Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “T7 DNA replication”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources
PubMed1985

Alterations of the bacteriophage T7 and T3 DNA packaging pathway in Escherichia coli mutant TSN B.

Data previously obtained indicate that, during assembly of the related bacteriophages T7 and T3, a DNA-free procapsid (capsid I) is produced and that subsequently capsid I: (1) binds to a longer than mature (concatemeric) DNA and then becomes structurally altered to a particle isolated as a capsid (capsid II) physically resembling the mature bacteriophage capsid more than the procapsid (initiation phase of packaging), (2) draws DNA to its interior (entry phase of packaging), (3) participates in cutting the concatemeric DNA to mature size. It was found that, after infection of Escherichia coli mutant tsnB (selected for a deficiency in plating T7; M. Chamberlin [1974], J. Virol. 14, 509-516), T7 and T3 capsid I is assembled at a rate not significantly different from its rate of assembly in the wild-type host. However, the conversion of capsid I to capsid II was slowed in E. coli tsnB, suggesting that the tsnB mutation interferes with the initiation of DNA packaging. Although some T3 and T7 DNA enters capsids and is cut to mature size in the tsnB mutant, the data further suggest that the entry rate of DNA into capsid II is lower in the tsnB mutant than it is in an unaltered host. T7 capsid II-concatemeric DNA complexes accumulate during infection of the tsnB mutant. These observations suggest that use of the tsnB mutant as a host will simplify studies of bacteriophage T7 and T3 DNA packaging.

Capsid↗
PubMed1994

Effect of abasic sites on bacteriophage T7 protein synthesis.

We have examined protein synthesis directed by bacteriophage T7 which had been alkylated with methyl methanesulfonate so as to produce apurinic sites in its DNA in vivo. Both repair-proficient and repair-deficient (xth nfo mutant) strains of Escherichia coli served as host cells. In repair-proficient cells, all three classes of phage proteins were synthesized, although with significant delays. In mutant cells, only class I proteins were produced and their synthesis was delayed and reduced, demonstrating a perturbation of protein synthesis and providing the first in vivo indication that transcription is inhibited by abasic sites. However, the proposed effects of abasic sites on transcription appear to be weaker than those on replication.

Alkylation↗
PubMed2003

Computer simulation studies of the fidelity of DNA polymerases.

Computer simulations can provide in principle quantitative correlation between the structures of DNA polymerases and the replication fidelity. This paper describes our progress in this direction. Using several theoretical approaches, including the free energy perturbation (FEP), linear response approximation (LRA), and the empirical valence bond (EVB) methods, we examined the stability of several mismatched base pairs in DNA duplex in aqueous solution, the contribution of binding energy to the fidelity of DNA polymerases beta and T7, and the mechanism and energetics of the polymerization reaction catalyzed by T7 DNA polymerase.

Computer Simulation↗
PubMed1998

Comparison of synonymous codon distribution patterns of bacteriophage and host genomes.

Synonymous codon usage patterns of bacteriophage and host genomes were compared. Two indexes, G + C base composition of a gene (fgc) and fraction of translationally optimal codons of the gene (fop), were used in the comparison. Synonymous codon usage data of all the coding sequences on a genome are represented as a cloud of points in the plane of fop vs. fgc. The Escherichia coli coding sequences appear to exhibit two phases, "rising" and "flat" phases. Genes that are essential for survival and are thought to be native are located in the flat phase, while foreign-type genes from prophages and transposons are found in the rising phase with a slope of nearly unity in the fgc vs. fop plot. Synonymous codon distribution patterns of genes from temperate phages P4, P2, N15 and lambda are similar to the pattern of E. coli rising phase genes. In contrast, genes from the virulent phage T7 or T4, for which a phage-encoded DNA polymerase is identified, fall in a linear curve with a slope of nearly zero in the fop vs. fgc plane. These results may suggest that the G + C contents for T7, T4 and E. coli flat phase genes are subject to the directional mutation pressure and are determined by the DNA polymerase used in the replication. There is significant variation in the fop values of the phage genes, suggesting an adjustment to gene expression level. Similar analyses of codon distribution patterns were carried out for Haemophilus influenzae, Bacillus subtilis, Mycobacterium tuberculosis and their phages with complete genomic sequences available.

Bacillus subtilis↗
PubMed2003

Type III intermediate filament proteins interact with four-way junction DNA and facilitate its cleavage by the junction-resolving enzyme T7 endonuclease I.

