T4 DNA Topoisomerase Research Articles

Bacteriophage T4 Proteins Replicate Plasmids with a Preformed R Loop at the T4 ori(uvsY) Replication Origin In Vitro

Bacteriophage T4 DNA replication proteins catalyze complete unidirectional replication of plasmids containing the T4 ori(uvsY) replication origin in vitro, beginning with a preformed R loop at the position of the origin R loop previously identified in vivo. T4 DNA polymerase, clamp, clamp loader, and 32 protein are needed for initial elongation of the RNA, which serves as the leading-strand primer. Normal replication is dependent on T4 41 helicase and 61 primase and is strongly stimulated by the 59 helicase loading protein. 59 protein slows replication without the helicase. As expected, leading-strand synthesis stalls prematurely in the absence of T4 DNA topoisomerase. A DNA unwinding element (DUE) is essential for replication, but the ori(uvsY) DUE can be replaced by other DUE sequences.

Bacteriophage T4 ribonucleoside diphosphate reductase: on the defect causing decreased formation of the β 293 subunit encoded by the nrdB93 mutant gene

Bacteriophage T4 ribonucleoside diphosphate reductase is composed of two proteins, α 2 and β 2, encoded by the nrdA and nrdB genes, respectively. The expression of nrdB is the limiting factor for the assembly of the enzyme. A recently described mutation, nrdB93, may give new insight into the regulation of synthesis of the β subunit encoded by nrdB. Infection by T4 nrdB93 produced only low concentrations of the β 2 93 protein. However, a site-specific mutation of phage T4 gene 39, encoding one of the subunits of T4 DNA topoisomerase, phenotypically suppressed the defect. The present work sought to characterize the nature of this defect. The mutation in nrdB93 was a single-base transition (G → A) resulting in a Gly 253 → Asp change. In vivo and in vitro studies provided no evidence of degradation of the β 2 93 protein. Furthermore, the decrease in β 2 93 formation was not caused by a delayed onset of transcription, neither by a decreased rate of mRNA formation from the nrdB promoter, nor by a defective intron splicing of the nrdB gene or in the transcription of the terminal segments of the message. These findings are consistent with the concept that the nrdB93 lesion produces a defect at the level of translation.

Sequence of the bacteriophage SP01 gene 30

The bacteriophage SP01 gene 30, whose function is essential for DNA synthesis, has been analyzed for its primary structural features. Conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame ( URF). The aa sequence deduced for gene 30 shares partial similarity with protein P of bacteriophage λ, which participated in λ DNA replication, and also with the exonuclease, gp46, of bacteriophage T4. A lysine-rich region of the hypothetical product of the URF shares similarity with both the T4 DNA topoisomerase and the θ29 gene 3-encoded protein; the latter codes for a terminal protein which participates in the priming of DNA elongation.

A nascent peptide is required for ribosomal bypass of the coding gap in bacteriophage T4 gene 60.

Bacteriophage T4 DNA topoisomerase gene 60 contains a 50 nucleotide untranslated region within the coding sequence of its mRNA. Translational bypass of this sequence by elongating ribosomes has been postulated for the mode of synthesis of an 18 kd polypeptide specified by the split coding segments. Ribosome bypass of the untranslated region also occurs when a segment of gene 60 is fused to lacZ and expressed in E. coli. The efficiency of bypass in these gene 60-lacZ fusions approaches 100%. Here, mutations that delete, insert, or substitute nucleotides from gene 60-lacZ fusions are examined. Essential features necessary for high level gap bypass emerging from this analysis are a cis-acting nascent peptide sequence, a short duplication bordering the gap, and a stop codon contained in a stem-loop structure at the 5′ junction of the gap.

