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PubMed1978

Regulation of transcription of the late genes of bacteriophage T7.

The transcription program of bacteriophage T7 in vivo was analyzed by hybridizing T7 mRNAs, labeled at intervals after infection, to Hpa I restriction fragments of T7 DNA. Transcripcion of the late genes is temporally regulated: class II genes are transcribed between 4 and 16 min after infection; most class III genes are transcribed from 8 min after infection until lysis. Genes 8--10 are transcribed as both class II and class III genes. The rate of T7 RNA synthesis decreases sharply at 10 min after infection. The rapid decrease in the rate of T7 RNA synthesis and the shutoff of class II RNA synthesis were not observed in cells infected with phage defective in gene 3.5 (lysozyme). Although the decrease in the rate of T7 RNA synthesis is independent of DNA replication, the failure to shut off class II RNA synthesis normally in 3.5-- -infected cells may reflect the role of T7 lysozyme in DNA replication. In vitro, the regions of T7 DNA transcribed by the phage RNA polymerase were found to be dependent upon ionic conditions.

Coliphages↗
PubMed1999

Crystal structure of the helicase domain from the replicative helicase-primase of bacteriophage T7.

Helicases that unwind DNA at the replication fork are ring-shaped oligomeric enzymes that move along one strand of a DNA duplex and catalyze the displacement of the complementary strand in a reaction that is coupled to nucleotide hydrolysis. The helicase domain of the replicative helicase-primase protein from bacteriophage T7 crystallized as a helical filament that resembles the Escherichia coli RecA protein, an ATP-dependent DNA strand exchange factor. When viewed in projection along the helical axis of the crystals, six protomers of the T7 helicase domain resemble the hexameric rings seen in electron microscopic images of the intact T7 helicase-primase. Nucleotides bind at the interface between pairs of adjacent subunits where an arginine is near the gamma-phosphate of the nucleotide in trans. The bound nucleotide stabilizes the folded conformation of a DNA-binding motif located near the center of the ring. These and other observations suggest how conformational changes are coupled to DNA unwinding activity.

Amino Acid Sequence↗
PubMed2000

A unique loop in the DNA-binding crevice of bacteriophage T7 DNA polymerase influences primer utilization.

The three-dimensional structure of bacteriophage T7 DNA polymerase reveals the presence of a loop of 4 aa (residues 401-404) within the DNA-binding groove; this loop is not present in other members of the DNA polymerase I family. A genetically altered T7 DNA polymerase, T7 polDelta401-404, lacking these residues, has been characterized biochemically. The polymerase activity of T7 polDelta401-404 on primed M13 single-stranded DNA template is one-third of the wild-type enzyme and has a 3'-to-5' exonuclease activity indistinguishable from that of wild-type T7 DNA polymerase. T7 polDelta401-404 polymerizes nucleotides processively on a primed M13 single-stranded DNA template. T7 DNA polymerase cannot initiate de novo DNA synthesis; it requires tetraribonucleotides synthesized by the primase activity of the T7 gene 4 protein to serve as primers. T7 primase-dependent DNA synthesis on single-stranded DNA is 3- to 6-fold less with T7 polDelta401-404 compared with the wild-type enzyme. Furthermore, the altered polymerase is defective (10-fold) in its ability to use preformed tetraribonucleotides to initiate DNA synthesis in the presence of gene 4 protein. The location of the loop places it in precisely the position to interact with the tetraribonucleotide primer and, presumably, with the T7 gene 4 primase. Gene 4 protein also provides helicase activity for the replication of duplex DNA. T7 polDelta401-404 and T7 gene 4 protein catalyze strand-displacement DNA synthesis at nearly the same rate as does wild-type polymerase and T7 gene 4 protein, suggesting that the coupling of helicase and polymerase activities is unaffected.

Bacteriophage M13↗
PubMed1997

Double-strand breaks increase the incidence of genetic deletion associated with intermolecular recombination in bacteriophage T7.

