Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis

The 3' to 5' exonuclease activity of bacteriophage T7 DNA polymerase (gene 5 protein) can be inactivated selectively by reactive oxygen species. Differences in the enzymatic properties between the two forms are exploited to show by a chemical screen that modification of a histidine residue reduces selectively the exonuclease activity. In vitro mutagenesis of the histidine at residue 123, and of the neighboring residues, results in varying reduction of the exonuclease activity, including mutant enzymes that have no detectable exonuclease activity; as a consequence their polymerase activity is increased up to 9-fold. T7 phage containing the mutant genes have a greatly reduced burst size and demonstrate up to a 14-fold increase in the spontaneous mutation rate.     

T7-induced DNA polymerase. Characterization of associated exonuclease activities and resolution into biologically active subunits.

Bacteriophage T7-induced DNA polymerase has been isolated by a procedure suitable for large scale use and which yields near homogeneous enzyme. In addition to previously described DNA polymerase activity and 3' to 5' exonucleolytic activity on single stranded DNA (Grippo, P., and Richardson, C. C. (1971) J. Biol. Chem. 246, 6867-6873), the enzyme also possesses a highly active exonuclease which hydrolyzes duplex substrates with 3' to 5' directionality. The native polymerase has been dissociated using 6 M guanidine HCl and resolved into biologically active subunits: T7 gene 5 protein and Escherichia coli thioredoxin. The phage-specified subunit obtained by this procedure is deficient in DNA polymerase and double strand exonuclease activities, with deficiencies in these activities being apparent at the level of a single turnover. However, it possesses near normal levels of a single strand hydrolytic activity which is identical to that associated with the native polymerase with respect to substrate specificity and suppression of hydrolysis by low levels of deoxyribonucleoside 5'-triphosphates. Thioredoxin forms a molecular complex with the T7 gene 5 protein, and addition of the host protein restores restores DNA polymerase and double strand exonuclease activities to near normal levels.     

Characterization of the 5'' to 3'' exonuclease associated with Thermus aquaticus DNA polymerase.

Thermus aquaticus DNA polymerase was shown to contain an associated 5' to 3' exonuclease activity. Both polymerase and exonuclease activities cosedimented with a molecular weight of 72,000 during sucrose gradient centrifugation. Using a novel in situ activity gel procedure to simultaneously detect these two activities, we observed both DNA polymerase and exonuclease in a single band following either nondenaturing or denaturing polyacrylamide gel electrophoresis: therefore, DNA polymerase and exonuclease activities reside in the same polypeptide. As determined by SDS-polyacrylamide gel electrophoresis this enzyme has an apparent molecular weight of 92,000. The exonuclease requires a divalent cation (MgCl2 or MnCl2), has a pH optimum of 9.0 and excises primarily deoxyribonucleoside 5'-monophosphate from double-stranded DNA. Neither heat denatured DNA nor the free oligonucleotide (24-mer) were efficient substrates for exonuclease activity. The rate of hydrolysis of a 5'-phosphorylated oligonucleotide (24-mer) annealed to M13mp2 DNA was about twofold faster than the same substrate containing a 5'-hydroxylated residue. Hydrolysis of a 5'-terminal residue from a nick was preferred threefold over the same 5'-end of duplex DNA. The 5' to 3' exonuclease activity appeared to function coordinately with the DNA polymerase to facilitate a nick translational DNA synthesis reaction.     

Deoxyribonucleic acid polymerase of bacteriophage T7. Characterization of the exonuclease activities of the gene 5 protein and the reconstituted polymerase.

Homogeneous gene 5 protein of bacteriophage T7, a subunit of T7 DNA polymerase, catalyzes the stepwise hydrolysis of single-stranded DNA in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The gene 5 protein itself does not hydrolyze duplex DNA. However, in the presence of Escherichia coli thioredoxin, the host-specified subunit of T7 DNA polymerase, duplex DNA is hydrolyzed in a 3' leads to 5' direction to yield nucleoside 5'-monophosphates. The apparent Km for thioredoxin in the reaction is 4.8 x 10(-8) M, a value similar to that for the apparent Km of thioredoxin in the complementation assay with gene 5 protein to restore T7 DNA polymerase activity. Both exonuclease activities require Mg2+ and a sulfhydryl reagent for optimal activity, and both activities are sensitive to salt concentration. Deoxyribonucleoside 5'-triphosphates inhibit hydrolysis by both exonuclease activities; hydrolysis of single-stranded DNA by the gene 5 protein is inhibited even in the absence of thioredoxin where there is less than 2% active T7 DNA polymerase. E. coli DNA binding protein (helix destabilizing protein) stimulates the hydrolysis of duplex DNA up to 9-fold under conditions where the hydrolysis of the single-stranded DNA is inhibited 4-fold.     

Polymerization activity of an alpha-like DNA polymerase requires a conserved 3''-5'' exonuclease active site.

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.     

Two forms of the DNA polymerase of bacteriophage T7

The DNA polymerase induced by bacteriophage T7 can be isolated in two different forms. The distinguishing properties are: 1) the specific activities of the associated 3' to 5' single- and double-stranded DNA exonuclease activities, 2) the ability to catalyze DNA synthesis and strand displacement at nicks, and 3) the degree of stimulation of DNA synthesis on nicked, duplex DNAs by the gene 4 protein of phage T7. Form I is obtained when purification is carried out in the absence of EDTA while Form II is obtained if all purification steps are carried out in the presence of 0.1 mM EDTA. Form I has low levels of both exonuclease activities, less than 5% of those of Form II. Form I can initiate DNA synthesis at nicks leading to strand displacement, a consequence of which is its ability to be stimulated manyfold by the helicase activity of gene 4 protein on nicked, duplex templates. On the other hand, Form II cannot initiate synthesis at nicks even in the presence of gene 4 protein. In keeping with its higher exonuclease activities, Form II of T7 DNA polymerase has higher turnover of nucleotides activity (5-fold higher than Form I) and exhibits greater fidelity of nucleotide incorporation, as indicated by the rate of incorporation of 2-aminopurine deoxynucleoside monophosphate. Both forms of T7 DNA polymerase exhibit higher fidelity of nucleotide incorporation than bacteriophage T4 DNA polymerase. In the absence of EDTA or in the presence of FeSO4 or CaCl2, Form II irreversibly converts to Form I. The physical difference between the two forms is not known. No difference in molecular weight can be detected between the corresponding subunits of each form of T7 DNA polymerase as measured by gel electrophoresis in the presence of sodium dodecyl sulfate.     

Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision.

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.     

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.     

Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, under the control of the baculovirus polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclonal antibody. The expressed EBV DNA polymerase catalytic polypeptide was purified from the cytosolic fraction of the recombinant virus-infected insect cells. The purified protein exhibited both DNA polymerase and 3'-to-5' exonuclease activities, which were neutralized by the anti-BALF5 protein-specific antibody. These results indicate that the 3'-to-5' exonuclease activity associated with the EBV DNA polymerase (T. Tsurumi, Virology 182:376-381, 1991) is an inherent feature of the polymerase catalytic polypeptide. The DNA polymerase and the exonuclease activities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purified from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase catalytic subunit was different from that for the polymerase complex. These observations strongly suggest that the presence of EBV DNA polymerase accessory protein, BMRF1 gene product, does influence the enzymatic properties of EBV DNA polymerase catalytic subunit.     

The proofreading 3'-->5' exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

The 3'-->5' exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase gamma (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3'-->5' exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity.     

Proofreading activity of DNA polymerase III responds like elongation activity to auxiliary subunits

A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted. This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction. At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP. E. coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III'. Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease. Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E. coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.     

Processive replication of single-stranded DNA templates by the herpes simplex virus-induced DNA polymerase

The DNA polymerase encoded by herpes simplex virus 1 consists of a single polypeptide of Mr 136,000 that has both DNA polymerase and 3'----5' exonuclease activities; it lacks a 5'----3' exonuclease. The herpes polymerase is exceptionally slow in extending a synthetic DNA primer annealed to circular single-stranded DNA (turnover number approximately 0.25 nucleotide). Nevertheless, it is highly processive because of its extremely tight binding to a primer terminus (Kd less than 1 nM). The single-stranded DNA-binding protein from Escherichia coli greatly stimulates the rate (turnover number approximately 4.5 nucleotides) by facilitating the efficient binding to and extension of the DNA primers. Synchronous replication by the polymerase of primed single-stranded DNA circles coated with the single-stranded DNA-binding protein proceeds to the last nucleotide of available 5.4-kilobase template without dissociation, despite the 20-30 min required to replicate the circle. Upon completion of synthesis, the polymerase is slow in cycling to other primed single-stranded DNA circles. ATP (or dATP) is not required to initiate or sustain highly processive synthesis. The 3'----5' exonuclease associated with the herpes DNA polymerase binds a 3' terminus tightly (Km less than 50 nM) and is as sensitive as the polymerase activity to inhibition by phosphonoacetic acid (Ki approximately 4 microM), suggesting close communication between the polymerase and exonuclease sites.     

The proofreading 3′→5′ exonuclease activity of DNA polymerases: a kinetic barrier to translesion DNA synthesis

The 3′→5′ exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase γ (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3′→5′ exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity.     

Selective immunoneutralization of the multiple activities of Escherichia coli DNA polymerase I supports the model for separate active sites and indicates a complex 5' to 3' exonuclease.

DNA polymerase I (pol I) from Escherichia coli has three well-defined activities: DNA polymerase, 3'-5' exonuclease, and 5'-3' exonuclease. We have raised monoclonal antibodies to pol I which selectively neutralize each of these three activities, thus supporting the model of separate active sites for each activity, heretofore exclusively demonstrated with proteolytic fragments of pol I. Antibodies from each class could bind pol I in the presence of antibodies of another class, indicating the existence of significant spatial separation between each of the three sites. In addition, several of the neutralizing antibodies were able to distinguish particular activities of the 5'-3' exonuclease. One of them, for example, inhibited the RNase H activity but not the DNase activity. Two other antibodies could, in addition to inhibiting the polymerase and the 3'-5' exonuclease, either stimulate or inhibit the 5'-3' exonuclease depending upon the assay conditions, particularly the ionic strength.     

Interaction of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage T7. The redox capacity of thioredoxin is not required for stimulation of DNA polymerase activity

DNA polymerase activity in Escherichia coli cells infected with bacteriophage T7 resides in a protein complex consisting of the T7 gene 5 protein and E. coli thioredoxin in a 1 to 1 stoichiometry. We have analyzed nine mutant thioredoxins, both in vivo and in vitro, for their ability to interact with the T7 gene 5 protein and stimulate the DNA polymerase and exonuclease activities inherent in gene 5 protein. The efficiency of plating of T7 on E. coli thioredoxin mutants depends strongly on the copy number of the respective mutant thioredoxin allele. Plating efficiencies at a constant copy number correlate well with the affinity of the purified mutant proteins for T7 gene 5 protein. The observed dissociation constant, Kobs, is increased between 5 and several hundredfold at 42 degrees C compared to wild-type thioredoxin. The maximum polymerase activity of the reconstituted gene 5 protein-thioredoxin complex at saturating concentrations of mutant thioredoxins, however, is reduced by less than 20%. Consequently, none of the mutant thioredoxins acts as a competitive inhibitor of wild-type thioredoxin. The active-site disulfide of thioredoxin is not essential for the activities of the gene 5 protein-thioredoxin complex. Both cysteines can be replaced without significantly affecting the maximum polymerase or exonuclease activities. Substitution or alkylation of either cysteine, however, reduces the affinity for gene 5 protein drastically, indicating that the active site is part of the thioredoxin surface involved in the protein-protein interaction.     

Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma

The high fidelity of chick embryo DNA polymerase-gamma (pol-gamma) observed during in vitro DNA synthesis (Kunkel, T. A. (1985) J. Biol. Chem. 260, 12866-12874) has led us to examine this DNA polymerase for the presence of an exonuclease activity capable of proofreading errors. Highly purified chick embryo pol-gamma preparations do contain exonuclease activity capable of digesting radiolabeled DNA in a 3'----5' direction, releasing deoxynucleoside 5'-monophosphates. The polymerase and exonuclease activities cosediment during centrifugation in a glycerol gradient containing 0.5 M KCl. In the absence of dNTP substrates, this exonuclease excises both matched and mismatched primer termini, with a preference for mismatched bases. Excision is inhibited by the addition of nucleoside 5'-monophosphates to the digestion reaction. In the presence of dNTP substrates to permit competition between excision and polymerization from the mismatched primer, the exonuclease excises mismatched bases from preformed terminal mispairs with greater than 98% efficiency. The preference for excision over polymerization can be diminished by addition of either high concentrations of dNTP substrates or nucleoside 5'-monophosphates to the exonuclease/polymerase reaction. To determine if this exonuclease is capable of proofreading misinsertions produced during a normal polymerization reaction, a sensitive base substitution fidelity assay was developed based on reversion of an M13mp2 lacZ alpha nonsense codon. In this assay using reaction conditions that permit highly active exonucleolytic proofreading, pol-gamma exhibits a fidelity of less than one error for every 260,000 bases polymerized. As for terminal mismatch excision, fidelity is reduced by the addition to the synthesis reaction of high concentrations of dNTP substrates or nucleoside 5'-monophosphates, both hallmarks of exonucleolytic proofreading by prokaryotic enzymes. Taken together, these observations suggest that the 3'----5' exonuclease present in highly purified chick embryo pol-gamma preparations proofreads base substitution errors during DNA synthesis. It remains to be determined if the polymerase and exonuclease activities reside in the same or different polypeptides.     

How DNA travels between the separate polymerase and 3'-5'-exonuclease sites of DNA polymerase I (Klenow fragment)

The polymerase and 3'-5'-exonuclease activities of the Klenow fragment of DNA polymerase I are located on separate structural domains of the protein, separated by about 30 A. To determine whether a DNA primer terminus can move from one active site to the other without dissociation of the enzyme-DNA complex, we carried out reactions on a labeled DNA substrate in the presence of a large excess of unlabeled DNA, to limit observations to a single enzyme-DNA encounter. The results indicated that while Klenow fragment is capable of intramolecular shuttling of a DNA substrate between the two catalytic sites, the intermolecular pathway involving enzyme-DNA dissociation can also be used. Thus, there is nothing in the protein structure or the reaction mechanism that dictates a particular means of moving the DNA substrate. Instead, the use of the intermolecular or the intramolecular pathway is determined by the competition between the polymerase or exonuclease reaction and DNA dissociation. When the substrate has a mispaired primer terminus, DNA dissociation seems generally more rapid than exonucleolytic digestion. Thus, Klenow fragment edits its own polymerase errors by a predominantly intermolecular process, involving dissociation of the enzyme-DNA complex and reassociation of the DNA with the exonuclease site of a second molecule of Klenow fragment.     

Characterization of 3''----5'' exonuclease associated with DNA polymerase of silkworm nuclear polyhedrosis virus.

3'----5' Exonuclease specific for single-stranded DNA copurified with DNA polymerase of nuclear polyhedrosis virus of silkworm Bombyx mori (BmNPV Pol). BmNPV Pol has no detectable 5'----3' exonuclease activity on single-stranded or duplex DNA. Analysis of the products of 3'----5' exonucleolytic reaction showed that deoxynucleoside monophosphates were released during the hydrolysis of single-stranded DNA. The exonuclease activity cosedimented with the polymerase activity during ultracentrifugation of BmNPV Pol in glycerol gradient. The polymerase and the exonuclease activities of BmNPV Pol were inactivated by heat with nearly identical kinetics. The mode of the hydrolysis of single-stranded DNA by BmNPV Pol-associated exonuclease was strictly distributive. The enzyme dissociated from single-stranded DNA after the release of a single dNMP and then reassociated with a next polynucleotide being degradated.     

Characterization of a 3''----5'' exonuclease activity in the phage phi 29-encoded DNA polymerase.

Purified protein p2 of phage phi 29, characterized as a specific DNA polymerase involved in the initiation and elongation of phi 29 DNA replication, contains a 3'----5' exonuclease active on single-stranded DNA, but not on double-stranded DNA. No 5'----3' exonuclease activity was found. The 3'----5' exonuclease activity was shown to be associated with the DNA polymerase since 1) the two activities were heat-inactivated with identical kinetics and 2) both activities, present in purified protein p2, cosedimented in a glycerol gradient.     

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.