The isolation from proliferating mouse and human embryo fibroblasts of SDS-stable crosslinkage products of vimentin with DNA fragments containing inverted repeats capable of cruciform formation under superhelical stress and the competitive effect of a synthetic Holliday junction on the binding of cytoplasmic intermediate filament (cIF) proteins to supercoiled DNA prompted a detailed investigation of the proteins' capacity to associate with four-way junction DNA and to influence its processing by junction-resolving endonucleases. Electrophoretic mobility shift analysis of reaction products obtained from vimentin and Holliday junctions under varying ionic conditions revealed efficient complex formation of the filament protein not only with the unstacked, square-planar configuration of the junctions but also with their coaxially stacked X-conformation. Glial fibrillary acidic protein (GFAP) was less efficient and desmin virtually inactive in complex formation. Electron microscopy showed binding of vimentin tetramers or octamers almost exclusively to the branchpoint of the Holliday junctions under physiological ionic conditions. Even at several hundredfold molar excess, sequence-related single- and double-stranded DNAs were unable to chase Holliday junctions from their complexes with vimentin. Vimentin also stimulated bacteriophage T7 endonuclease I in introducing single-strand cuts diametrically across the branchpoint and thus in the resolution of the Holliday junctions. This effect is very likely due to vimentin-induced structural distortion of the branchpoint, as suggested by the results of hydroxyl radical footprinting of Holliday junctions in the absence and the presence of vimentin. Moreover, vimentin, and to a lesser extent GFAP and desmin, interacted with the cruciform structures of inverted repeats inserted into a supercoiled vector plasmid, thereby changing their configuration via branch migration and sensibilizing them to processing by T7 endonuclease I. This refers to both plasmid relaxation caused by unilateral scission and, particularly, linearization via bilateral scission at primary and cIF protein-induced secondary cruciform branchpoints that were identified by T7 endonuclease I footprinting. cIF proteins share these activities with a variety of other architectural proteins interacting with and structurally modulating four-way DNA junctions. In view of the known and hypothetical functions of four-way DNA junctions and associated protein factors in DNA metabolism, cIF proteins as complementary nuclear matrix proteins may play important roles in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription, with special emphasis on both the preservation and evolution of the genome.

Bacteriophage T7↗
PubMed2002

Effect of the O6 substituent on misincorporation kinetics catalyzed by DNA polymerases at O(6)-methylguanine and O(6)-benzylguanine.

Misincorporation at a DNA-carcinogen adduct may contribute to formation of mutations if a polymerase proceeds past the lesion, compromising fidelity, as in the G:C to A:T mutations caused by O(6)-alkylguanine. Replication of primer/templates containing guanine (G), O(6)-methylguanine (O(6)-MeG), or O(6)-benzylguanine (O(6)-BzG) was assessed using T7 DNA polymerase exo(-) (T7(-)) and HIV-1 reverse transcriptase (RT). The steady-state parameters indicated that T7(-) and RT preferentially incorporated dTTP opposite O(6)-MeG and O(6)-BzG. The incorporation efficiencies (k(cat)/K(m)) were less for O(6)-BzG than O(6)-MeG for both dCTP and dTTP insertion. Pre-steady-state analysis indicated that the product formed during the burst phase, i.e., the burst amplitude, differed significantly between the unmodified 24-mer/36-G-mer and the O(6)-alkylG-containing substrates. Extension of the O(6)-BzG-containing duplexes was much more difficult for both polymerases as compared to O(6)-MeG, except when RT easily extended the O(6)-BzG:T base pair. The for binding of dCTP or dTTP to a RT*DNA complex containing O(6)-MeG was 8-fold greater than for dNTP binding to a complex containing unmodified DNA. The for a RT*DNA complex containing O(6)-BzG was 50-fold greater. In conclusion, the bulkier O(6)-BzG is a greater block to polymerization by T7(-) and RT than is O(6)-MeG, but some polymerization does occur with an O(6)-BzG substrate. Pre-steady-state analysis indicates that neither dCTP nor dTTP insertion is strongly preferred during polymerization of O(6)-BzG-containing DNA, unlike the case of O(6)-MeG. These results and others regarding polymerase stalling opposite O(6)-MeG and O(6)-BzG are discussed in the following paper in this issue [Woodside, A. M., and Guengerich, F. P. (2002) Biochemistry 41, 1039-1050].

Antineoplastic Agents↗
PubMed1996

Exonucleolytic proofreading during replication of repetitive DNA.

We are attempting to understand the processes required to accurately replicate the repetitive DNA sequences whose instability is associated with several human diseases. Here we test the hypothesis that the contribution of exonucleolytic proofreading to frameshift fidelity during replication of repetitive DNA sequences diminishes as the number of repeats in the sequence increases. The error rates of proofreading-proficient T7, T4, and Pyrococcus furiosis DNA polymerases are compared to their exonuclease-deficient derivatives, for +1 and -1 base errors in homopolymeric repeat sequences of three to eight base pairs. All three exonuclease-deficient polymerases produce frameshift errors during synthesis at rates that increase as a function of run length, suggesting the involvement of misaligned intermediates. Their wild-type counterparts are all much more accurate, suggesting that the majority of the intermediates are corrected by proofreading. However, the contribution of the exonuclease to fidelity decreases substantially as the length of the homopolymeric run increases. For example, the exonuclease enhances the frameshift fidelity of T7 DNA polymerase in a run of three A.T base pairs by 160-fold, similar to its contribution to base substitution fidelity. However, in a run of eight consecutive A.T base pairs, the exonuclease only enhances frameshift fidelity by 7-fold. A similar pattern was observed with T4 and Pfu DNA polymerases. Thus, both polymerase selectivity and exonucleolytic proofreading efficiency are diminished during replication of repetitive sequences. This may place an increased relative burden on post-replication repair processes to reduce rates of addition and deletion mutations in organisms whose genome contains abundant simple repeat DNA sequences.

