Mutants of S. CEREVISIAE Defective in the Maintenance of Minichromosomes

We have isolated yeast mutants that are defective in the maintenance of circular minichromosomes. The minichromosomes are mitotically stable plasmids, each of which contains a different ARS (autonomously replicating sequence), a centrometeric sequence, CEN5, and two yeast genes, LEU2 and URA3. Forty minichromosome maintenance-defective (Mcm-) mutants were characterized. They constitute 16 complementation groups. These mutants can be divided into two classes, specific and nonspecific, by their differential ability to maintain minichromosomes with different ARSs. The specific class of mutants is defective only in the maintenance of minichromosomes that carry a particular group of ARSs irrespective of the centromeric sequence present. The nonspecific class of mutants is defective in the maintenance of all minichromosomes tested irrespective of the ARS or centromeric sequence present. The specific class may include mutants that do not initiate DNA replication effectively at specific ARSs present on the minichromosomes; the nonspecific class may include mutants that are affected in the segregation and/or replication of circular plasmids in general.     

Distribution of minichromosomes in individual Escherichia coli cells: implications for replication control

A novel method was devised to measure the number of plasmids in individual Escherichia coli cells. With this method, involving measurement of plasmid-driven expression of the green fluorescent protein gene by flow cytometry, the copy number distribution of a number of different plasmids was measured. Whereas natural plasmids had fairly narrow distributions, minichromosomes, which are plasmids replicating only from a cloned oriC copy, have a wide distribution, suggesting that there is no copy number control for minichromosomes. When the selection pressure (kanamycin concentration) for minichromosomes was increased, the copy number of minichromosomes was also increased. At up to 30 minichromosomes per host chromosome, replication and growth of the host cell was unaffected. This is evidence that there is no negative element for initiation control in oriC and that there is no incompatibility between oriC located on the chromosome and minichromosome. However, higher copy numbers led to integration of the minichromosomes at the chromosomal oriC and to initiation asynchrony of the host chromosome. At a minichromosome copy number of approximately 30, the cell's capacity for synchronous initiation is exceeded and free minichromosomes will compete out the chromosome to yield inviable cells, unless the minichromosomes are incorporated into the chromosome.     

Failure to relax negative supercoiling of DNA is a primary cause of mitotic hyper-recombination in topoisomerase-deficient yeast cells

In the yeast Saccharomyces cerevisiae, DNA topoisomerases I and II can functionally substitute for each other in removing positive and negative DNA supercoils. Yeast Delta top1 top2(ts) mutants grow slowly and present structural instability in the genome; over half of the rDNA repeats are excised in the form of extrachromosomal rings, and small circular minichromosomes strongly multimerize. Because these traits can be reverted by the extrachromosomal expression of either eukaryotic topoisomerase I or II, their origin is attributed to the persistence of unconstrained DNA supercoiling. Here, we examine whether the expression of the Escherichia coli topA gene, which encodes the bacterial topoisomerase I that removes only negative supercoils, compensates the phenotype of Delta top1 top2(ts) yeast cells. We found that Delta top1 top2(ts) mutants expressing E. coli topoisomerase I grow faster and do not manifest rDNA excision and minichromosome multimerization. Furthermore, the recombination frequency in repeated DNA sequences, which is increased by nearly two orders of magnitude in Delta top1 top2(ts) mutants relative to the parental TOP+ cells, is restored to normal levels when the bacterial topoisomerase is expressed. These results indicate that the suppression of mitotic hyper-recombination caused by eukaryotic topoisomerases I and II is effected mainly by the relaxation of negative rather than positive supercoils; they also highlight the potential of unconstrained negative supercoiling to promote homologous recombination.     

Yeast plasmids have fewer superhelical turns than predicted by the nucleosome repeat of yeast DNA

The superhelical density of three Saccharomyces cerevisiae plasmids was determined with respect to a defined reference state during vegetative growth and stationary phase. The levels of supercoiling determined were approximately 20% lower than predicted by comparisons with SV40 DNA and reconstituted minichromosomes using histones from higher eukaryotes. In two different plasmids with the ARS1 origin of replication, the level of supercoiling changed substantially as the host cells entered stationary phase. Supercoiling of the endogenous 2-microns plasmid during vegetative growth was lower than in the ARS1-containing plasmids but did not change significantly upon entry of the cells into stationary phase.     

