Changes In DNA Supercoiling Research Articles

Role of DNA Superhelicity in Regulation of Bacteroid-Associated Functions of Bradyrhizobium sp. Strain 32H1

Bradyrhizobium sp. strain 32H1 cells express a number of bacteroid-associated functions and repress some functions related to the free-living state when grown ex planta under conditions of low (0.2%) oxygen tension and relatively high levels (>8 mM) of medium K. Expression of the bacteroid-associated phenotype was blocked by the DNA gyrase inhibitor novobiocin. Because the degree of negative supercoiling of DNA is the result of the activities of both DNA gyrase and topoisomerase I, we measured these enzymes in cells grown under nitrogen-fixing (low O(2), high K) and non-nitrogen-fixing conditions (low O(2), low [50 muM] K or high O(2), high K). Lower topoisomerase I activities were seen in extracts from nitrogen-fixing cells than in those from non-nitrogen-fixing cells. In contrast, DNA gyrase levels were lower in high-O(2)-grown cells than under the other conditions tested. These differences are consistent with an increase in DNA superhelicity associated with growth under low-O(2), high-K conditions. A spontaneous mutant resistant to the DNA gyrase inhibitor ciprofloxacin was found to be constitutive with respect to the K requirement, because it expressed the bacteroid-associated phenotype when grown under low-O(2), low-K conditions. The mutant cells gave rise to effective nodules on Macroptilium atropurpureum and possessed the low topoisomerase I activities and high DNA gyrase levels of low-O(2)-, high-K-grown wild-type cells. Our data suggest that changes in DNA supercoiling resulting from low O(2) tension and a high K concentration exert a major influence on the expression of the bacteroid-associated phenotype.

DNA supercoiling and the anaerobic and growth phase regulation of tonB gene expression

We show that several interacting environmental factors influence the topology of intracellular DNA. Negative supercoiling of DNA in vivo is increased by anaerobic growth and is also influenced by growth phase. The tonB promoter of Escherichia coli and Salmonella typhimurium was found to be highly sensitive to changes in DNA supercoiling. Expression was increased by novobiocin, an inhibitor of DNA gyrase, and was decreased by factors which increase DNA superhelicity. Expression of the plasmid-encoded tonB gene was enhanced by gamma delta insertions in cis in a distance- and orientation-independent fashion. Both the res site and the TnpR protein of gamma delta, which is known to function as a type I topoisomerase, were required for this activation. tonB expression increased during the growth cycle and was reduced by anaerobiosis. There was excellent correlation between tonB expression from a plasmid and the level of supercoiling of that plasmid under a wide range of conditions. The chromosomal tonB gene was regulated in a manner identical to that of the plasmid-encoded gene. Thus, the physiological regulation of tonB expression in response to anaerobiosis and growth phase appears to be mediated by environmentally induced changes in DNA superhelicity.

Topoisomerase II inhibitors influence simian virus 40 chromatin structure in vivo accompanied with inhibition of replication, transcription and changes in DNA supercoiling.

The association of topoisomerase II enzymes with papovavirus-chromatin has been described recently. We used cells infected with simian virus 40 (SV40) to investigate the in vivo effect of the topoisomerase II inhibitors nalidixic acid and novobiocin on the viral chromatin template. Blocking of topoisomerases inhibits DNA and RNA synthesis. The block with the inhibitors resulted in a conversion of a 95- to 75-svedberg chromatin to a 180- to 150-svedberg component accompanied with a variation in the topological linking number of the DNA. The condensed chromatin fractions (180-150 S) from inhibited cells resemble encapsidation intermediates and their DNA has a linking number as the DNA extracted from purified virions. Moreover, the virion DNA has a higher superhelical density compared to intracellular chromatin isolated from untreated cells and argues for a supercoiling activity in the cell.

Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli.

The supercoiling levels of plasmid DNA were determined from Escherichia coli which was grown in ways that are known to alter global patterns of gene expression and metabolism. Changes in DNA supercoiling were shown to occur during several types of these nutrient upshifts and downshifts. The most dramatic change in supercoiling was seen in starved cells, in which two populations of differentially relaxed plasmids were shown to coexist. Thus, some changes in the external nutritional environment that cause the cells to reorganize their global metabolism also cause accompanying changes in DNA supercoiling. Results of experiments with dinitrophenol suggested that the observed relaxations were probably not due to reduced pools of ATP. When rifampin was used to release supercoils restrained by RNA polymerase, the cellular topoisomerases responded by removing these new, unrestrained supercoils. We interpret these results as implying that the cellular topological machinery maintains a constant superhelical energy in the DNA except during certain growth transitions, when changes in metabolism and gene expression are accompanied by changes in DNA supercoiling.

Changes in DNA supercoiling during repair of gamma-radiation-induced damage.

Enriched human B-lymphocyte and T-lymphocyte subpopulations were isolated by means of two Percoll density gradient centrifugation steps. Strand-break repair kinetics of 60Co gamma-irradiation-damaged DNA were obtained by applying a modified nucleoid sedimentation technique. Although no variations in the strand-break repair patterns of B-cells and T-cells within an individual could be detected, variations between individuals were evident. Changes in DNA supercoiling during the repair process were studied by means of ethidium bromide intercalation. The supercoiled conformation of DNA in peripheral lymphocyte subpopulations displayed similar undulating patterns within an individual, variations occurring between individuals. The results of this study indicate that alterations to the higher-order structure (supercoiling) of DNA are integral to the repair process.

DNA topoisomerase I mutants: Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon. replicon.

The usefulness of viscometric analysis for detecting minimal levels DNA fragmentation.

Basic requirements for a method used to study the genotoxic potential of xenobiotic agents in the intact animal are sensitivity and capability of detecting the effect of the compound under test in various tissues. A new viscometric technique, which has been found capable of measuring DNA damage in liver, kidney, and lung of rats treated with small single doses of 10 chemical carcinogens, seems to possess such requirements. Single-strand breaks and probably other types of lesions indeed cause changes in DNA supercoiling which can be sensitively measured by monitoring time-dependent changes of DNA viscosity. The main advantage of this technique is that clear-cut modifications of viscometric parameters can be obtained with doses of various carcinogens markedly lower than those found to be the minimal effective ones in other commonly employed short-term in vivo tests. The importance of studying the genotoxic effects of pharmacologically meaningful doses is discussed.