The topoisomerase I gene from Ustilago maydis: sequence, disruption and mutant phenotype.

The Ustilago maydis genomic TOP1 gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction, and using this fragment to search a genomic DNA library by hybridization. The predicted peptide sequence exhibited 30-40% identity to other eukaryotic TOP1 genes, yet differed in several features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second, an atypical carboxy-terminal 'tail', absent in other TOP1 genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 gene, with a top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident in wild-type U.maydis, and displayed a subtle coloration phenotype evident during cell senescence.     

Genome rearrangement in top3 mutants of Saccharomyces cerevisiae requires a functional RAD1 excision repair gene.

Saccharomyces cerevisiae cells that are mutated at TOP3, a gene that encodes a protein homologous to bacterial type I topoisomerases, have a variety of defects, including reduced growth rate, altered gene expression, blocked sporulation, and elevated rates of mitotic recombination at several loci. The rate of ectopic recombination between two unlinked, homologous loci, SAM1 and SAM2, is sixfold higher in cells containing a top3 null mutation than in wild-type cells. Mutations in either of the two other known topoisomerase genes in S. cerevisiae, TOP1 and TOP2, do not affect the rate of recombination between the SAM genes. The top3 mutation also changes the distribution of recombination events between the SAM genes, leading to the appearance of novel deletion-insertion events in which conversion tracts extend beyond the coding sequence, replacing the DNA flanking the 3' end of one SAM gene with nonhomologous DNA flanking the 3' end of the other. The effects of the top3 null mutation on recombination are dependent on the presence of an intact RAD1 excision repair gene, because both the rate of SAM ectopic gene conversion and the conversion tract length were reduced in rad1 top3 mutant cells compared with top3 mutants. These results suggest that a RAD1-dependent function is involved in the processing of damaged DNA that results from the loss of Top3 activity, targeting such DNA for repair by recombination.     

Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae

We have examined the roles of eukaryotic DNA topoisomerases I and II in DNA replication by the use of a set of four isogenic strains of Saccharomyces cerevisiae that are TOP1+ TOP2+, TOP1+ top2 ts, delta top1 TOP2+, and delta top1 top2 ts. Cells synchronized by treatment with the alpha-mating factor, or by cycles of feeding and starvation, were released from cell-cycle arrest, and the size distribution of DNA chains that were synthesized after the cells reentered the S-phase was determined as a function of time. The results indicate that synthesis of short DNA chains several thousand nucleotides in length can initiate in the absence of both topoisomerases, but their further elongation requires at least one of the two topoisomerases. Inactivation of DNA topoisomerase II does not alter significantly the time dependence of the patterns of nascent DNA chain synthesis, which is consistent with the notion that the requirement of this enzyme for viability is due to its essential role during mitosis, when pairs of intertwined newly replicated chromosomes are being segregated. The absence of DNA topoisomerase I leads to a temporary delay in the extension of the short DNA chains; this delay in chain elongation is also reflected in the rate of total DNA synthesis in the delta top1 mutant during the early S-phase. Thus, in wild-type cells, DNA topoisomerase I is probably the major replication swivel. The patterns of DNA synthesis in asynchronously grown delta top1 top2 ts cells at permissive and non-permissive temperatures are also consistent with the above conclusions.     

Functional and physical interaction between Sgs1 and Top3 and Sgs1-independent function of Top3 in DNA recombination repair

A mutant allele of SGS1 of Saccharomyces cerevisiae was identified as a suppressor of the slow-growth phenotype of top3 mutants. We previously reported the involvement of Top3 via the interaction with the N-terminal region of Sgs1 in the complementation of methylmethanesulfonate (MMS) sensitivity and the suppression of hyper recombination of a sgs1 mutant. In this study, we found that several amino acids residues in the N-terminal region of Sgs1 between residues 4 and 33 were responsible for binding to Top3 and essential for complementing the sensitivity to MMS of sgsl cells. Two-hybrid assays suggested that the region of Top3 responsible for the binding to Sgs1 was bipartite, with portion in the N- and C-terminal domains. Although disruption of the SGS1 gene suppressed the semi-lethality of the top3 mutant of strain MR, the sgsl-top3 double mutant grew more slowly and was more sensitive to MMS than the sgsl single mutant, indicating that Top3 plays some role independently of Sgs1. The DNA topoisomerase activity of Top3 was required for the Top3 function to repair DNA damages induced by MMS, as shown by the fact that the TOP3 gene carrying a mutation (Phe for Tyr) at the amino acid residue essential for its activity (residue 356) failed to restore the MMS sensitivity of sgs1-top3 to the level of that of the sgs1 single mutant. Epistatic analysis using the sgs1-top3 double mutant, rad52 mutant and sgs1-top3-rad52 triple mutant indicated that TOP3 belongs to the RAD52 recombinational repair pathway.     

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 2 mu 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 2 mu circle-directed marker loss method. Results from standard meiotic mapping experiments indicate that TOP2 is about 16 centi-Morgans 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.     

Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC

Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.     

Topoisomerase II Suppresses the Temperature Sensitivity of Saccharomyces cerevisiae pds5 Mutants,but not the Defect in Sister Chromatid Cohesion

Sister chromatid cohesion enables chromosomes to achieve bipolar attachment to the mitotic spindle and its dissolution is required for chromosome segregation. The cohesin complex serves as the primary molecular glue responsible for cohesion. Pds5p binds to the same chromosomal loci as the cohesin complex but plays a distinct role as a regulator of cohesion maintenance. Catenation between sister chromatids must also be removed by Topoisomerase II (Top2p) enzymatic activity to enable chromosome segregation. We identified TOP2 as a high-copy suppressor of the temperature sensitivity of pds5 mutants. TOP2 suppression is specific for pds5 mutants as it does not suppress mutants in the cohesin complex. TOP2 suppresses mini-chromosome loss in pds5 mutants indicating that it rescues a chromosome segregation defect. Surprisingly, TOP2 over-expression fails to suppress the cohesion defect of pds5 mutants, suggesting that it suppresses an additional and as yet uncharacterized defect in pds5 mutants that is essential for viability. A catalytically dead TOP2 allele suppresses pds5 temperature sensitivity, suggesting that suppression is unrelated to Top2p enzymatic function. Consistent with this idea, when the pds5 mutant is combined with the top2-4 mutant, which accumulates DNA catenanes due to defective enzymatic activity, the double mutants exhibit synthetic sickness indicating that increased catenation is toxic to pds5 cells. Our results suggest that Pds5p and Top2p cooperate to promote proper chromosome segregation by a mechanism unrelated to either cohesion or catenation/decatenation.     

Mitotic recombination in the rDNA of S. cerevisiae is suppressed by the combined action of DNA topoisomerases I and II

We have found that mitotic recombination within the S. cerevisiae rDNA cluster (200 tandemly repeated 9.1 kb units) is strongly suppressed and that this suppression requires the combined action of DNA topoisomerases I and II. Strains with a null mutation in the TOP1 gene (encoding topoisomerase I) or a ts mutation in the TOP2 gene (encoding topoisomerase II) grown at a semipermissive temperature show 50- to 200-fold higher frequencies of mitotic recombination in rDNA relative to TOP+ controls. Suppression of recombination is specific to the rDNA because the recombination frequency at another tandem array, the CUP1 locus, at a simple HIS4 duplication, or among dispersed repeats (MAT and HML or HMR) is not elevated in top1 or top2 mutants. The high frequency of mitotic recombination within the rDNA cluster in topoisomerase mutants shows that both TOP1 and TOP2 are required for suppression of recombination in this region of the genome.     

Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase.

The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E. coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R. (1989) Cell 58, 409-419). We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter. The purified protein possesses a distinct DNA topoisomerase activity: similar to E. coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA. Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA. A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined. These sequences differ from those recognized by E. coli DNA topoisomerase I but resemble those recognized by E. coli DNA topoisomerase III. Based on these results, the yeast TOP3 gene product can formally be termed S. cerevisiae DNA topoisomerase III. Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.     

