mobile multiprotein DNA replication-priming apparatus that requires seven Escherichia coli proteins (replication factor Y (protein n'), proteins n and n", and the products of the dnaB, dnaC, dnaT, and dnaG genes) for assembly at a specific site (termed a primosome assembly site) on single-stranded DNA binding protein-coated single-stranded DNA. Two of the protein components of the primosome have intrinsic DNA helicase activity. The DNA B protein acts in the 5'----3' direction, whereas factor Y acts in the 3'----5' direction. The primosome complex has DNA helicase activity when present at a replication fork in conjunction with the DNA polymerase III holoenzyme. In this report, evidence is presented that the multiprotein primosome per se can act as a DNA helicase in the absence of the DNA polymerase III holoenzyme. The primosome DNA helicase activity can be manifested in either direction along the DNA strand. The directionality of the primosome DNA helicase activity is modulated by the concentration and type of nucleoside triphosphate present in the reaction mixture. This DNA helicase activity requires all the preprimosomal proteins (the primosomal proteins minus the dnaG-encoded primase). Preprimosome complexes must assemble at a primosome assembly site in order to be loaded onto the single-stranded DNA and act subsequently as a DNA helicase. The 5'----3' primosome DNA helicase activity requires a 3' single-stranded tail on the fragment to be displaced, while the 3'----5' activity does not require a 5' single-stranded tail on the fragment to be displaced. Multienzyme preprimosomes moving in either direction are capable of associating with the primase to form complete primosomes that can synthesize RNA primers.     

A single subunit MCM6 from pea forms homohexamer and functions as DNA helicase

The initiation of DNA replication starts from origins and is controlled by a multiprotein complex, which involves about twenty protein factors. One of the important factors is hetrohexameric minichromosome maintenance (MCM2-7) protein complex which is evolutionary conserved and functions as essential replicative helicase for DNA replication. Here we report the isolation and characterization of a single subunit of pea MCM protein complex, the MCM6. The deduced amino acid (827) sequence contains all the known canonical MCM motifs including zinc finger, MCM specific Walker A and Walker B and arginine finger. The purified recombinant protein contains ATP-dependent 3′–5′ DNA helicase, ATP-binding and ATPase activities. The helicase activity was stimulated by replication fork like substrate and anti-MCM6 antibodies curtail all the enzyme activities of MCM6 protein. In vitro it self-interacts and forms a homohexamer which is active for DNA helicase and ATPase activities. The complete protein is required for self-interaction as the truncated MCM6 proteins were unable to self-interact. Western blot analysis and in vivo immunostaining followed by confocal microscopy showed the localization of MCM6 both in the nucleus and cytosol. These findings provide first direct evidence that single subunit MCM6 contains DNA helicase activity which is unique to plant MCM6 protein, as this activity was only reported for heteromultimers of MCM proteins in animal system. This discovery should make an important contribution to a better understanding of DNA replication in plants.     

Domain structure of phage P4 alpha protein deduced by mutational analysis.

Bacteriophage P4 DNA replication depends on the product of the alpha gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the alpha gene. Sequence analysis of the entire gene predicted a domain structure for the alpha polypeptide chain (777 amino acid residues, M(r) 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the alpha polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for alpha mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.     

The three-component helicase/primase complex of herpes simplex virus-1

Herpes simplex virus type 1 (HSV-1) is one of the nine herpesviruses that infect humans. HSV-1 encodes seven proteins to replicate its genome in the hijacked human cell. Among these are the herpes virus DNA helicase and primase that are essential components of its replication machinery. In the HSV-1 replisome, the helicase–primase complex is composed of three components including UL5 (helicase), UL52 (primase) and UL8 (non-catalytic subunit). UL5 and UL52 subunits are functionally interdependent, and the UL8 component is required for the coordination of UL5 and UL52 activities proceeding in opposite directions with respect to the viral replication fork. Anti-viral compounds currently under development target the functions of UL5 and UL52. Here, we review the structural and functional properties of the UL5/UL8/UL52 complex and highlight the gaps in knowledge to be filled to facilitate molecular characterization of the structure and function of the helicase–primase complex for development of alternative anti-viral treatments.     

Homogenous assays for Escherichia coli DnaB-stimulated DnaG primase and DnaB helicase and their use in screening for chemical inhibitors

Escherichia coli DnaG primase is a single-stranded DNA-dependent RNA polymerase. Primase catalyzes the synthesis of a short RNA primer to initiate DNA replication at the origin and to initiate Okazaki fragment synthesis for synthesis of the lagging strand. Primase activity is greatly stimulated through its interaction with DnaB helicase. Here we report a 96-well homogeneous scintillation proximity assay (SPA) for the study of DnaB-stimulated E. coli primase activity and the identification of E. coli primase inhibitors. The assay uses an adaptation of the general priming reaction by employing DnaG primase, DnaB helicase, and ribonucleotidetriphosphates (incorporation of [(3)H]CTP) for in vitro primer synthesis on single-stranded oligonucleotide and M13mp18 DNA templates. The primase product is captured by polyvinyl toluene-polyethyleneimine-coated SPA beads and quantified by counting by beta-scintography. In the absence of helicase as a cofactor, primer synthesis is reduced by 85%. The primase assay was used for screening libraries of compounds previously identified as possessing antimicrobial activities. Primase inhibitory compounds were then classified as direct primase inhibitors or mixed primase/helicase inhibitors by further evaluation in a specific assay for DnaB helicase activity. By this approach, specific primase inhibitors could be identified.     

A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein

We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A' helicase-primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein-DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction.