Topoisomerase Genes Research Articles

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.

Identification and DNA sequence of the shope fibroma virus DNA topoisomerase gene

The Shope fibroma virus (SFV) DNA topoisomerase gene has been identified and mapped to the BamHl D fragment near the midpoint of the genome. The DNA sequence of the SFV BamHl S fragment together with the contiguous BamHI- Clal subfragment of BamHl D which encompasses the topoisomerase gene and two flanking genes has been determined and analyzed. Both the SFV DNA topoisomerase and the two flanking genes are closely related in terms of sequence and spatial organization to the homologous sequences from the midpoint of the vaccinia virus genome, indicating that these proteins are conserved not only in their sequence but also by position within the poxvirus genome. To confirm the assignment of the SFV gene, the putative SFV DNA topoisomerase has been expressed as an active fusion protein in Escherichia coli and this system should be useful in the analysis of topoisomerase function following the introduction of targeted mutations into the topoisomerase gene. The results of this work shed further light on the evolutionary relationship of the different poxvirus genera and indicate that central unique regions of the poxvirus genomes contain many of the essential viral genes and are thus highly conserved.

Identification of Pneumocystis carinii chromosomes and mapping of five genes

Pulsed field gel electrophoresis was used to identify the chromosome-size DNA of Pneumocystis carinii, a major pathogen of immunocompromised patients. Thirteen chromosomes of rodent Pneumocystis carinii, ranging in size from 300 to 700 kilobases (kb), were identified. The minimum genome size for P. carinii, estimated on the basis of the sizes of chromosomes, is 7,000 kb. Genetic heterogeneity among different P. carinii isolates was documented by demonstration of chromosomal size variability. By hybridization studies, the genes for topoisomerase I, dihydrofolate reductase, rRNA, actin, and thymidylate synthase were mapped to single chromosomes of approximately 650, 590, 550, 460, and 350 kb, respectively. Hybridization studies further confirmed the genetic heterogeneity of P. carinii.

Bacterial topoisomerases and the control of DNA supercoiling

DNA in bacterial cells is under negative superbelical tension, a feature that facilitates many of the activities of DNA. Supercoiling is introduced enzymatically by DNA gyrase, and the accumulation of excessively high levels is prevented by the relaxing activity of DNA topoisomerase I. Among the factors likely to influence supercoiling are topoisomerase gene expression, the ratio of ATP to ADP concentration, and processes such as transcription that unwind DNA and then translocate along it.

Expression of the type I DNA topoisomerase gene in adenovirus-5 infected human cells

The amount of topoisomerase I specific mRNA increases three- to fivefold during the early phase of infection of HeLa cells with adenovirus-5. The observed increase in specific mRNA is mainly due to an increased rate of transcription of the gene. In human 293 cells, which constitutively express the viral E1A and E1B genes, we determined an elevated level of topoisomerase I mRNA, comparable to the amount of mRNA present in HeLa cells early after infection with adenovirus. In contrasts, in HeLa cells infected with adenovirus dI312, a mutant were the E1A region had been deleted, the amount of topoisomerase I mRNA remained constant, unless the cells were superinfected with wild type virus. Our experiments indicate that the topoisomerase I gene is transactivated by an early adenovirus protein product coded by the E1A region. In contrast to the increase in mRNA synthesis, the amount of topoisomerase I protein and the topoisomerase I activity remain constant up to 24 hours after infection.

Localization of the active type I DNA topoisomerase gene on human chromosome 20q11.2-13.1, and two pseudogenes on chromosomes 1q23-24 and 22q11.2-13.1

Different subfragments of a cDNA coding for DNA topoisomerase I were used as probes to determine the chromosomal localization of topoisomerase I sequences in human cells. Southern blotting of restricted DNA from a panel of rodent-human somatic cell hybrids revealed the localization of the complete gene on chromosome 20 and the presence of two truncated topoisomerase I pseudogene sequences on chromosomes 1 and 22. In situ chromosome hybridization experiments confirmed these results showing the location of the complete gene on band q11.2-13.1 of chromosome 20, and the location of the pseudogene sequences on band q23-24 of chromosome 1 and q11.2-13.1 of chromosome 22.

