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PubMed1991

Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction.

We present a kinetic partitioning mechanism by which the highly efficient 3'----5' exonuclease activity of T7 DNA polymerase maximizes its contribution to replication fidelity with minimal excision of correctly base-paired DNA. The elementary rate constants for the proposed mechanism have been measured directly from single-turnover experiments by using rapid chemical quench-flow techniques. The exonuclease activity of T7 DNA polymerase toward single-stranded DNA is quite fast (kx greater than 700 s-1). This rapid exonuclease is restrained with double-stranded DNA by a kinetic partitioning mechanism that favors the binding of the DNA to the polymerase site to prevent the rapid degradation of matched DNA and yet allows selective removal of mismatched DNAs. Both matched and mismatched DNAs bind tightly to the polymerase site, with approximately equal affinities, Kdp = 20 and 10 nM, respectively. Selective removal of the mismatch is governed by the rate of transfer of the DNA from the polymerase to the exonuclease site (kp----x). The rapid excision of matched DNA is limited by a slow transfer rate (kp----x = 0.2 s-1) from the polymerase to the exonuclease site relative to the rate of polymerization [kp = 300 s-1; Patel et al. (1991) Biochemistry (first of three papers in this issue)]. Removal of mismatched DNA is facilitated by its faster transfer rate (kp----x = 2.3 s-1) to the exonuclease site relative to the slow rate of polymerization over a mismatch [kpi = 0.012 s-1; Wong et al. (1991) Biochemistry (second of three papers in this issue)].(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1998

Replication inhibition and miscoding properties of DNA templates containing a site-specific cis-thymine glycol or urea residue.

Oligodeoxynucleotides modified site-specifically with cis-thymine glycol or urea residue, two ionizing radiation/oxidation damages, were used as templates in primer extension reactions catalyzed by 3' --> 5' exonuclease-deficient Klenow fragment, human DNA polymerase beta, AMV reverse transcriptase, and a modified T7 DNA polymerase (Sequenase). Both lesions blocked DNA replication one nucleotide before and opposite the lesion site, but a significant fraction of full-length product was obtained after prolonged incubation. Hill plot analysis of the results on both thymine glycol- and urea- containing templates by 3' --> 5' exonuclease-deficient Klenow fragment for incorporation of either dATP or dGTP gave linear plots with Hill coefficients much less than 1. This suggests that the dNTP concentration influences the termination of DNA synthesis at multiple steps of the catalytic process. The specificity of nucleotide incorporation opposite these lesions and chain extension by the same polymerase was determined by a steady-state kinetic analysis. The kinetic studies established that the rate of nucleotide incorporation and chain extension was highest with deoxyadenosine opposite both these lesions. However, the efficiency of forming a G.T pair relative to an A.T pair for the control at a level of 1/10(9) was enhanced to approximately 1/160 for thymine glycol and 1/20 for urea, although the former lesion was more bypassable than the latter lesion. On the basis of these in vitro results, we conclude that both these DNA damages are impediments of DNA synthesis and that a urea residue, in particular, has the potential to miscode.

DNA↗
PubMed2005

Thermodynamic and in vitro replication studies of an intrastrand G[8-5]C cross-link lesion.

We recently identified, from the gamma-irradiation mixture of duplex DNA, a new intrastrand G[8-5]C cross-link lesion, in which the C8 atom of guanine and the C5 atom of its 3' neighboring cytosine are covalently bonded, and carried out in vitro replication studies for the lesion-bearing substrate with a translesion synthesis polymerase, yeast polymerase eta. Here we extended the in vitro replication studies to two replicative polymerases, exonuclease-deficient bacteriophage T7 DNA polymerase (T7(-)) and HIV reverse transcriptase (HIV-RT). Primer extension assays showed that both polymerases stopped synthesis after incorporating a nucleotide opposite the 3'-cytosine in the G[8-5]C lesion. Steady-state kinetic measurements for nucleotide incorporation opposite the 3'-cytosine of the lesion showed that both T7(-) and HIV-RT preferentially incorporated the correct nucleotide, dGMP. We also examined the thermal stabilities and base pairing properties of G[8-5]C in d(ATGGCG[8-5]CGCTAT). The G[8-5]C lesion destabilizes the duplex form by approximately 4 kcal/mol in free energy at 25 degrees C relative to the undamaged parent duplex, and the thermally most stable duplex has natural bases opposite the lesion.