Influence of the growth rate on the macromolecular composition of Acinetobacter calcoaceticus in carbon-limited chemostat culture

Abstract Infectious phage particles can be formed in vitro when extracts of T1-infected cells are incubated with T1 DNA. The DNA packaging system is based on mixtures of complementing extracts from Escherichia coli sup⁰ cells infected with the amber mutants am 4 (gene 16) or am 10 (gene 13). Gene 16 mutants are defective in the formation of DNA-filled heads but make proheads; gene 13 mutants are defective in prohead formation. Three forms of DNA have been packaged: (1) endogenous concatemeric DNA present in mixtures of am 4 and am 10 mutant extracts; (2) concatemeric DNA; (3) virion DNA both when supplied exogenously to mixtures of am 4 · am 20 and am 10 · am 20 double mutant extracts ( am 20 inhibits T1 DNA synthesis). The reaction requires added ATP, Mg²⁺ and spermidine for optimum efficiency and produces about 1.5 × 10³ pfu/ μ g and about 1 × 10⁴ pfu/ μ g for exogenous concatemeric and virion DNA, respectively.     

Plasmid transformation in Bacillus subtilis: Fate of plasmid DNA

Summary Only multimeric, and not monomeric forms of B. subtilis plasmids can transform B. subtilis cells (Canosi et al. 1978). This finding prompted us to study the physico-chemical fate of plasmid DNA in transformation. Competent cells of B. subtilis were exposed to either unfractionated preparations or to preparations of multimeric plasmid DNA. Plasmid DNA was re-extracted from such cells and then analyzed by sedimentation and isopycnic centrifugation and also defined by its sensitivity to nuclease S1 degradation. No double-stranded plasmid DNA could be recovered from cells transformed with unfractionated plasmid preparations which contained predominantly monomeric covalently closed circular (CCC) DNA, Re-extracted plasmid DNA was single-stranded, had a molecular weight considerably smaller than monomer length DNA and had been subject to degradation to acid soluble products. However, when transformations were performed with multimeric DNA (constructed by in vitro ligation of linearized pC194 DNA), both double-stranded and partially double-stranded DNA could be recovered in addition to single-stranded DNA.We assume that plasmid DNA is converted to a single-stranded form in transformation, irrespective of its molecular structure. Double-stranded and partially double-stranded DNAs found in transformation with multimeric DNA would be the products of intramolecular annealing.Some of these results were presented at the 5th European Meeting on Bacterial Transformation and Transfection, September 1980, Florence     

Biochemical and genetic characterization of the DNA ligase encoded by Saccharomyces cerevisiae open reading frame YOR005c, a homolog of mammalian DNA ligase IV.

Here we demonstrate that the Saccharomyces cerevisiae DNA ligase activity, which we previously designated DNA ligase II, is encoded by the genomic DNA sequence YOR005c. Based on its homology with mammalian LIG4, this yeast gene has been named DNL4 and the enzyme activity renamed Dnl4. In agreement with others, we find that DNL4 is not required for vegetative growth but is involved in the repair of DNA double-strand breaks by non-homologous end joining. In contrast to a previous report, we find that a dnl4 null mutation has no effect on sporulation efficiency, indicating that Dnl4 is not required for proper meiotic chromosome behavior or subsequent ascosporogenesis in yeast. Disruption of the DNL4 gene in one strain, M1-2B, results in temperature-sensitive vegetative growth. At the restrictive temperature, mutant cells progressively lose viability and accumulate small, nucleated and non-dividing daughter cells which remain attached to the mother cell. This novel temperature-sensitive phenotype is complemented by retransformation with a plasmid-borne DNL4 gene. Thus, we conclude that the abnormal growth of the dnl4 mutant strain is a synthetic phenotype resulting from Dnl4 deficiency in combination with undetermined genetic factors in the M1-2B strain background.     

Only one of the origin binding forms of SV40 T antigen has helicase activity

SV40 T antigen exists in monomeric and multimeric forms. We have separated the individual components by glycerol gradient centrifugation. Helicase activity is found to be associated with monomeric forms only. Dimers and other multimeric forms have no discernable helicase activity. However, results obtained from DNA binding experiments carried out with separated forms of T antigen indicate that both monomers and dimers bind to region I and region II of SV40 origin of replication. Possibly monomeric T antigen unwinds DNA at the replication fork while both monomeric and dimeric forms are utilized for positioning of T antigen at the origin of replication.     

