DNA translocation activity of the multifunctional replication protein ORF904 from the archaeal plasmid pRN1

The replication protein ORF904 from the plasmid pRN1 is a multifunctional enzyme with ATPase-, primase- and DNA polymerase activity. Sequence analysis suggests the presence of at least two conserved domains: an N-terminal prim/pol domain with primase and DNA polymerase activities and a C-terminal superfamily 3 helicase domain with a strong double-stranded DNA dependant ATPase activity. The exact molecular function of the helicase domain in the process of plasmid replication remains unclear. Potentially this motor protein is involved in duplex remodelling and/or origin opening at the plasmid replication origin. In support of this we found that the monomeric replication protein ORF904 forms a hexameric ring in the presence of DNA. It is able to translocate along single-stranded DNA in 3′–5′ direction as well as on double-stranded DNA. Critical residues important for ATPase activity and DNA translocation activity were identified and are in agreement with a homology model of the helicase domain. In addition we propose that a winged helix DNA-binding domain at the C-terminus of the helicase domain could assist the binding of the replication protein specifically to the replication origin.     

Effect of spermine on interaction of DNA polymerase α from the loach (Misgurnus fossilis) eggs with DNA

Polyamines (putrescine, spermidine and spermine) cause a marked increase in the activity of the loach Misgurnus fossilis DNA polymerase α on activated (gapped) DNA. The stimulatory effect increases in the order: putrescine, spermidine, spermine. Kinetic analysis shows that spermine does not change the affinity of the polymerase for dTTP, but it decreases the enzyme affinity for DNA. The apparent K_m of the polymerase for activated DNA progressively increases from 14 to 1200 μM (nucleotide), if the concentration of spermine rises up to 2 mM, while V_max reaches a maximum at 0.5 mM spermine and then drops at higher polyamine concentrations. Native calf thymus DNA and especially single-stranded DNA from phage M13 appear to be inhibitors of α-polymerase activity on gapped DNA. Dixon plots suggest simple competitive inhibition of the polymerase activity by single- or double-stranded DNA and absence of cooperativity in the interaction of the polymerase with DNA. Hill-plot analysis is compatible with the interpretation that there is only one DNA binding site on each DNA polymerase α molecule. Spermine, even at low concentrations, decreases sharply the affinity of the enzyme for double-stranded DNA, while the enzyme affinity for single-stranded DNA changes insignificantly. Another result of spermine action is the destabilization of the polymerase-DNA complex. The ratio of the ‘static affinity’ of the enzyme to its ‘kinetic affinity’ decreases 2.2-fold in the presence of 0.5 mM spermine. As a result, the sensitivity of DNA synthesis to 3′-deoxy-3′-aminothymidine 5′-triphosphate and to 1-β-d-arabinofuranosylcytidine 5′-triphosphate decreases in the presence of the polyamine. Both spermine effects, the decrease in the ‘nonproductive binding’ of the polymerase to double-stranded regions in DNA and the destabilization of the polymerase-DNA complex, presumably account for the increase in the activity of the loach α-polymerase on activated DNA.     

The N-terminal domain of TWINKLE contributes to single-stranded DNA binding and DNA helicase activities

The TWINKLE protein is a hexameric DNA helicase required for replication of mitochondrial DNA. TWINKLE displays striking sequence similarity to the bacteriophage T7 gene 4 protein (gp4), which is a bi-functional primase-helicase required at the phage DNA replication fork. The N-terminal domain of human TWINKLE contains some of the characteristic sequence motifs found in the N-terminal primase domain of the T7 gp4, but other important motifs are missing. TWINKLE is not an active primase in vitro and the functional role of the N-terminal region has remained elusive. In this report, we demonstrate that the N-terminal part of TWINKLE is required for efficient binding to single-stranded DNA. Truncations of this region reduce DNA helicase activity and mitochondrial DNA replisome processivity. We also find that the gp4 and TWINKLE are functionally distinct. In contrast to the phage protein, TWINKLE binds to double-stranded DNA. Moreover, TWINKLE forms stable hexamers even in the absence of Mg²⁺ or NTPs, which suggests that an accessory protein, a helicase loader, is needed for loading of TWINKLE onto the circular mtDNA genome.     

Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A.

DNA primase has been partially purified from wheat germ. This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication. However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha. Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A. In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A. Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined. The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate. The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion. Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF). M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory. Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA.     

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.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

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.     

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader

Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.     

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.     

DNA primase associated with 10 S DNA polymerase α from calf thymus

Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d₂TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

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.     

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.     

The Escherichia coli dnaB replication protein is a DNA helicase

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.     

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.     

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

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

S1 nuclease of Aspergillus oryzae: a glycoprotein with an associated nucleotidase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae has been purified 1600-fold by a procedure designed to remove traces of contaminating phosphatases. The nearly homogeneous enzyme was found to be a glycoprotein with a carbohydrate content of 18%. At pH 4.5 the enzyme preparation hydrolyzed single-stranded DNA, RNA, 3′-AMP, and 2′-AMP at relative rates of 100, 52, 13, and 0.05, respectively. The 3′-nucleotidase activity of this single-strand specific nuclease is inhibited by single-stranded DNA but not by double-stranded DNA. Three forms of the enzyme, with isoelectric points of 3.35, 3.53, and 3.67, were observed on electrofocusing, and each form exhibited the same relative activity on single-stranded DNA and 3′-AMP. Enzymatic hydrolysis of nucleotides occurred over a broad range of pH, with maximal activity at pH 6–7. Ribonucleotides were hydrolyzed approximately 100-fold more rapidly than deoxyribonucleotides. A high degree of base specificity was not observed. The 3′-nucleotidase activity was stimulated by Zn²⁺, but not by other divalent cations tested.     

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3′- and 5′-ends were immobilized within the 100 × 100 × 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T_m) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T_m. The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.