Thioredoxin,the Processivity Factor,Sequesters an Exposed Cysteine in the Thumb Domain of Bacteriophage T7 DNA Polymerase

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase. It achieves processivity by binding to Escherichia coli thioredoxin (trx). gp5/trx complex binds tightly to a primer-DNA template enabling the polymerization of hundreds of nucleotides per binding event. gp5 contains 10 cysteines. Under non-reducing condition, exposed cysteines form intermolecular disulfide linkages resulting in the loss of polymerase activity. No disulfide linkage is detected when Cys-275 and Cys-313 are replaced with serines. Cys-275 and Cys-313 are located on loop A and loop B of the thioredoxin binding domain, respectively. Replacement of either cysteine with serine (gp5-C275S, gp5-C313S) drastically decreases polymerase activity of gp5 on dA₃₅₀/dT₂₅. On this primer-template gp5/trx in which Cys-313 or Cys-275 is replaced with serine have 50 and 90%, respectively, of the polymerase activity observed with wild-type gp5/trx. With single-stranded M13 DNA as a template gp5-C275S/trx retains 60% of the polymerase activity of wild-type gp5/trx. In contrast, gp5-C313S/trx has only one-tenth of the polymerase activity of wild-type gp5/trx on M13 DNA. Both wild-type gp5/trx and gp5-C275S/trx catalyze the synthesis of the entire complementary strand of M13 DNA, whereas gp5-C313S/trx has difficulty in synthesizing DNA through sites of secondary structure. gp5-C313S fails to form a functional complex with trx as measured by the apparent binding affinity as well as by the lack of a physical interaction with thioredoxin during hydroxyapatite-phosphate chromatography. Small angle x-ray scattering reveals an elongated conformation of gp5-C313S in comparison to a compact and spherical conformation of wild-type gp5.     

Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7

Bacteriophage T7 gene 5 protein has been purified to apparent homogeneity from cells overexpressing its gene several hundred-fold. Gene 5 protein is a DNA polymerase with low processivity; it dissociates from the primer-template after catalyzing the incorporation of 1-50 nucleotides, depending on the salt concentration. Escherichia coli thioredoxin, a host protein that is tightly associated with the gene 5 protein in phage-infected cells, is not required for this activity. Thioredoxin acts as an accessory protein to bestow processivity on the polymerizing reaction; DNA synthesis catalyzed by the gene 5 protein-thioredoxin complex on a single-stranded DNA template can polymerize thousands of nucleotides without dissociation. Conditions that increase the stability of secondary structures in the template (i.e., low temperature or high ionic strength) decrease the processivity. E. coli single-stranded DNA-binding protein stimulates both the rate of elongation and the processivity of the gene 5 protein-thioredoxin complex.     

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.     

Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.     

Processivity of the DNA polymerase alpha-primase complex from calf thymus

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.     

A unique region in bacteriophage t7 DNA polymerase important for exonucleolytic hydrolysis of DNA

A crystal structure of the bacteriophage T7 gene 5 protein/Escherichia coli thioredoxin complex reveals a region in the exonuclease domain (residues 144-157) that is not present in other members of the E. coli DNA polymerase I family. To examine the role of this region, a genetically altered enzyme that lacked residues 144-157 (T7 polymerase (pol) Delta144-157) was purified and characterized biochemically. The polymerase activity and processivity of T7 pol Delta144-157 on primed M13 DNA are similar to that of wild-type T7 DNA polymerase implying that these residues are not important for DNA synthesis. The ability of T7 pol Delta144-157 to catalyze the hydrolysis of a phosphodiester bond, as judged from the rate of hydrolysis of a p-nitrophenyl ester of thymidine monophosphate, also remains unaffected. However, the 3'-5' exonuclease activity on polynucleotide substrates is drastically reduced; exonuclease activity on single-stranded DNA is 10-fold lower and that on double-stranded DNA is 20-fold lower as compared with wild-type T7 DNA polymerase. Taken together, our results suggest that residues 144-157 of gene 5 protein, although not crucial for polymerase activity, are important for DNA binding during hydrolysis of polynucleotides.     