Base Sequence↗
PubMed1997

Analysis of nucleotide insertion and extension at 8-oxo-7,8-dihydroguanine by replicative T7 polymerase exo- and human immunodeficiency virus-1 reverse transcriptase using steady-state and pre-steady-state kinetics.

Pre-steady-state kinetics of incorporation of dCTP and dATP opposite site-specific 8-oxo-7,8-dihydroguanine (8-oxoGua), in contrast to dCTP insertion opposite G, were examined as well as extension beyond the lesion using the replicative enzymes bacteriophage polymerase T7 exo- (T7-) and HIV-1 reverse transcriptase (RT). These results were compared to previous findings for Escherichia coli repair polymerases I (KF-) and II (pol II-) exo- [Lowe, L. G., & Guengerich, F. P. (1996) Biochemistry 35, 9840-9849]. HIV-1 RT showed a very high preference for insertion of dATP opposite 8-oxoGua, followed by pol II-, T7-, and KF-. Steady-state assays showed k(cat) consistently lower than pre-steady-state polymerization rates (k(p)) for insertion of dCTP opposite G or 8-oxoGua and insertion of dATP opposite 8-oxoGua. Pre-steady-state kinetic curves for the addition of dCTP opposite 8-oxoGua or G by KF-, pol II-, and T7- were all biphasic, with a rapid initial single-turnover burst followed by a slower multiple turnover rate, while addition of dATP opposite 8-oxoGua by these polymerases did not display burst kinetics. With HIV-1 RT, addition of dATP opposite 8-oxoGua displayed burst kinetics while addition of dCTP did not. Analyses of the chemical step by substitution of phosphorothioate analogs for normal dNTPs suggest that the chemistry is rate-limiting during addition of dCTP and dATP opposite 8-oxoGua by KF-, pol II-, and T7-; HIV- RT did not show a chemical rate-limiting step during addition of dATP opposite 8-oxoGua. Kinetic assays performed with various dCTP concentrations indicate that dCTP has a higher Kd and lower k(p) for incorporation opposite 8-oxoGua compared to G with all four enzymes. The K(d,app)dATP values for KF-, pol II-, and T7- incorporation of dATP opposite 8-oxoGua, estimated in competition assays, were found to be 3-10-fold greater than the K(d)dCTP. Likewise, the K(d,app)dCTP for HIV-1 RT incorporation of dCTP opposite 8-oxoGua was found to be 10-fold greater than the K(d)dATP. The repair enzymes (KF- and pol II-) efficiently extended the 8-oxoGua x A pair; extension of 8-oxoGua x C was severely impaired, whereas the replicative enzymes (T7- and HIV-1 RT) extended both pairs, with faster rates for the extension of the 8-oxoGua x A pair. On the basis of these findings, the fidelity of all four enzymes during replication of 8-oxoGua depends on contributions from the apparent Kd, the ease of base pair extension, and either the rate of conformational change before chemistry or the rate of bond formation.

Bacteriophage T7↗
PubMed1992

Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.

T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.

Base Sequence↗
PubMed1997

Substrate and mispairing properties of 5-formyl-2'-deoxyuridine 5'-triphosphate assessed by in vitro DNA polymerase reactions.

5-Formyluracil (fU) is one of the thymine lesions produced by reactive oxygen radicals in DNA and its constituents. In this work, 5-formyl-2'-deoxyuridine 5'-triphosphate (fdUTP) was chemically synthesized and extensively purified by HPLC. The electron withdrawing 5-formyl group facilitated ionization of fU. Thus, p K a of the base unit of fdUTP was 8.6, significantly lower than that of parent thymine (p K a = 10.0 as dTMP). fdUTP efficiently replaced dTTP during DNA replication catalyzed by Escherichia coli DNA polymerase I (Klenow fragment), T7 DNA polymerase (3'-5'exonuclease free) and Taq DNA polymerase. fU-specific cleavage of the replication products by piperidine revealed that when incorporated as T, incorporation of fU was virtually uniform, suggesting minor sequence context effects on the incorporation frequency of fdUTP. fdUTP also replaced dCTP, but with much lower efficiency than that for dTTP. The substitution efficiency for dCTP increased with increasing pH from 7.2 to 9.0. The parallel correlation between ionization of the base unit of fdUTP (p K a = 8.6) and the substitution efficiency for dCTP strongly suggests that the base-ionized form of fdUTP is involved in mispairing with template G. These data indicate that fU can be specifically introduced into DNA as unique lesions by in vitro DNA polymerase reactions. In addition, fU is potentially mutagenic since this lesion is much more prone to form mispairing with G than parent thymine.