An in vitro DNA replication system based on extracts prepared from Escherichia coli cells infected with bacteriophage T7 was used to study deletion associated with the repair of double-strand breaks. The gene for T7 ligase was interrupted by a DNA insert which included 17-bp direct repeats. Deletion between the repeats restored the reading frame of the gene, and these DNA molecules could be detected by their ability to give rise to ligase-positive phage after in vitro packaging. T7 genomes that had a pre-existing double-strand break located between the direct repeats were incubated together with intact genomes which had the same direct repeats. Genetic markers placed on either side of the insert in the ligase gene allowed identification of the source of DNA molecules that underwent deletion between the direct repeats. This allowed an assessment of the participation of the molecules with strand breaks in the deletion process, under conditions where any mechanism could contribute to deletion. Approximately three-quarters of the T7 molecules that had lost the region between the direct repeats contained one or both of the partial genomes originally introduced into the reactions. About 50% of the genomes which had undergone deletion had recombined markers between the partial and intact genomes. The data demonstrate that double-strand breaks substantially enhance the contribution of intermolecular recombination to deletion.

Bacteriophage T7↗
PubMed1987

Sequence of a conditionally essential region of bacteriophage T3, including the primary origin of DNA replication.

The 3526 base-pair nucleotide sequence from near the end of bacteriophage T3 gene 1 to within the coding sequence of gene 2.5 is given. It includes the complete coding sequences for nine known or presumptive proteins, most of which are only conditionally essential for phage growth. The sequence includes five promoters for the phage RNA polymerase, the terminator for early (host enzyme-catalyzed) transcription, and two recognition sites for RNAase III. The primary origin of T3 DNA replication that is utilized by the phage in vivo has been localized to a 142 base-pair region. It has several features in common with the phage T7 origin of DNA replication, and exhibits considerable homology to recognition sites for the mRNA processing enzyme RNAase III. It is proposed that the primary origin of T3 DNA replication may have evolved directly from an RNAase III recognition site. The deletions present in a number of T3 mutant strains and the location of the nucleotide changes in several T3 strains that are defective in their ability to grow on F+-containing strains or on optA mutant hosts have been determined. We discuss how T3 may have become genetically isolated from its relatives in the T7-T3 group and simultaneously acquired novel biological and biochemical properties.

Amino Acid Sequence↗
PubMed2004

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7.

Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on single-stranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp/s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.

Bacteriophage T7↗
PubMed2004

The fidelity of replication of the three-base-pair set adenine/thymine, hypoxanthine/cytosine and 6-thiopurine/5-methyl-2-pyrimidinone with T7 DNA polymerase.

With the goal of constructing a genetic alphabet consisting of a set of three base pairs, the fidelity of replication of the three base pairs T(H) (5-methyl-2-pyrimidinone)/H(S) (6-thiopurine; thiohypoxanthine), C/H (hypoxanthine) and T/A was evaluated using T7 DNA polymerase, a polymerase with a strong 3'-->5' exonuclease activity. An evaluation of the suitability of a new base pair for replication should include both the contribution of the fidelity of a polymerase activity and the contribution of proofreading by a 3'-->5' exonuclease activity. Using a steady-state kinetics method that included the contribution of the 3'-->5' exonuclease activity, the fidelity of replication was determined. The method determined the ratio of the apparent rate constant for the addition of a deoxynucleotide to the primer across from a template base by the polymerase activity and the rate constant for removal of the added deoxynucleotide from the primer by the 3'-->5' exonuclease activity. This ratio was designated the eni (efficiency of net incorporation). The eni of the base pair C/H was equal to or greater than the eni of T/A. The eni of the base pair T(H)/H(S) was 0.1 times that of A/T for T(H) in the template and 0.01 times that of A/T for H(S) in the template. The ratio of the eni of a mismatched deoxynucleotide to the eni of a matched deoxynucleotide was a measure of the error frequency. The error frequencies were as follows: thymine or T(H) opposite a template hypoxanthine, 2x10(-6); H(S) opposite a template cytosine, <3x10(-4). The remaining 24 mismatched combinations of bases gave no detectable net incorporation. Two mismatches, hypoxanthine opposite a template thymine or a template T(H), showed trace incorporation in the presence of a standard dNTP complementary to the next template base. T7 DNA polymerase extended the primer beyond each of the matched base pairs of the set. The level of fidelity of replication of the three base pairs with T7 DNA polymerase suggests that they are adequate for a three-base-pair alphabet for DNA replication.

Adenine↗
PubMed2005

Functional evidence for a small and rigid active site in a high fidelity DNA polymerase: probing T7 DNA polymerase with variably sized base pairs.