Nonproductive Rearrangement of DNA Topoisomerase I and II Genes: Correlation With Resistance to Topoisomerase Inhibitors

Topoisomerase inhibitors comprise an important group of agents that is used in cancer treatment. Because the development of resistance to cancer chemotherapeutic agents represents a major limitation of cancer chemotherapy, we investigated the mechanism of resistance by murine P388 leukemia to camptothecin (topoisomerase I inhibitor) or amsacrine (topoisomerase II inhibitor). The resistant cells contained reduced levels of topoisomerase activity and messenger RNA. The topoisomerase gene of these cells was rearranged (only in one allele) and hypermethylated. These topoisomerase gene alterations probably resulted in reduced transcription and, thus, enzyme production, which was correlated with resistance to the topoisomerase inhibitor.

A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase

A hyper-recombination mutation was isolated that causes an increase in recombination between short repeated δ sequences surrounding the SUP4-o gene in S. cerevisiae. The wild-type copy of this gene was cloned by complementation of one of its pleiotropic phenotypes, slow growth. DNA sequence of the clone revealed a 656 amino acid open reading frame capable of encoding a protein homologous to the bacterial type I topoisomerase. No homology was detected with previously identified eukaryotic topoisomerases. Construction of double mutants with either of the two known yeast topoisomerase genes revealed synergistic effects on growth suggesting overlapping functions. Expression of bacterial topoisomerase I in yeast can fully complement the slow growth defect of a null mutation. We have named this locus TOP3 and suggest that it defines a novel eukaryotic topoisomerase gene.

A subthreshold level of DNA topoisomerases leads to the excision of yeast rDNA as extrachromosomal rings

In a yeast DNA topoisomerase double mutant TG205 (Δ top1 top2–4), over half of the rDNA is present as extrachromosomal rings containing one 9 kb unit of the rDNA gene or tandem repeats of it. Expression of a plasmid-borne TOP1 or TOP2 gene in the strain leads to the integation of the extrachromosomal rDNA rings back into the chromosomal rDNA cluster. When the plasmid-borne topoisomerase gene is expressed from an inducible promoter of the GAL1 gene, repression of the gene by dextrose leads to reappearance of the extrachromosomal rDNA rings. The DNA topoisomerase-dependent excision/integration of rDNA is discussed in terms of the possibility of rDNA supercoiling by transcription and the effects of DNA topology on intra- and interchromosomal recombination.

Insertional mutagenesis of the vaccinia virus gene encoding a type I DNA topoisomerase: Evidence that the gene is essential for virus growth

Vaccinia virus encodes a type I DNA topoisomerase whose function in virus replication is not known. To determine whether topoisomerase is required for growth of vaccinia in cell culture, we attempted to isolate null mutations in the topoisomerase gene through insertional mutagenesis. Plasmids containing mutant topoisomerase alleles were constructed by intragenic insertion of the Escherichia coli gpt gene. Recombinant viruses containing the gpt insertion were isolated by selection for growth in the presence of mycophenolic acid. Analysis of the genome structures of drug-resistant viruses revealed that in every case ( n = 22) both the wild-type and the gpt-inserted allele were present in viral DNA. We interpret the retention of the wild-type allele as indicative of the essential nature of the topoisomerese gene for vaccinia virus growth.

Vaccinia DNA topoisomerase I promotes illegitimate recombination in Escherichia coli.

Vaccinia virus encapsidates a Mr 32,000 type IDNA topoisomerase. Although the vaccinia gene encoding the topoisomerase is essential for virus growth, the role of the enzyme in vivo remains unclear. In the present study, the physiologic consequences of vaccinia topoisomerase action have been examined in a heterologous system, Escherichia coli. The vaccinia topoisomerase gene was inducibly expressed in an int-lambda lysogen BL21(DE3) using a T7 RNA polymerase-based transcription system. Expression of active topoisomerase in this context resulted in recA-dependent lysogenic induction as well as cell lysis. Surprisingly, topoisomerase expression also effected a 200-fold increase in the titer of infectious lambda phage, apparently by promoting int-independent prophage excision. This effect was not observed during lysogenic induction with nalidixic acid. Restriction analysis of genomic DNA from plaque-purified excisants revealed (in 10 of 10 cases) gross alterations of the DNA structure around the att site relative to the structure of the parental phage DE3. It is construed therefore that vaccinia DNA topoisomerase I acts to promote illegitimate recombination in E. coli.

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.

Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA

Yeast strains with mutations in the genes for DNA topoisomerases I and II have been identified previously in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The topoisomerase II mutants (top2) are conditional-lethal temperature-sensitive (ts) mutants. They are defective in the termination of DNA replication and the segregation of daughter chromosomes, but otherwise appear to replicate and transcribe DNA normally. Topoisomerase I mutants (top1), including strains with null mutations are viable and exhibit no obvious growth defects, demonstrating that DNA topoisomerase I is not essential for viability in yeast. In contrast to the single mutants, top1 top2 ts double mutants from both Schizosaccharomyces pombe and Saccharomyces cerevisiae grow poorly at the permissive temperature and stop growth rapidly at the non-permissive temperature. Here we report that DNA and ribosomal RNA synthesis are drastically inhibited in an S. cerevisiae top1 top2 ts double mutant at the restrictive temperature, but that the rate of poly(A)+ RNA synthesis is reduced only about threefold and transfer DNA synthesis remains relatively normal. The results suggest that DNA replication and at least ribosomal RNA synthesis require an active topoisomerase, presumably to act as a swivel to relieve torsional stress, and that either topoisomerase can perform the required function (except in termination of DNA replication where topoisomerase II is required).

Molecular cloning and genetic mapping of the DNA topoisomerase II gene of Saccharomyces cerevisiae

The structural gene for DNA topoisomerase II from the yeast Saccharomyces cerevisiae has been cloned. The clones were selected from a YEp13 plasmid bank of yeast DNA by complementing a temperature-sensitive mutation ( top2-1) in the topoisomerase II gene, TOP2. Chromosomal integrants of the clone were derived by homologous recombination in strains lacking the 2μ circle plasmid. Genetic analysis of these integrants indicates that we have cloned the TOP2 gene and not an extragenic suppressor. A YEp13-TOP2 hybrid plasmid integrant was used to localize the TOP2 gene to the left arm of chromosome XIV by the 2μ circle-directed marker loss method. Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centiMorgans to the centromere proximal side of MET4. Northern blot analysis of TOP2 RNA isolated from a wild-type strain and from an rna2 mutant shows the RNA to be 4.5 kb long in both cases, thus indicating that the TOP2 gene has no large introns.

Molecular characterization of chromosomal genes affecting double-stranded RNA replication in Saccharomyces cerevisiae.

We cloned MAK11, MAK18, and MKT1 utilizing their genetic map positions. The MAK11 gene is close to CDC16 on chromosome XI. Both genes were cloned on a single 7-kb fragment, and both have now been sequenced. The MAK18 gene is located close to PET3 on chromosome VIII. A large plasmid carrying PET3 was obtained from R. Elder and R.E. Esposito and was found to also have the MAK18 gene. The MAK16 gene has been subcloned and sequenced starting with a clone provided by J. Crowley and D. Kaback. The MKT1 gene was mapped near the gene for topoisomerase II. The topoisomerase II clone was used as the starting point for chromosome-walking to isolate MKT1. A deletion-insertion mutation (disruption) of MKT1 results in an inability to maintain M2, but does not affect M1 or L-A maintenance. Clones of SKI3 and SKI8 were selected using the cold sensitivity for cell growth of ski- M1 strains. The SKI8 gene was disrupted and found to be nonessential for cell growth in the absence of M double-stranded RNA (dsRNA). The SKI3 and SKI8 genes were mapped using these clones. We have also obtained other clones suppressing the pathology caused by the high M titer in ski- strains. These clones are not the SKI genes themselves but somehow avoid the growth defect without repressing M copy number.

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.

The Escherichia coli supX locus is topA, the structural gene for DNA topoisomerase I.

Mutations in the supX locus, which result in the absence of DNA topoisomerase I enzyme activity in both Salmonella typhimurium and Escherichia coli, are all selected as suppressors of the leu-500 promoter mutation in S. typhimurium. To determine whether the supX locus is the structural gene topA for the DNA topoisomerase I enzyme or is a positive-acting regulator/activator gene for a nearby topA structural gene, nonsense mutations were selected in the E. coli supX gene carried on an F' episome in S. typhimurium cells. The cysB-topA region of the episomes with nonsense-mutant supX alleles were then cloned onto plasmid pBR322 and transformed into E. coli cells lacking a chromosomal supX gene. Three such E. coli strains, each carrying cloned DNA from episomes with different nonsense-mutant supX alleles, all lacked DNA topoisomerase I activity but expressed antigenic determinants specific to the enzyme; control cells lacked both enzyme activity and antigenic determinants. Maxicell studies of plasmid-coded proteins demonstrated the absence of the DNA topoisomerase I protein (100 kDa) in the three strains but the appearance of a new smaller peptide in each (36, 47, and 64 kDa). These new peptides must represent fragments of the enzyme resulting from translation termination at the supX nonsense codons and confirm the interpretation that the supX gene is topA, the structural gene for DNA topoisomerase I.

Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020.

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.