Base Composition↗
PubMed1986

Expression of the unassembled capsid protein during infection of Shigella sonnei by bacteriophage T7 results in DNA damage that is repairable by bacteriophage T3, but not T7, DNA ligase.

The abortive infection of bacteriophage T7 in Shigella sonnei D2 371-48 is characterized by a premature inhibition of phage DNA replication and nucleolytic breakdown of all phage DNA. Mutations in T7 gene 10 which are recessive to the presence of the wild-type allele can alleviate the restriction of phage growth. Phage T3 productively infects S. sonnei D2 371-48, as does a T7-T3 hybrid phage that contains, in particular, a gene 10 of T7 origin. It is the presence of T3 DNA ligase that allows phage growth on S. sonnei D2 371-48, and this enzyme can also rescue wild-type T7 from the abortive infection. T7+ is therefore functionally ligase deficient during the infection of S. sonnei D2 371-48; this deficiency is a result of the expression of the phage capsid protein, but it is independent of the assembly of the protein into a procapsid or other morphogenetic structure.

Capsid↗
PubMed1998

The herpes simplex virus type 1 helicase-primase. Analysis of helicase activity.

The rate of unwinding of duplex DNA by the herpes simplex virus type 1 (HSV-1)-encoded helicase-primase (primosome) was determined by measuring the rate of appearance of single strands from a circular duplex DNA containing a 40-nucleotide 5' single-stranded tail, i.e. a preformed replication fork, in the presence of the HSV-1 single strand DNA-binding protein, infected cell protein 8 (ICP8). With this substrate, the rate at low ionic strength was highly sensitive to Mg2+ concentration. The Mg2+ dependence was a reflection of both the requirement for ICP8 for helicase activity and the ability of ICP8 to reverse the helicase reaction as a consequence of its capacity to anneal homologous single strands at Mg2+ concentrations in excess of 3 mM. The rate of unwinding of duplex DNA by the HSV-1 primosome was also determined indirectly by measuring the rate of leading strand synthesis with a preformed replication fork as template in the presence of the T7 DNA polymerase. The value of 60-65 base pairs unwound/s by both methods is consistent with the rate of 50 base pairs/s estimated for the rate of fork movement in vivo during replication of pseudorabies virus, another herpesvirus. Interaction with the helicase-primase did not increase its helicase activity.

Base Sequence↗
PubMed2003

Dissociative properties of the proteins within the bacteriophage T4 replisome.

DNA replication is a highly processive and efficient process that involves the coordination of at least eight proteins to form the replisome in bacteriophage T4. Replication of DNA occurs in the 5' to 3' direction resulting in continuous replication on the leading strand and discontinuous replication on the lagging strand. A key question is how a continuous and discontinuous replication process is coordinated. One solution is to avoid having the completion of one Okazaki fragment to signal the start of the next but instead to have a key step such as priming proceed in parallel to lagging strand replication. Such a mechanism requires protein elements of the replisome to readily dissociate during the replication process. Protein trapping experiments were performed to test for dissociation of the clamp loader and primase from an active replisome in vitro whose template was both a small synthetic DNA minicircle and a larger DNA substrate. The primase, clamp, and clamp loader are found to dissociate from the replisome and are continuously recruited from solution. The effect of varying protein concentrations (dilution) on the size of Okazaki fragments supported the protein trapping results. These findings are in accord with previous results for the accessory proteins but, importantly now, identify the primase as dissociating from an active replisome. The recruitment of the primase from solution during DNA synthesis has also been found for Escherichia coli but not bacteriophage T7. The implications of these results for RNA priming and extension during the repetitive synthesis of Okazaki fragments are discussed.

Adenosine Triphosphatases↗
PubMed1995

Isolation of transcriptionally active mutants of T7 RNA polymerase that do not support phage growth.