Escherichia coli minichromosomes: random segregation and absence of copy number control

Minichromosomes, i.e. plasmids that can replicate from an integrated oriC, have been puzzling because of their high copy numbers compared to that of the chromosomal oriC, their lack of incompatibility with the chromosome and their high loss frequencies. Using single cell resistance to tetracycline or ampicillin as an indicator of copy number we followed the development of minichromosome distributions in Escherichia coli cells transformed with minichromosomes and then allowed to grow towards the steady state. The final copy number distribution was not reached within 15 to 20 generations. If the minichromosome carried the sop (partitioning) genes from plasmid F, the development of the copy number distribution was further drastically delayed. We conclude that E. coli cells have no function that directly controls minichromosomal copy numbers, hence the absence of incompatibility in the sense of shared copy number control. We suggest that minichromosomes are subject to the same replication control as the chromosome but segregate randomly in the absence of integrated partitioning genes. This, combined with evidence that the lowest copy number classes are normally present despite high average copy numbers, can account for the high loss frequencies.     

Replication and segregation of a miniF plasmid during the division cycle of Escherichia coli.

Replication of the miniF plasmid pML31 was examined during the division cycle of Escherichia coli growing with doubling times between 40 and 90 min at 37 degrees C and compared to the replication of plasmid pBR322 and the minichromosome pAL70. The replication pattern of pML31 was indistinguishable from that of pBR322 at all growth rates and very different from the cell-cycle-specific replication of the minichromosome. It is concluded that both pML31 and pBR322 plasmids can replicate at all stages of the division cycle, with a probability of replication that increases gradually, but perhaps not exponentially, during the cycle. In contrast, the modes of segregation of pML31 and pBR322 plasmids into daughter cells at division appeared to differ, raising the possibility that pML31 may segregate in a nonrandom fashion similar to that of chromosomes and minichromosomes.     

Mutations in the DNA gyrB gene that are temperature sensitive for lambda site-specific recombination, Mu growth, and plasmid maintenance.

We report the isolation of two mutations in the gyrB gene of Escherichia coli K12 obtained from an initial selection for resistance to coumermycin A1 and a subsequent screening for bacteria that fail to support site-specific recombination of phage lambda, i.e., Him-. These two mutations have a temperature-sensitive Him- phenotype, supporting site-specific recombination efficiently at low temperature, but inefficiently at high temperatures. Like other Him mutants, the gyrB-him mutants fail to plate phage Mu; again this defect is observed only at high temperatures. Additional thermally sensitive characteristics have also been observed; growth of lambda as well as maintenance of the plasmids pBR322 and F' gal are reduced at high temperature. Restriction of foreign DNA imposed by a P1 prophage is also reduced in these mutants. The temperature-sensitive phenotypic characteristics imposed by both the gyrB-him-230(Ts) and gyrB-him-231(Ts) mutations correlate with in vitro studies that show decreased gyrase activity, especially at higher temperatures, and in vivo studies showing reduced supercoiling of lambda DNA in the mutants at high temperature.     

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.     

A novel type of E. coli mutants with increased chromosomal copy number

We have isolated E. coli mutants which can grow at 30 degrees C but not at 42 degrees C and are able to harbor the oriC plasmid (minichromosome) at a higher copy number than the parental wild-type strain at the permissive temperature. The mutants were found to contain higher amounts of chromosomal DNA per mg protein than the wild-type, whether or not they harbor the plasmid. Experimental results suggest that the higher amount of chromosomal DNA is due to a higher copy number of chromosomes and not to a larger amount of DNA per chromosome. These properties in each of the mutants are caused by a single mutation at the rpoB or rpoC gene that code for the beta or beta' subunit of RNA polymerase, respectively. The mutations are thought to affect the regulation of replication of oriC-bearing replicons, that is, the E. coli chromosome and oriC plasmids, but not the miniF plasmid.     

Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli

The closely related Escherichia coli genes, hupA, hupB, himA and himD (hip), encode the bacterial histone-like protein subunits, HU-2, HU-1, IHF chi and IHF beta, respectively. We report here that E. coli minichromosomes [plasmids (2.7-12.2 kb) with oriC] carrying the intact mioC region were unable to transform mutants deficient in both HU and integration host factor (IHF), whereas they could transform mutants deficient in either HU or IHF as efficiently as the wild-type strain. Minichromosomes carrying a deletion of the proximal part of mioC or a DnaA box just upstream from mioC could not transform cells deficient in IHF, but could transform cells deficient in HU. These results suggested that HU and IHF participate in minichromosomal replication from oriC in E. coli.     

Replication restart in gyrB Escherichia coli mutants

Gyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases. Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected. We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants. The helicase function of PriA is not essential. The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains. The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co-lethal in the presence of a DeltatopA mutation. Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate. We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA-dependent primosome assembly, directly on the in-activated replication forks, without the formation of a recombination intermediate.     

Different effects of mioC transcription on initiation of chromosomal and minichromosomal replication in Escherichia coli.