Function of the loop residue Thr792 in human DNA topoisomerase II alpha

We studied the mutation effect of one of the putative loop residues Thr792 in human DNA topoisomerase II alpha (TOP2 alpha). Thr792 mutants were expressed from high or low copy plasmids in a temperature sensitive yeast strain deficient in TOP2 (top2-1). When expressed from a high copy plasmid, mutants with small side chains complemented the yeast defect; however, from a low copy plasmid, only wild-type, Ser, and Cys substitution mutants complemented the yeast defect. Interestingly, at the permissive temperature other mutants (e.g., Val, Gly, and Glu substitutions) showed the dominant negative effect to the top2-1 allele, which was not observed by the control alpha 4-helix mutants. T792E mutant was 10-fold less active than wild-type and the T792P had no decatenation activity in vitro. These results suggest that Thr792 in human TOP2 alpha is involved in enzyme catalysis.     

A DNase from the trypanosomatid Crithidia fasciculata.

We have purified to homogeneity a DNase from a Crithidia fasciculata crude mitochondrial lysate. The enzyme is present in two forms, either as a 32 kDa polypeptide or as a multimer containing the 32 kDa polypeptide in association with a 56 kDa polypeptide. Native molecular weight measurements indicate that these forms are a monomer and possibly an alpha 2 beta 2 tetramer, respectively. The monomeric and multimeric forms of the enzyme are similar in their catalytic activities. Both digest double-stranded DNA about twice as efficiently as single-stranded DNA. They introduce single-strand breaks into a supercoiled plasmid but do not efficiently make double-strand breaks. They degrade a linearized plasmid more efficiently than a nickel plasmid. Both enzymes degrade a 5'-32P-labeled double-stranded oligonucleotide to completion, with the 5'-terminal nucleotide ultimately being released as a 5'-mononucleotide. One difference between the monomeric and multimeric forms of the enzyme, demonstrated by a band shift assay, is that the multimeric form binds tightly to double-stranded DNA, possibly aggregating it.     

A novel family of TRF (DNA topoisomerase I-related function) genes required for proper nuclear segregation.

We recently reported the identification of a gene, TRF4 (for DNA topoisomerase related function), in a screen for mutations that are synthetically lethal with mutations in DNA topoisomerase I (top1). Here we describe the isolation of a second member of the TRF4 gene family, TRF5. Overexpression of TRF5 complements the inviability of top1 trf4 double mutants. The predicted Trf5 protein is 55% identical and 72% similar to Trf4p. As with Trf4p, a region of Trf5p is homologous to the catalytically dispensable N-terminus of Top1p. The TRF4/5 function is essential as trf4 trf5 double mutants are inviable. A trf4 (ts) trf5 double mutant is hypersensitive to the anti-microtubule agent thiabendazole at a semi-permissive temperature, suggesting that TRF4/5 function is required at the time of mitosis. Examination of nuclear morphology in a trf4 (ts) trf5 mutant at a restrictive temperature reveals the presence of many cells undergoing aberrant nuclear division, as well as many anucleate cells, demonstrating that the TRF4/5 function is required for proper mitosis. Database searches reveal the existence of probable Schizosaccharomyces pombe and human homologs of Trf4p, indicating that TRF4 is the canonical member of a gene family that is highly conserved evolutionarily.     

Mutant isolation of mouse DNA topoisomerase II alpha in yeast.

For characterizing in vivo functions of a mammalian protein, it is informative to obtain conditional mutations and apply them to the mouse genetic system. However, the isolation of conditional mutations has been quite difficult in cultured cells. We report here that functional expression of a heterologous mammalian gene in the yeast Saccharomyces cerevisiae provides a system for isolating mutated genes. We found that the cloned mouse TOP2 alpha cDNA, which encodes mouse DNA topoisomerase II (topo II) alpha, could rescue the lethal phenotype caused by yeast top2 null mutation. In order to generate and select temperature-sensitive mouse topo II alpha, an expression plasmid was mutagenized in vitro and was transformed, using the plasmid shuffling method, into the yeast strain, in which the endogenous TOP2 gene had been disrupted. We observed that one of such clone of yeast cells harboring a mutagenized mouse TOP2 alpha showed temperature-sensitive growth. Enzymatic assays and sequencing analysis revealed that this phenotype was caused by the thermosensitive nature of the mutant mouse protein, which has isoleucine at amino acid 961 instead of threonine. Therefore we have isolated the first conditional mutation in the mouse TOP2 alpha.