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.

Exposure of E. coli to hydrogen peroxide induces the transcription of a small RNA denoted oxyS. The oxyS RNA is stable, abundant, and does not encode a protein. oxyS activates and represses the expression of numerous genes in E. coli, and eight targets, including genes encoding the transcriptional regulators FhlA and sigma(S), were identified. oxyS expression also leads to a reduction in spontaneous and chemically-induced mutagenesis. Our results suggest that the oxyS RNA acts as a regulator that integrates adaptation to hydrogen peroxide with other cellular stress responses and helps to protect cells against oxidative damage.

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...

The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (؉)-⌬Lk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (؉)-⌬Lk were not supercoiled nor were the replicated arms interwound in precatenanes. The electrophoretic mobility of these molecules indicated that they have no net writhe. Instead, the (؉)-⌬Lk is absorbed by a regression of the replication fork. As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at the replication fork, which is locally identical to a Holliday junction in recombination. We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visualized the structure with scanning force microscopy. We discuss possible physiological implications of the junction for stalled replication in vivo.