Mutants of bacteriophage T7 RNA polymerase defective in functions other than transcription were sought by random chemical mutagenesis of the cloned gene and selection for inability to support the growth of a T7 mutant whose growth is dependent on T7 RNA polymerase supplied by the host cell. About half of the mutant clones appeared unable to make full-length T7 RNA polymerase, many of them producing a truncated protein. Among 116 mutants expressing full-length protein, two-thirds were severely impaired in transcription, but a surprisingly high one-third were able to direct significant transcription in vivo. Both types of mutation were distributed across much of the gene, as determined by a rapid genetic mapping procedure that allows the lethal mutation in each clone to be localized. One mutation (isolated twice) allowed normal gene expression but prevented the formation of mature ends of T7 DNA from concatemers, which normally happens during packaging into phage particles. Thirty-seven of the mutations appeared to increase the sensitivity of the polymerase to inhibition by T7 lysozyme; all were suppressed by mutations in the lysozyme gene, including one suppressor constructed to retain full amidase activity but to be unable to bind T7 RNA polymerase. The two lysozyme-hypersensitive polymerase mutants analyzed in detail showed premature cessation of transcription during infection. Early proteins and those late proteins specified by genes as far right in T7 DNA as genes 8-9 appeared to be produced normally, but expression of genes farther to the right was strongly depressed. DNA replication was depressed about 50% in one of these mutants and 90% in the other, even though the T7 replication proteins were made in normal amounts at the normal time.

Amino Acid Sequence↗
PubMed1979

Replication of the colicin E1 plasmid in extracts of Escherichia coli: uncoupling of leading strand from lagging strand synthesis.

The replication of the ColEl plasmid was studied in extracts from E. coli dnaG mutants. It was found that the synthesis of the complementary strands of ColEl DNA can be carried out in these extracts in two consecutive steps: (1) synthesis of the leading L strand independent of the dnaG function, and (2) synthesis of the lagging H strand depending upon addition of wild-type dnaG protein. In contrast to L strand synthesis, the latter reaction is insensitive to rifampicin and novobiocin. Both synthetic pathways are however blocked by antiserum directed against dnaB protein. This indicates an additional role of the dnaB protein in duplex DNA replication besides assisting the dnaG protein in the priming of lagging strand synthesis. The T7 gene-4 protein acting in conjunction with T7 DNA polymerase can substitute for both the function of the dnaB and dnaG protein. It is concluded that plasmid replication proceeds by a semi-discontinuous mechanism.

Bacteriocin Plasmids↗
PubMed1995

Differences in the mechanism of stimulation of T7 DNA polymerase by two binding modes of Escherichia coli single-stranded DNA-binding protein.

Escherichia coli single-stranded DNA-binding protein (Eco SSB) has been shown previously to display several DNA binding modes depending on the ionic conditions. To determine what effect these various binding modes have on DNA replication, we have studied DNA synthesis by the T7 DNA polymerase under ionic conditions where Eco SSB interacts with either 72 or 91 nucleotides of M13 DNA. These forms presumably correspond to the previously described (SSB)56 and (SSB)65 (Lohman and Ferrari, 1994) that were determined using the binding of SSB to homopolymers. Here we report the stimulation induced by (SSB)91 to be 4-fold greater than that produced by (SSB)72 under conditions where the template is in large excess. Surprisingly, when the polymerase level is raised so that it is in molecular excess, (SSB)91 no longer stimulates synthesis while (SSB)72 affords a 4-fold stimulation, which is the same level of stimulation as when the template was in excess. Both SSB forms increase the rate of DNA synthesis and were found to stimulate synthesis by relieving template secondary structures. However, (SSB)72 specifically increases strand displacement synthesis, while (SSB)91 stimulates synthesis by increasing the affinity of the polymerase for the template.

Base Sequence↗
PubMed2025

An orthogonal T7 replisome for continuous hypermutation and accelerated evolution in E. coli.

Systems that perform continuous hypermutation of designated genes without compromising the integrity of the host genome can substantially accelerate the evolution of new or enhanced protein functions. We describe an orthogonal DNA replication system in Escherichia coli based on the controlled expression of the replisome of bacteriophage T7 (T7-ORACLE). The system replicates circular plasmids that enable high transformation efficiencies and seamless integration into standard molecular biology workflows. Engineering of T7 DNA polymerase yielded variant proteins with mutation rates of 1.7 × 10-5 substitutions per base in vivo-100,000-fold above the genomic mutation rate. We demonstrated continuous evolution using the T7 replisome by expanding the substrate scope of TEM-1 β-lactamase and increasing activity 5000-fold against clinically relevant monobactam and cephalosporin antibiotics in less than 1 week.

Bacteriophage T7↗
PubMed1992

Transcription dependence of DNA packaging of bacteriophages T3 and T7.