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.     

Mutation of Phe102 to Ser in the carboxyl terminal helix of Escherichia coli thioredoxin affects the stability and processivity of T7 DNA polymerase

Processivity of T7 DNA polymerase relies on the coupling of its cofactor Escherichia coli thioredoxin (Trx) to gene 5 protein (gp5) at 1:1 stoichiometry. We designed a coexpression system for gp5 and Trx that allows in vivo reconstitution of subunits into a functional enzyme. The properties of this enzyme were compared with the activity of commercial T7 DNA polymerase. Examination of purified enzymes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the thioredoxin subunit of the two enzymes did not comigrate. To our surprise, we identified a mutation (Phe102 to Ser) in the Trx component from the commercial T7 DNA polymerase (gp5/TrxS102) that was not in the enzyme from the coexpression system (wild type gp5/Trx). A comparison of polymerase activity of the T7 DNA polymerases shows that both enzymes possessed similar specific activity but they were different in their residual activity at 37 degrees C. The half-life of gp5/TrxS102 was 7 min at 37 degrees C and 12 min for gp5/Trx. gp5/TrxS102 polymerase activity was reduced by fourfold with 3'-5' exonuclease activity as the prominent activity detected after 10 min of heat inactivation at 37 degrees C. Supplementation of reaction mixtures containing gp5/TrxS102 with exogenous nonmutant thioredoxin restored the enzyme activity levels. Pulse proteolysis was used to demonstrate that TrxS102 unfolded at lower urea concentrations than wild type thioredoxin. Thus, Ser substitution at position 102 affected the structural stability of thioredoxin resulting in a reduced binding affinity for gp5 and loss of processivity.     

Functional interaction between the herpes simplex-1 DNA polymerase and UL42 protein

The herpes simplex virus 1 (HSV-1) UL42 protein, one of seven herpes-encoded polypeptides that are required for the replication of the HSV-1 genome, is found in a 1:1 complex with the HSV-1 DNA polymerase (Crute, J. J., and Lehman, I. R. (1989) J. Biol. Chem. 264, 19266-19270). To obtain herpes DNA polymerase free of UL42 protein, we have cloned and overexpressed the Pol gene in a recombinant baculovirus vector and purified the recombinant DNA polymerase to near homogeneity. Replication of singly primed M13mp18 single-stranded DNA by the recombinant enzyme in the presence of the herpes encoded single-stranded DNA-binding protein ICP8 yields in addition to some full-length product a distribution of intermediate length products by a quasi-processive mode of deoxynucleotide polymerization. Addition of the purified UL42 protein results in completely processive polymerization and the generation of full-length products. Similar processivity is observed with the HSV-1 DNA polymerase purified from herpes-infected Vero cells. Processive DNA replication by the DNA polymerase isolated from HSV-1-infected Vero cells or the recombinant DNA polymerase-UL42 protein complex requires that the single-stranded DNA be coated with saturating levels of ICP8. ICP8 which binds single-stranded DNA in a highly cooperative manner is presumably required to melt out regions of secondary structure in the single-stranded DNA template, thereby potentiating the processivity enhancing action of the UL42 protein.     

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.     

Mechanism of stimulation of T7 DNA polymerase by Escherichia coli single-stranded DNA binding protein (SSB)

