The circular chromosome of Escherichia coli is organized into independently supercoiled loops, or topological domains. We investigated the organization and size of these domains in vivo and in vitro. Using the expression of >300 supercoiling-sensitive genes to gauge local chromosomal supercoiling, we quantitatively measured the spread of relaxation from double-strand breaks generated in vivo and thereby calculated the distance to the nearest domain boundary. In a complementary approach, we gently isolated chromosomes and examined the lengths of individual supercoiled loops by electron microscopy. The results from these two very different methods agree remarkably well. By comparing our results to Monte Carlo simulations of domain organization models, we conclude that domain barriers are not placed stably at fixed sites on the chromosome but instead are effectively randomly distributed. We find that domains are much smaller than previously reported, ∼10 kb on average. We discuss the implications of these findings and present models for how domain barriers may be generated and displaced during the cell cycle in a stochastic fashion.

The unwinding of the parental DNA duplex during replication causes a positive linking number difference, or superhelical strain, to build up around the elongating replication fork. The branching at the fork and this strain bring about different conformations from that of (؊) supercoiled DNA that is not being replicated. The replicating DNA can form (؉) precatenanes, in which the daughter DNAs are intertwined, and (؉) supercoils. Topoisomerases have the essential role of relieving the superhelical strain by removing these structures. Stalled replication forks of molecules with a (؉) superhelical strain have the additional option of regressing, forming a four-way junction at the replication fork. This four-way junction can be acted on by recombination enzymes to restart replication. Replication and chromosome folding are made easier by topological domain barriers, which sequester the substrates for topoisomerases into defined and concentrated regions. Domain barriers also allow replicated DNA to be (؊) supercoiled. We discuss the importance of replicating DNA conformations and the roles of topoisomerases, focusing on recent work from our laboratory.A thorough understanding of DNA replication and recombination requires knowledge of the conformations and topology of replicating DNA. These are different from those of nonreplicating DNA. The action of DNA helicases, interruptions in replicated strands, and, most importantly, the uniquely branched structure of the replication fork itself, all contribute to these differences. In this review, we illustrate the major conformational differences between replicating and nonreplicating DNA and their physiological importance. We highlight the evidence for each structure in vitro and in vivo. In addition, we address how the links originally residing in the double helix of the parental duplex are fully resolved in bacteria by two type-2 topoisomerases, DNA gyrase and topoisomerase (topo) IV, to form two separate daughter molecules. Although we emphasize the situation in bacteria, we will also make generalizations applicable to the eukarya and archaea.We begin by defining a few basic terms that form the language of DNA topology (1). The topology we will focus on are the links between the complementary Watson and Crick strands of an intact, topologically constrained piece of DNA. The simplest example is a closed circular DNA, as is found in plasmids and viruses, but the results can be generalized to linear chromosomes because of their organization into closed domains or loops. The intertwining of the complementary strands is described by the linking number (Lk), which is one-half of the signed number of times one strand crosses the other in any projection. According to the sign convention, the crossings in ordinary B-type DNA are (ϩ). The crossings, or nodes, of the complementary strands can result from the local intertwining of the double helix itself, in which case they are measured by a parameter called twist (Tw). Alternatively, nodes result from one segment of the double heli...

SummaryChromosomes are divided into topologically independent regions, called domains, by the action of uncharacterized barriers. With the goal of identifying domain barrier components, we designed a genetic selection for mutants with reduced negative supercoiling of the Escherichia coli chromosome. We employed a strain that contained two chromosomally located reporter genes under the control of a supercoiling-sensitive promoter and used transposon mutagenesis to generate a wide range of mutants. We subjected the selected mutants to a series of secondary screens and identified five proteins as modulators of chromosomal supercoiling in vivo . Three of these proteins: H-NS, Fis and DksA, have clear ties to chromosome biology. The other two proteins, phosphoglucomutase (Pgm) and transketolase (TktA), are enzymes involved in carbohydrate metabolism and have not previously been shown to affect DNA. Deletion of any of the identified genes specifically affected chromosome topology, without affecting plasmid supercoiling. We suggest that at least H-NS, Fis and perhaps TktA assist directly in the supercoiling of domains by forming topological barriers on the E. coli chromosome.

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase

Telomerase is a reverse transcriptase responsible for adding simple sequence repeats to chromosome 3-ends. The template for telomeric repeat synthesis is carried within the RNA component of the telomerase ribonucleoprotein complex. Telomerases can copy their internal templates with repeat addition processivity, reusing the same template multiple times in the extension of a single primer. For some telomerases, optimal repeat addition processivity requires high micromolar dGTP concentrations, a much higher dGTP concentration than required for processive nucleotide addition within a repeat. We have investigated the requirements for dGTP-dependent repeat addition processivity using recombinant Tetrahymena telomerase. By altering the template sequence, we show that repeat addition processivity retains the same dGTP-dependence even if dGTP is not the first nucleotide incorporated in the second repeat. Furthermore, no dNTP other than dGTP can stimulate repeat addition processivity, even if it is the first nucleotide incorporated in the second repeat. Using structural variants of dGTP, we demonstrate that the stimulation of repeat addition processivity is specific for dGTP base and sugar constituents but requires only a single phosphate group. However, all nucleotides that stimulate repeat addition processivity also inhibit or compete with dGTP incorporation into product DNA. By assaying telomerase complexes reconstituted with a variety of altered templates, we find that repeat addition processivity has an unanticipated template or product sequence specificity. Finally, we show that a novel, nascent product DNA binding site establishes dGTPdependent repeat addition processivity.The ends of chromosomes in most eukaryotes are capped with tandem simple sequence repeats. These telomeric repeats and their associated proteins are necessary and sufficient to distinguish a stable linear chromosome end from a highly unstable DNA break (reviewed in Ref. 1). However, telomeres are incompletely replicated by DNA-dependent DNA polymerases. The resulting loss of telomeric repeats with cell proliferation induces senescence or apoptosis of cultured human primary cells (reviewed in Ref. 2). Telomeric repeats eroded by proliferation can be restored by the enzyme telomerase, a specialized reverse transcriptase (RT) 1 that uses a defined region within its integral RNA component to template telomeric repeat synthesis (reviewed in Refs. 3, 4). Although telomerases in most organisms recognize only established telomeres as substrates, ciliate telomerases also recognize nontelomeric sites of developmentally programmed chromosome fragmentation. This ciliate telomerase chromosome healing activity is required to generate a transcriptionally competent macronucleus containing thousands of amplified, telomere-capped minichromosomes (reviewed in Ref. 5).Most biochemical characterization of telomerase has been done in ciliate systems because of the relative abundance of enzyme (reviewed in Ref. 6). Ciliate telomerases have been shown to catalyze at l...