Base Sequence↗
PubMed1994

A functional chimeric DNA primase: the Cys4 zinc-binding domain of bacteriophage T3 primase fused to the helicase of bacteriophage T7.

Two colinear bacteriophage T7 gene 4 proteins provide helicase and primase functions in vivo. T7 primase differs from T7 helicase by an additional 63 residues at the amino terminus. This terminal domain contains a zinc-binding motif which mediates an interaction with the basic primase recognition sequence 3'-CTG-5'. We have generated a chimeric primase in which the 81 amino-terminal residues are derived from the primase of phage T3 and the 484 carboxyl-terminal residues are those of phage T7 helicase. The amino-terminal domain of T3 primase is 50% homologous with that of T7 primase. The resulting T3/T7 chimeric protein is a functional primase in vivo. While the primase activity of the purified protein is about one-third that of T7 primase, the recognition sites used and the oligoribonucleotides synthesized from these sites are identical. We conclude that the residues responsible for the interaction with the sequence 3'-CTG-5' are conserved between the chimeric and T7 proteins.

Amino Acid Sequence↗
PubMed1997

Template recognition and ribonucleotide specificity of the DNA primase of bacteriophage T7.

The 63-kDa gene 4 DNA primase of phage T7 catalyzes the synthesis of oligoribonucleotides on single-stranded DNA templates. At the sequence, 5'-GTC-3', the primase synthesizes the dinucleotide pppAC; the cytidine residue of the recognition sequence is cryptic. Only tetraribonucleotides function as primers, but the specificity for the third and fourth position is not as stringent with a preference of CMP > AMP >> UMP > GMP. The predominant recognition sites on M13 DNA are 5'-(G/T)GGTC-3' and 5'-GTGTC-3'. Synthesis is usually limited to tetranucleotides, but T7 primase can synthesize longer oligoribonucleotides on templates containing long stretches of guanosine residues 5' to the recognition sequence. The specificity beyond the first two positions of the primer increases as the length of the template on the 3'-side of 5'-GTC-3' increases. On an oligonucleotide having 20 3'-flanking cytidine residues GMP is incorporated at the third position; incorporation is reduced 4-fold when the flanking sequence reaches 65 residues, and little is incorporated on M13 templates. The presence of the 56-kDa gene 4 helicase decreases the incorporation of GMP on long templates. We propose that pausing is required for the incorporation of less preferred nucleotides and that pausing is decreased by the ability of the primase to translocate 5' to 3' on templates having long 3'-flanking sequences.

Bacteriophage T7↗
PubMed1975

The mechanism of template activation by exonuclease V.

In crude extracts from Escherichia coli cells the ATP-dependent exonuclease V was found to be most active in converting double-stranded DNA into a suitable template for DNA polymerase. This phenomenon was studied in some detail with isolated exonuclease V and T7 DNA polymerase. We found that, at ATP concentrations arount 1 mM, the exonuclease produces a broad spectrum of DNA fragments. One class of fragments is largely single stranded with hydrogen-bonded small primer sequences. These structures allow the synthesis of remarkably homogeneous polynucleotide strands by T7 DNA polymerase.

Adenosine Triphosphate↗
PubMed1994

Acidic carboxyl-terminal domain of gene 2.5 protein of bacteriophage T7 is essential for protein-protein interactions.

The product of gene 2.5 protein of bacteriophage T7, a single-stranded DNA-binding protein, physically interacts with phage encoded DNA polymerase and primase/helicase proteins. A truncated gene 2.5 protein (GP2.5-delta 21C) was constructed by in vitro mutagenesis and lacks the 21 carboxyl-terminal amino acids found in wild-type gene 2.5 protein, 15 of which are acidic. GP2.5-delta 21C cannot substitute for wild-type gene 2.5 protein in vivo; the phage are not viable and exhibit less than 1% of the DNA synthesis observed in wild-type phage-infected cells. GP2.5-delta 21C has been purified to apparent homogeneity from cells overexpressing its cloned gene and has a conformation that differs from that of the wild-type gene 2.5 protein as judged by its circular dichroism spectra. Purified GP2.5-delta 21C retains its ability to bind to single-stranded DNA; the association constant of the protein for single-stranded DNA, determined by nitrocellulose filter binding, is 3.2 x 10(6) M-1 and is identical to that determined for wild-type gene 2.5 protein. However, GP2.5-delta 21C is a monomer in solution, whereas the wild-type protein exists as a dimer. GP2.5-delta 21C does not physically interact with T7 DNA polymerase as measured by affinity chromatography and fluorescent emission anisotropy. The mutant protein cannot stimulate T7 DNA polymerase activity on primed single-stranded DNA templates.

Bacteriophage T7↗