Single-stranded DNA binding protein is a key component in growth of bacteriophage T7. In addition, DNA synthesis by the purified in vitro replication system is markedly stimulated when the DNA template is coated with Escherichia coli single-stranded DNA binding protein (SSB). In an attempt to understand the mechanism for this stimulation, we have studied the effect of E. coli SSB on DNA synthesis by the T7 DNA polymerase using a primed single-stranded M13 DNA template which serves as a model for T7 lagging strand DNA synthesis. Polyacrylamide gel analysis of the DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase pauses at many specific sites, some stronger than others. By comparing the position of pausing with the DNA sequence of this region and by using a DNA template that contains an extremely stable hairpin structure, it was found that many, but not all, of these pause positions correspond to regions of potential secondary structure. The presence of SSB during synthesis resulted in a large reduction in the frequency of hesitations at many sites that correspond to these secondary structures. However, the facts that a large percentage of the pause sites remain unaffected even at saturating levels of SSB and that SSB stimulates synthesis on a singly primed poly(dA) template suggested that other mechanisms also contribute to the stimulation of DNA synthesis caused by SSB. Using a sucrose gradient analysis, we found that SSB increases the affinity of the polymerase for single-stranded DNA that this increased binding is only noticed when the polymerase concentration is limiting. The effect of this difference in polymerase affinity was clearly observed by a polyacrylamide gel analysis of the product DNA synthesized during a limited DNA synthesis reaction using conditions where only two nucleotides are added to the primer. Under these circumstances, where the presence of hairpin structures should not contribute to the stimulatory effect of SSB, we found that the extension of the primer is stimulated 4-fold if the DNA template is coated with SSB. Furthermore, SSB had no effect on this synthesis at large polymerase to template ratios.     

Two Modes of Interaction of the Single-stranded DNA-binding Protein of Bacteriophage T7 with the DNA Polymerase-Thioredoxin Complex

The DNA polymerase encoded by bacteriophage T7 has low processivity. Escherichia coli thioredoxin binds to a segment of 76 residues in the thumb subdomain of the polymerase and increases the processivity. The binding of thioredoxin leads to the formation of two basic loops, loops A and B, located within the thioredoxin-binding domain (TBD). Both loops interact with the acidic C terminus of the T7 helicase. A relatively weak electrostatic mode involves the C-terminal tail of the helicase and the TBD, whereas a high affinity interaction that does not involve the C-terminal tail occurs when the polymerase is in a polymerization mode. T7 gene 2.5 single-stranded DNA-binding protein (gp2.5) also has an acidic C-terminal tail. gp2.5 also has two modes of interaction with the polymerase, but both involve the C-terminal tail of gp2.5. An electrostatic interaction requires the basic residues in loops A and B, and gp2.5 binds to both loops with similar affinity as measured by surface plasmon resonance. When the polymerase is in a polymerization mode, the C terminus of gene 2.5 protein interacts with the polymerase in regions outside the TBD. gp2.5 increases the processivity of the polymerase-helicase complex during leading strand synthesis. When loop B of the TBD is altered, abortive DNA products are observed during leading strand synthesis. Loop B appears to play an important role in communication with the helicase and gp2.5, whereas loop A plays a stabilizing role in these interactions.     

Analysis of secondary structures in M13mp8 (+) single-stranded DNA by the pausing of DNA polymerase alpha

The pausing of DNA replication has been used as a tool for analyzing secondary structures in a single-stranded DNA. M13mp8 (+) single-stranded DNA was replicated in vitro by the DNA polymerase alpha from calf thymus. The positions of pausing were determined from DNA sequencing gels. All experimentally observed pausing sites could be correlated with computer-predicted secondary structures of the M13 single-stranded DNA. In the computer calculations of the secondary structures, long-range base-pairing, G.T mispairs and loop-out of bases were allowed. By using six different primers, the pausing site pattern and the corresponding secondary structure map of a region comprising 1400 nucleotides of the M13 genome has been established. Our experiments indicate that the M13 DNA is highly structured. Most of the stable structures are clustered around the origin of replication. With fragments of the M13 DNA, we show that long-range base-pairing exists in the M13 single-stranded genome and we present evidence for tertiary structure interactions. Furthermore we observe structures that form newly during the course of replication. The Escherichia coli single-stranded DNA-binding protein facilitates replication through the barriers.     

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.     

Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.     