Hypotheses on the origins of high fidelity in replicative DNA polymerases have recently focused on the importance of geometric or steric effects in this selectivity. Here we reported a systematic study of the effects of base pair size in T7 DNA polymerase (pol), the replicative enzyme for bacteriophage T7. We varied base pair size in very small (0.25 A) increments by use of a series of nonpolar thymidine shape mimics having gradually increasing size. Steady-state kinetics were evaluated for the 5A7A exonuclease-deficient mutant in a 1:1 complex with thioredoxin. For T7 pol, we studied insertion of natural nucleotides opposite variably sized T analogs in the template and, conversely, for variably sized dTTP analogs opposite natural template bases. The enzyme displayed extremely high selectivity for a specific base pair size, with drops in efficiency of as much as 280-fold for increases of 0.4 A beyond an optimum size approximating the size of a natural pair. The enzyme also strongly rejected pairs that were smaller than the optimum by as little as 0.3 A. The size preferences with T7 DNA pol were generally smaller, and the steric rejection was greater than DNA pol I Klenow fragment, correlating with the higher fidelity of the former. The hypothetical effects of varied active site size and rigidity are discussed. The data lend direct support to the concept that active site tightness is a chief determinant of high fidelity of replicative polymerases and that a less rigid (looser) and larger active site can lead to lower fidelity.

Bacteriophage T7↗
PubMed1977

Bacteriophage-T7-induced DNA-priming protein. A novel enzyme involved in DNA replication.

The T7gene-4 protein has been purified to near homogeneity using a complementation assay in vitro, and it is designated T7 DNA-priming protein (DNA primase). The purified enzyme enables T7 DNA polymerase to initate DNA synthesis on various circular single-stranded DNA templates by a mechanism which involes the synthesis of a very short RNA primer. The oligoribonucleotide, which is linked to the product DNA via a 3':5'-phosphodiester bond, starts with pppA-C and terminates predominantly with AMP. When only ATP and CPT are precursors, the RNA primer is found to be primarily a tetranucleotide of the sequence pppA-C-C-A. Using oligoribonucleotides in place of ribonucleoside triphosphates as chain initators, T7 DNA-priming protein drastically increases the efficiency with which T7 DNA polymerase can utilize particular tetranucleotide primers containing A and C residues. T7 DNA-priming protein also enables T7 DNA polymerase to make use of native or nicked duplex T7 DNA as template-primer. This reaction does not require ribonucleoside triphosphates, although their addition enhances DNA synthesis 2--4 fold. The product formed in their absence is covalently attached to the template DNA and is found to contain a few long branches when examined by electron microscopy. In the presence of ribonucleoside triphosphates most of the newly made product arises from imitation of DNA chains de novo. Incubation of three proteins: T7 DNA-priming protein, T7 DNA polymerase, and T7 DNA-binding protein, with ribonucleoside and deoxyribonucleoside triphosphates, and with phiX174DNA as template leads to the generation of 'rolling circle-like' structures as visualized in the electron microscope. Single-stranded regions at the tail-circle junction indicate that initations can occur de novo on the displaced complementary strand. This is consistent with a discontinuous mode of 'lagging' strand synthesis and suggests that the same proteins may also be responsible for fork propagation in vivo.

Bacterial Proteins↗
PubMed1979

The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis.

The nucleotide sequence recognized and cleaved by the restriction endonuclease MboI is 5' GATC and is identical to the central tetranucleotide of the restriction sites of BamHI and BglII. Experiments on the restriction of DNA from Escherichia coli dam and dam+ confirm the notion that GATC sequences are adenosyl-methylated by the dam function of E. coli and thereby are made refractory to cleavage by MboI. On the basis of this observation the degree of dam methylation of various DNAs was examined by cleavage with MboI and other restriction endonucleases. In plasmid DNA essentially all of the GATC sequences are methylated by the dam function. The DNA of phage lambda is only partially methylated, extended methylation is observed in the DNA of a substitution mutant of lambda, lambda gal8bio256, and in the lambda derived plasmid, lambdadv93, which is completely methylated. In contrast, phage T7 DNA is not methylated by dam. A suppression of dam methylation of T7 DNA appears to act only in cis dam. A suppression of dam methylation of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely methylated. The results are discussed with respect to the participation of the dam methylase in different replication systems.

Base Sequence↗
PubMed1977

Thermodynamic limits on the size and size distribution of nucleic acids synthesized in vitro: the role of pyrophosphate hydrolysis.