Mutational alteration of the breakage/resealing subunit of bacteriophage T4 DNA topoisomerase confers resistance to antitumor agent m-AMSA

Bacteriophage T4 provides a simple model system in which to examine the mechanism of action of antitumor agents that have been proposed to attack type II DNA topoisomerases. Prior results demonstrated that T4 type II DNA topoisomerase is the target of antitumor agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in phage-infected Escherichia coli: a point mutation in topoisomerase structural gene 39 was shown to confer both m-AMSA-resistant phage growth and m-AMSA-insensitive topoisomerase activity. We report here that a point mutation in T4 topoisomerase structural gene 52 can also independently render both phage growth and topoisomerase activity resistant to m-AMSA. The DNA relaxation and DNA cleavage activities of this newly isolated mutant topoisomerase were significantly insensitive to m-AMSA. The drug-resistance mutation in gene 52, as well as that in gene 39, alters the DNA cleavage site specificity of wild-type T4 topoisomerase. This finding is consistent with a mechanism of drug action in which both topoisomerase and DNA participate in formation of the drug-binding site.

Common Sites for Recombination and Cleavage Mediated by Bacteriophage T4 DNA Topoisomerase in vitro

We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends.

Effect of bacteriophage T4 DNA topoisomerase gene 39 on level of beta chain of ribonucleoside diphosphate reductase in a T4 nrdB mutant.

Bacteriophage T4 ribonucleoside diphosphate reductase consists of alpha 2 and beta 2 subunits encoded by genes nrdA and nrdB, respectively, and plays a central role in the T4-induced deoxyribonucleotide synthetase complex. The accompanying paper describes the decreased rate of synthesis of deoxyribonucleotides after infection by the T4 mutant, nrdB93, and the suppression of this defect by a second mutation in gene 39, coding for one of the three protein chains of T4 DNA topoisomerase. In this study we examined these effects at the protein level. On infection by nrdB93 not only was the beta 93 protein chain altered, as shown by its migration relative to the wild type protein in electrophoretic gels and by its temperature sensitivity, but the infected cells showed very low levels of the protein. However, on infection with the double mutant of nrdB93 and 39-01 (gene 39) the concentration of beta 93 chain returned to the values of beta protein found with wild type phage. A double mutant bearing nrdB93 and an amber mutation of gene 39 also suppressed the nrdB93 defect. By contrast, a temperature-sensitive mutant of gene 39, A41, did not show suppression at either 30 or 41 degrees C. Amber mutations in the two other genes coding for T4 DNA topoisomerase, 52 and 60, did not suppress the defect. We propose that the deficiency in the quantity of beta 93 chain and the suppression of this defect occur at the transcriptional or translational expression of the nrdB93 gene and that a specific domain of the gene 39 protein, not acting in the capacity of T4 DNA topoisomerase, inhibits the expression.

Defect in synthesis of deoxyribonucleotides by a bacteriophage T4 nrdB mutant is suppressed on mutation of T4 DNA topoisomerase gene.

Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication. The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB. Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60. We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01. After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures. The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01. However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded. Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression. The accompanying paper (Cook, K. S., Wirak, D. O., Seasholtz, A. F., and Greenberg, G. R. (1988) J. Biol. Chem. 263, 6202-6208) examines the nature of the suppression process at the molecular level.

Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.

Mutagenicity of topoisomerase‐active agents in bacteriophage T4

Recently, the antitumor agent 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) was shown to revert a frameshift mutant of T4 (rFC11), and its mutagenicity was shown to be mediated by T4 DNA topoisomerase II [Ripley et al.: J Mol Biol 200: 665-680, 1988]. Here we report dose-response data on the mutagenicity and toxicity of m-AMSA in T4 rFC11. We find that m-AMSA is among the most potent frameshift mutagens observed in T4, inducing a 10-fold increase in mutant frequency in the absence of toxicity and a 500-fold increase in mutant frequency at 31% survival. In addition to m-AMSA, the topoisomerase-active agents ellipticine, oxolinic acid, and nalidixic acid also reverted rFC11; however, they required concentrations 10-100 times greater than those required by m-AMSA in order to be mutagenic, and they did not produce mutant frequencies as high as those produced by m-AMSA. Unlike m-AMSA, all three agents were mutagenic only at toxic doses. The other agents evaluated--actinomycin D, adriamycin, 9-aminoellipticine, 9-methoxyellipticine, teniposide (VM-26), and novobiocin--were toxic but not mutagenic to T4 rFC11. Thus, m-AMSA appears to be distinctly different from the other topoisomerase-active agents in exhibiting such potent mutagenic activity in T4 rFC11. Because E. coli DNA gyrase may substitute for T4 topoisomerase II, we examined the ability of two inhibitors of E. coli DNA gyrase, novobiocin and nalidixic acid, to inhibit m-AMSA's mutagenicity. Both agents substantially reduced the mutagenicity of m-AMSA in T4 rFC11, further suggesting that topoisomerase mediates the mutagenicity of m-AMSA.

Topoisomerization of plasmid DNA in Escherichia coli infected with bacteriophage T4

The degradation of host DNA, and the block to transcription of cytosine-containing DNA, which are a part of the normal course of infection by bacteriophage T4, can be eliminated in an appropriate T4 genetic background (designated as our reference type, or r.t.), so that T4 late promoters carried on plasmid DNA can function. The changes of topoisomer distribution that ensue when phage T4 r.t. infect Escherichia coli carrying a plasmid containing a T4 late promoter were analyzed. The linking number of the covalently closed circular plasmid DNA increased (implying relaxation) at the same time as the distribution of topoisomers became much broader. The relaxation of plasmid DNA was primarily, but not exclusively, due to T4 DNA topoisomerase II. The bacterial DNA topoisomerase II (gyrase) continued to function after phage infection to maintain some degree of superhelicity in plasmid DNA. When the DNA gyrase was inhibited by coumermycin or oxolinic acid, the topoisomer distribution became distinctly bimodal, part of the DNA remaining highly negatively supercoiled. It is argued that the observed post-infection topological changes involve relaxation of torsional stress and changes of binding by proteins that topologically constrain the plasmid DNA.

Illegitimate recombination mediated by T4 DNA topoisomerase in vitro

Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage lambda DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage lambda and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross.

Nucleotide sequence of a type II DNA topoisomerase gene. Bacteriophage T4 gene 39.

T4 DNA topoisomerase is a type II enzyme and is thought to be required for normal T4 DNA replication T4 gene 39 codes for the largest of the three subunits of T4 DNA topoisomerase. I have determined the nucleotide sequence of a region of 2568 nucleotides of T4 DNA which includes gene 39. The location of the gene was established by the identification of the first fifteen amino acids in the large open reading frame in the DNA sequence as those found at the amino-terminus of the purified 39-protein. The coding region of gene 39 has 1560 bases, and it is followed by two in-frame stop codons. The gene is preceded by a typical Shine-Dalgarno sequence as well as possible promoter sequences for E. coli RNA polymerase. T4 39-protein consists of 520 amino acids, and it has a calculated molecular weight of 58,478. By comparing the amino acid sequences, T4 39-protein is found to share homology with the gyrB subunit of DNA gyrase. This suggests that these topoisomerase subunits may be equivalent functionally. Some of the characteristics of the 39-protein and its structural features predicted from the DNA sequence data are discussed.

Relationship between bacteriophage T4 and T6 DNA topoisomerases. T6 39-protein subunit is equivalent to the combined T4 39- and 60-protein subunits.

T6 DNA topoisomerase has been purified from bacteriophage T6 infected Escherichia coli. Unlike the T4 DNA topoisomerase which has three subunits, it consists of two subunits of molecular weights 75,000 and 51,000. They are the products of T6 genes 39 and 52, respectively. The purified T6 enzyme can stimulate in vitro T6 DNA replication. It has an ATP-dependent DNA relaxation activity similar to the T4 enzyme. Either ATP or dATP can be used in both reactions. Using a "Western blotting" and radioimmuno-detection methods, we show that T6 39 subunit contains protein sequences specified by both the T4 39 and 60 genes. The 52-proteins of both phages appear to be identical. The T4 and T6 topoisomerase genes represent a naturally occurring example of gene separation or fusion.

Identification of the breakage-reunion subunit of T4 DNA topoisomerase.

The antitumor drug 4'-(9-acridinylamino)methanesulfon-m-anisidide which stimulates the cleavable complex formation between mammalian DNA topoisomerase II and DNA also stimulates the cleavable complex formation between bacteriophage T4-induced DNA topoisomerase and DNA. In the presence of 4'-(9-acridinylamino)methanesulfon-m-anisidide, T4 DNA topoisomerase and DNA form a "cleavable complex" which is characterized by its sensitivity to protein-denaturant treatment. Upon protein-denaturant treatment, the phosphodiester bond of DNA is cleaved, and the gene 52 protein subunit of the topoisomerase becomes covalently linked to the 5'-end of the broken DNA. The covalent protein-DNA linkage has been determined by both paper electrophoresis and thin layer chromatography to be tyrosyl phosphate.

Identification of bacteriophage T4 gene 60 product and a role for this protein in DNA topoisomerase.

Bacteriophage T4 DNA topoisomerase has been isolated and shown to contain the proteins coded by the DNA-delay genes 39 and 52 (Liu, L. F., Liu, C.-C., and Alberts, B. M. (1979) Nature (Lond.) 281, 456-461 and Stetler, G. L., King, G. J., and Huang, W. M. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3737-3741). From complementation measurements in vitro and from earlier genetic evidence, these workers suggested that the product of gene 60 (p60) was also a component of the DNA topoisomerase complex. This paper now establishes the identity of p60 and unequivocally shows that this protein is a component of the enzyme complex. T4 DNA topoisomerase was purified by a simplified two-column procedure and found to be a stable complex of p39, p52, and a protein with a relative molecular weight of 18,000. The 18,000-dalton chain has been unambiguously shown to be the product of gene 60 through the use of an amber mutant of gene 60 with Sup+ and Sup- hosts and analyses by two-dimensional gel electrophoresis. While p39 and p52 were tightly associated in the wild type enzyme complex, they were readily separated on a hydroxylapatite column from extracts of cells infected by an amber mutant of gene 60. These findings suggest that p60 plays a structural/functional role in the enzyme complex by holding the larger p39 and p52 in juxtaposition.

Characteristics of a bacteriophage T4-induced complex synthesizing deoxyribonucleotides.

A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.

A homogeneous type II DNA topoisomerase from HeLa cell nuclei.

Using kinetoplast DNA networks as a substrate in a decatenation assay, we have purified to apparent homogeneity a type II DNA topoisomerase from HeLa cell nuclei. The most pure preparations contain a single polypeptide of 172,000 daltons as determined by sodium dodecyl sulfate-gel electrophoresis. The molecular weight of the native protein, based on sedimentation and gel filtration analyses, is estimated to be 309,000. These results suggest that the enzyme is a dimer of 172,0090-dalton subunits. The enzyme is a type II topoisomerase as demonstrated by its ability to change the linking number of DNA circles in steps of two and to decatenate or unknot covalently closed DNA circles. No gyrase activity is detectable. ATP is required for the relaxation, decatenation, and unknotting of DNA, and a DNA-dependent ATPase activity is present in the most pure fractions. ATP is hydrolyzed to ADP in this properties to T4 DNA topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1979) Nature 281, 456-461).

Type II DNA topoisomerases: Enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.

T4 DNA topoisomerase: a new ATP-dependent enzyme essential for initiation of T4 bacteriophage DNA replication

A novel ATP-dependent DNA topoisomerase which makes reversible double-strand breaks in the DNA double helix has been purified to near homogeneity from T4 bacteriophage-infected Escherichia coli cells. Genetic data suggest that this activity is essential for initiating T4 DNA replication forks in vivo.