DNA-thumb interactions and processivity of T7 DNA polymerase in comparison to yeast polymerase eta

The replicative polymerase of bacteriophage T7 is structurally and mechanistically well characterized. The crystal structure of T7 DNA polymerase or gene 5 protein complexed to its processivity factor, Escherichia coli thioredoxin, a primer-template, and a dideoxynucleotide reveals how this enzyme interacts with the 3'-end of the primer-template, but does not show how thioredoxin confers processivity to the polymerase. In the crystal structure highly conserved amino acids Asn(335) and Ser(338) of the thumb subdomain of T7 DNA polymerase are seen to interact with phosphates 7 and 8 of the DNA template strand. Results with a mutant T7 DNA polymerase in which aliphatic residues are substituted for these amino acids and experiments with different length and methylphosphonate-modified primer-templates demonstrate that these interactions are essential for processive synthesis and d(A.T)(n) tract bypass. Our data with methylphosphonate-modified DNA suggests that thioredoxin confers processivity to T7 DNA polymerase in part by causing an interaction with the phosphate backbone or minor groove of DNA. Residues Asn(335) and Ser(338) may also function with a nearby helix-loop-helix motif located at residues 339-372 to enclose the DNA during processive synthesis. Our results suggest that this structure must be held close to the DNA by ionic interactions to function. These interactions also allow for DNA sliding but physically block the passage of a 3T bulge in the template. In contrast, yeast polymerase eta, a polymerase that non-mutagenically repairs cis-syn thymidine dimers, allows the same bulge to slide past its thumb subdomain during synthesis. A relaxed thumb interaction with the DNA could account for the notably low processivity of polymerase eta.     

1H-NMR studies of calmodulin. The nature of the Ca2+-dependent conformational change

The replication of M13 single-stranded DNA by the 9S DNA polymerase alpha from calf thymus has been studied in vitro. Priming conditions, the nature of the replication products and conditions for optimal elongation have been investigated. Oligonucleotides comprising only four nucleotides can serve as primers. Both ribo and deoxy oligonucleotides can be elongated. Priming by the short oligonucleotides occurs at multiple sites on the M13 genome. If replication is primed at single sites with a specific pentadecamer or with RNA in the origin of replication, specific pausing sites are observed. These pausing sites can partly be correlated with secondary structures in the template DNA. Addition of Escherichia coli single-stranded DNA binding protein leads to a weakening of pausing sites and to the synthesis of longer products. The 9S enzyme is able to proceed through most of the pausing sites resulting in the synthesis of product molecules as long as 6600 nucleotides. The 9S DNA polymerase alpha contains a potent DNA primase activity which enables it to initiate replication on a single-stranded template in the presence of the four NTPs . However, priming is also possible in the presence of ATP alone. The priming sites are not randomly distributed over the M13 DNA.     

Initiation, elongation and pausing of in vitro DNA synthesis catalyzed by immunopurified yeast DNA primase: DNA polymerase complex.

Yeast DNA primase and DNA polymerase I can be purified by immunoaffinity chromatography as a multipeptide complex which can then be resolved into its functional components and further reassembled in vitro. Isolated DNA primase synthesizes oligonucleotides of a preferred length of 9-10 nucleotides and multiples thereof on a poly(dT) template. In vitro reconstitution of the DNA primase:DNA polymerase complex allows the synthesis of long DNA chains covalently linked to RNA initiators shorter than those synthesized by DNA primase alone. The SS (single-stranded) circular DNA of phage M13mp9 can also be replicated by the DNA primase:DNA polymerase complex. Priming by DNA primase occurs at multiple sites and the initiators are utilized by the DNA polymerase moiety of the complex, so that almost all the SS template is converted into duplex form. The rate of DNA synthesis catalyzed by isolated yeast DNA polymerase I on the M13mp9 template is not constant and is characterized by distinct pausing sites, which partly correlate with secondary structures on the template DNA. Thus, replication of M13mp9 SS DNA with the native primase:polymerase complex gives rise to a series of DNA chains with significantly uniform termini specified by the primase start sites and the polymerase stop sites.     

Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro.

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.