The free-energy change of phosphodiester bond formation from nucleoside triphosphates is more favorable than with nucleoside diphosphates as substrates. Base-stacking interactions can make significant contributions to both delta G degrees ' values. Pyrophosphate hydrolysis when it accompanies the former reaction dominates all thermodynamic considerations. Three experimental situations are discussed in which high-molecular-weight polynucleotides are synthesized without a strong driving force for covalent bond formation. For one of these, a kinetic scheme is presented which encompasses an early narrow Poisson distribution of chain lengths with ultimate passage to a disperse equilibrium population of chain sizes. Hydrolytic removal of pyrophosphate expands the time scale for this undesirable process by a factor of 10(9), while it enormously elevates the thermodynamic ceiling for the average degrees of polymerization in the other two examples. The electron micrographically revealed broad size population from an early study of partial replication of a T7 DNA template is found to adhere (fortuitously) to a disperse most probable representation. Some possible origins are examined for the branched structures in this product, as well as in a later investigation of replication of this nucleic acid. The achievement of both very high molecular weights and sharply peaked size distributions in polynucleotides synthesized in vitro will require coupling to inorganic pyrophosphatase action as in vivo.

Calorimetry↗
PubMed1996

Termination within oligo(dT) tracts in template DNA by DNA polymerase gamma occurs with formation of a DNA triplex structure and is relieved by mitochondrial single-stranded DNA-binding protein.

Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.

Base Composition↗
PubMed1987

T7 lysozyme inhibits transcription by T7 RNA polymerase.

The selectivity of T7 RNA polymerase for its own promoters is used to direct all transcription and replication to bacteriophage T7 DNA during infection. We now find that T7 lysozyme, which is known to cut a bond in the peptidoglycan layer of the cell wall, forms a specific complex with T7 RNA polymerase and inhibits transcription. Mutations that weaken this interaction have been found in the coding sequence for T7 RNA polymerase; an affinity column containing wildtype polymerase selectively binds T7 lysozyme, but a similar column containing mutant polymerase does not. The lysozyme-polymerase interaction ensures a controlled burst of late transcription during infection, and could possibly have some direct role in replication and/or control of lysis.

Cloning, Molecular↗
PubMed2004

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.

Amino Acid Substitution↗
PubMed1993

Deletion between directly repeated DNA sequences measured in extracts of bacteriophage T7-infected Escherichia coli.

An in vitro system based upon extracts of bacteriophage T7 infected Escherichia coli was used to study genetic deletions and to examine the importance of DNA replication in the deletion process. When T7 genomes with gene 1.3 inactivated by a 43-bp insert of random sequence DNA bracketed by 11-bp direct repeats were replicated in vitro the inserts were deleted with a frequency of about 10(-5) deletions per genome replication. Under conditions where deletion could take place only by recombination between direct repeats on distinct DNA molecules deletion frequency was at least an order of magnitude lower. These data demonstrate the utility of the in vitro system as a means for examining deletion mechanisms and underscore the importance of DNA replication in deletions.

Bacteriophage T7↗
PubMed2001

Replisome-mediated DNA replication.

The elaborate process of genomic replication requires a large collection of proteins properly assembled at a DNA replication fork. Several decades of research on the bacterium Escherichia coli and its bacteriophages T4 and T7 have defined the roles of many proteins central to DNA replication. These three different prokaryotic replication systems use the same fundamental components for synthesis at a moving DNA replication fork even though the number and nature of some individual proteins are different and many lack extensive sequence homology. The components of the replication complex can be grouped into functional categories as follows: DNA polymerase, helix destabilizing protein, polymerase accessory factors, and primosome (DNA helicase and DNA primase activities). The replication of DNA derives from a multistep enzymatic pathway that features the assembly of accessory factors and polymerases into a functional holoenzyme; the separation of the double-stranded template DNA by helicase activity and its coupling to the primase synthesis of RNA primers to initiate Okazaki fragment synthesis; and the continuous and discontinuous synthesis of the leading and lagging daughter strands by the polymerases. This review summarizes and compares and contrasts for these three systems the types, timing, and mechanism of reactions and of protein-protein interactions required to initiate, control, and coordinate the synthesis of the leading and lagging strands at a DNA replication fork and comments on their generality.

Bacterial Proteins↗