The mioC gene, which neighbors the chromosomal origin of replication (oriC) in Escherichia coli, has in a number of studies been implicated in the control of oriC initiation on minichromosomes. The present work reports on the construction of cells carrying different mioC mutations on the chromosome itself. Flow cytometry was employed to study the DNA replication control and growth pattern of the resulting mioC mutants. All parameters measured (growth rate, cell size, DNA/cell, number of origins per cell, timing of initiation) were the same for the wild type and all the mioC mutant cells under steady state growth and after different shifts in growth medium and after induction of the stringent response. It may be concluded that the dramatic effects of mioC mutations reported for minichromosomes are not observed for chromosomal replication and that the mioC gene and gene product is of little importance for the control of initiation. The data demonstrate that a minichromosome is not necessarily a valid model for chromosomal replication.     

A mutant that affects the function of autonomously replicating sequences in yeast

We previously reported the isolation of a series of mcm mutants that are defective in the maintenance of minichromosomes in yeast. These minichromosomes are circular plasmids, each containing an autonomously replicating sequence (ARS) and a centromere. One of the mcm mutants, mcm2, has the following phenotype: at room temperature it affects the stability of only some minichromosomes depending on the ARS present, while at high temperature it affects all minichromosomes tested irrespective of the ARS present. Here we show that the mcm defect as well as its temperature-dependent specificity for ARSs can be demonstrated with circular as well as linear plasmids that do not contain centromeric sequences. Larger chromosomes containing multiple ARSs are also unstable in this mutant. Further analyses indicate that the mcm2 mutation causes the loss, rather than the aberrant segregation, of the circular minichromosomes. In addition, this mutation appears to stimulate mitotic recombination frequencies. These properties of the mcm2 mutant are consistent with the idea that the mcm2 mutation results in a defect in the initiation of DNA replication at ARSs, the putative chromosomal replication origins in yeast.     

Purification and characterization of DNA topoisomerase IV in Escherichia coli.

The subunits of topoisomerase IV (topo IV), the ParC and ParE proteins in Escherichia coli, were purified to near homogeneity from the respective overproducers. They revealed type II topoisomerase activity only when they were combined with each other. In the presence of Mg2+ and ATP, topo IV was capable of relaxing a negatively or positively supercoiled plasmid DNA or converting the knotted P4 phage DNA, whether nicked or ligated, to a simple ring. However, supercoiling activity was not detected. The topoisomerase activity was not detectable when the purified ParC and ParE proteins were combined with the purified GyrB and GyrA proteins, respectively. This is consistent with the result that neither a parC nor a parE mutation was compensated by transformation with a plasmid carrying either the gyrA or the gyrB gene. Simultaneous introduction of both the gyrA and gyrB plasmids corrected the phenotypic defect of parC and parE mutants. The results suggest that DNA gyrase can substitute for topo IV at least in some part of the function for chromosome partitioning. Antisera were prepared against the purified ParC, ParE, GyrA, and GyrB proteins and used to investigate cellular localization of these gene products. ParC protein was found to be specifically associated with inner membranes only in the presence of DNA. This result suggests that one of the functions of topo IV might be to anchor chromosomes on membranes as previously proposed for eukaryotic topoisomerase II.     

Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region.

Initiation of chromosomal replication in Escherichia coli is dependent on availability of the initiator protein DnaA. We have introduced into E. coli cells plasmids carrying the chromosomal locus datA, which has a high affinity for DnaA. To be able to monitor oriC initiation as a function of datA copy number, we introduced a minichromosome which only replicates from oriC, using a host cell which replicates its chromosome independently of oriC. Our data show that a moderate increase in datA copy number is accompanied by increased DnaA protein synthesis that allows oriC initiation to occur normally, as measured by minichromosome copy number. As datA gene dosage is increased dnaA expression cannot be further derepressed, and the minichromosome copy number is dramatically reduced. Under these conditions the minichromosome was maintained by integration into the chromosome. These findings suggest that the datA locus plays a significant role in regulating oriC initiation, by its capacity to bind DnaA. They also suggest that auto regulation of the dnaA gene is of minor importance in regulation of chromosome initiation.     

New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.

In Escherichia coli K-12 mutants which had a new nalidixic acid resistance mutation at about 82 min on the chromosome map, cell growth was resistant to or hypersusceptible to nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, and novobiocin. Deoxyribonucleic acid gyrase activity as tested by supercoiling of lambda phage deoxyribonucleic acid inside the mutants was similarly resistant or hypersusceptible to the compounds. The drug concentrations required for gyrase inhibition were much higher than those for cell growth inhibition but similar to those for inhibition of lambda phage multiplication. Transduction analysis with lambda phages carrying the chromosomal fragment of the tnaA-gyrB region suggested that one of the mutations, nal-31, was located on the gyrB gene.