T3 and T7 phages package homologous DNA more efficiently than heterologous DNA and recombinant plasmids carrying DNA sequences necessary for DNA packaging (pac sequence). The pac sequence contains a promoter for phage RNA polymerase and transcription from the promoter is necessary for DNA packaging. T3 and T7 RNA polymerases are stringently specific for their own promoters. To examine the relationship between DNA packaging and transcription, we constructed a cleared in vitro system for packaging T3 or T7 DNA containing an ammonium sulfate fractionate of a high-speed supernatant of phage-infected cells. In the system, DNA packaging required GTP and was inhibited by the 3'-deoxy analog of GTP, ATP, or CTP. The DNA packaging activity paralleled the transcriptional activity, assayed by incorporation of [32P]UTP into acid-insoluble material. In the system, homologous DNA was packaged more efficiently than heterologous DNA, but heterologous DNA was packaged as efficiently as homologous DNA by the addition of heterologous phage RNA polymerase, demonstrating that the transcriptional specificity determines the DNA packaging specificity of T3 and T7.

Bacteriophage T7↗
PubMed1997

Role of the bacteriophage T7 and T4 single-stranded DNA-binding proteins in the formation of joint molecules and DNA helicase-catalyzed polar branch migration.

Bacteriophage T7 gene 2.5 single-stranded DNA-binding protein and gene 4 DNA helicase together promote pairing of two homologous DNA molecules and subsequent polar branch migration (Kong, D., and Richardson, C. C. (1996) EMBO J. 15, 2010-2019). In this report, we show that gene 2.5 protein is not required for the initiation or propagation of strand transfer once a joint molecule has been formed between the two DNA partners, a reaction that is mediated by the gene 2.5 protein alone. A mutant gene 2.5 protein, gene 2.5-Delta21C protein, lacking 21 amino acid residues at its C terminus, cannot physically interact with gene 4 protein. Although it does bind to single-stranded DNA and promote the formation of joint molecule via homologous base pairing, subsequent strand transfer by gene 4 helicase is inhibited by the presence of the gene 2.5-Delta21C protein. Bacteriophage T4 gene 32 protein likewise inhibits T7 gene 4 protein-mediated strand transfer, whereas Escherichia coli single-stranded DNA-binding protein does not. The 63-kDa gene 4 protein of phage T7 is also a DNA primase in that it catalyzes the synthesis of oligonucleotides at specific sequences during translocation on single-stranded DNA. We find that neither the rate nor extent of strand transfer is significantly affected by concurrent primer synthesis. The bacteriophage T4 gene 41 helicase has been shown to catalyze polar branch migration after the T4 gene 59 helicase assembly protein loads the helicase onto joint molecules formed by the T4 UvsX and gene 32 proteins (Salinas, F., and Kodadek, T. (1995) Cell 82, 111-119). We find that gene 32 protein alone forms joint molecules between partially single-stranded homologous DNA partners and that subsequent branch migration requires this single-stranded DNA-binding protein in addition to the gene 41 helicase and the gene 59 helicase assembly protein. Similar to the strand transfer reaction, strand displacement DNA synthesis catalyzed by T4 DNA polymerase also requires the presence of gene 32 protein in addition to the gene 41 and 59 proteins.

Bacteriophage T4↗
PubMed2003

A covalent linkage between the gene 5 DNA polymerase of bacteriophage T7 and Escherichia coli thioredoxin, the processivity factor: fate of thioredoxin during DNA synthesis.

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA.

Bacteriophage T7↗
PubMed2006

Mechanisms of a ring shaped helicase.

Bacteriophage T7 helicase (T7 gene 4 helicase-primase) is a prototypical member of the ring-shaped family of helicases, whose structure and biochemical mechanisms have been studied in detail. T7 helicase assembles into a homohexameric ring that binds single-stranded DNA in its central channel. Using RecA-type nucleotide binding and sensing motifs, T7 helicase binds and hydrolyzes several NTPs, among which dTTP supports optimal protein assembly, DNA binding and unwinding activities. During translocation along single stranded DNA, the subunits of the ring go through dTTP hydrolysis cycles one at a time, and this probably occurs also during DNA unwinding. Interestingly, the unwinding speed of T7 helicase is an order of magnitude slower than its translocation rate along single stranded DNA. The slow unwinding rate is greatly stimulated when DNA synthesis by T7 DNA polymerase is coupled to DNA unwinding. Using the T7 helicase as an example, we highlight critical findings and discuss possible mechanisms of helicase action.

Bacteriophage T7↗