Martin Gellert

Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyrase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.

Mechanistic Aspects of DNA Topoisomerases

Publisher Summary Topoisomerases are a diverse and important group of enzymes. Although attention has until recently been focused on their ability to interconvert DNA topoisomers, this does not necessarily constitute the primary biological function for all of them. DNA topoisomerases are enzymes that catalyze changes in the topology of circular DNA. With a closed-circular double-stranded DNA, one type of reaction alters the number of times the two strands are wound around each other and thus changes the degree of supercoiling. Supercoiled DNA molecules are prevalent in cells, and enzymes that can modify this property are important in DNA metabolism. Reactions involving other topological isomers of DNA are also known; various topoisomerases can form or resolve knotted or catenated structures in circular duplex DNA, or form knots in single-stranded circular DNA. Some of these reactions also have biological importance; for instance, replication of a circular-duplex DNA often produces two catenated circles, which then have to be separated. Cells of all organisms examined to date have been found to contain DNA topoisomerases; commonly there are several distinct types in a cell. Where a genetic test has been feasible, the presence of at least one topoisomerase has been found to be essential for cell growth. This chapter discusses various lines of information bearing on the enzymatic mechanisms of topoisomerases.

Cruciform structures in palindromic DNA are favored by DNA supercoiling

Abstract A totally palindromic circular DNA has been prepared by head-to-head ligation of a restriction fragment of plasmid pBR322 DNA. When negatively supercoiled, this DNA readily converts to a cruciform structure, as seen by either electron microscopy or gel electrophoresis. If the DNA is further supercoiled by DNA gyrase after hairpin formation has been initiated, as much as 80% of the molecular length can be extruded into hairpins. The rate of formation of the cruciform structure is strongly temperature-dependent; it is at least five-fold slower at 25 °C than at 35 °C. The palindromic DNA, although it contains all the necessary genetic information, is unable to transform Escherichia coli. We suggest that the intracellular formation of large cruciform structures is incompatible with survival of the DNA species.

Genetics and function of DNA ligase in Escherichia coli.

The characterization of two classes of DNA ligase mutants in Escherichia coli is described. The first class consists of three mutations coding for a temperature-sensitive ligase and defines the structural gene for DNA ligase (lig). The second class of mutants (lop) overproduces an apparently wild-type enzyme; a genetic diploid analysis implies that these are promoter or operator mutations, lig and lop are cotransduced by phage P1 and map at 46 minutes on the E. coli map. Detailed studies of two lig mutants (lig4 and lig ts7) are reported, lig ts7 is a conditionally lethal mutation, proving the essential nature of the ligase gene product. Neither mutant has a major defect in recombination or ultraviolet-repair, but both show retarded sealing of 10 S pulse-labeled DNA (Okazaki fragments).

Interactions of CcdB with DNA Gyrase INACTIVATION OF GyrA, POISONING OF THE GYRASE-DNA COMPLEX, AND THE ANTIDOTE ACTION OF CcdA

The F plasmid-carried bacterial toxin, the CcdB protein, is known to act on DNA gyrase in two different ways. CcdB poisons the gyrase-DNA complex, blocking the passage of polymerases and leading to double-strand breakage of the DNA. Alternatively, in cells that overexpress CcdB, the A subunit of DNA gyrase (GyrA) has been found as an inactive complex with CcdB. We have reconstituted the inactive GyrA-CcdB complex by denaturation and renaturation of the purified GyrA dimer in the presence of CcdB. This inactivating interaction involves the N-terminal domain of GyrA, because similar inactive complexes were formed by denaturing and renaturing N-terminal fragments of the GyrA protein in the presence of CcdB. Single amino acid mutations, both in GyrA and in CcdB, that prevent CcdB-induced DNA cleavage also prevent formation of the inactive complexes, indicating that some essential interaction sites of GyrA and of CcdB are common to both the poisoning and the inactivation processes. Whereas the lethal effect of CcdB is most probably due to poisoning of the gyrase-DNA complex, the inactivation pathway may prevent cell death through formation of a toxin-antitoxin-like complex between CcdB and newly translated GyrA subunits. Both poisoning and inactivation can be prevented and reversed in the presence of the F plasmid-encoded antidote, the CcdA protein. The products of treating the inactive GyrA-CcdB complex with CcdA are free GyrA and a CcdB-CcdA complex of approximately 44 kDa, which may correspond to a (CcdB)2(CcdA)2 heterotetramer.

Involvement of supertwisted DNA in integrative recombination of bacteriophage lambda

Abstract Negatively supertwisted closed circular DNA is the primary substrate for integrative recombination of phage λ DNA in vitro . Closed circular λ DNA without supertwists must be converted to the supertwisted form by the action of Escherichia coli DNA gyrase before efficient recombination can occur. When negatively supertwisted substrate is provided, E. coli DNA gyrase and its cofactors are dispensable components of recombination reaction mixtures. In the absence of DNA gyrase activity, circular DNA considerably less negatively twisted than naturally occurring supercoils is an effective substrate, but positively supertwisted DNA appears to be an ineffective substrate. The predominant products of integrative recombination in vitro are covalently closed circles. The closure of the recombined sites appears to occur without appreciable DNA synthesis and without the action of E. coli DNA ligase. No detectable difference can be observed between the degree of supertwisting of product DNA and that of unrecombined DNA. These facts suggest that the resealing of broken DNA strands is an integral part of the recombination reaction mechanism and is closely coupled with the breakage and realignment steps of recombination.

Catenation and supercoiling in the products of bacteriophage λ integrative recombination in vitro

Abstract Catenanes (interlocked circular DNA molecules) are the exclusive products of the bacteriophage λ integrative recombination reaction in vitro when the substrate is a supercoiled DNA molecule containing both the att P and att B sites. It is proposed that the catenation results from the superhelical form of the substrate DNA. We also show that both circular DNA products of a single recombination event can be recovered as superhelical molecules with a superhelical density approximately that of the substrate DNA. The recombination reaction must therefore occur as a coupled process which does not permit free rotation around single-strand breaks at any stage.

Mechanism of illegitimate recombination: Common sites for recombination and cleavage mediated by E. coli DNA gyrase

SummaryIllegitimate recombination dependent on DNA gyrase in a cell-free system has previously been described. We have now mapped DNA gyrase cleavage sites in the vicinity of known recombination sites in pBR322. Among five recombination sites examined, three were found to coincide with a DNA gyrase cleavage site. This result suggests that the cleavage of DNA by DNA gyrase has a central role in the recombination process.

ORGANIZATION OF DNA IN BACTERIOPHAGE T4.

A quantitative evaluation of the degree of preferred orientation of the DNA in bacteriophage T4 has been carried out, principally by the method of flow birefringence. The results indicate that the degree of preferred orientation is small, with about 9% of the DNA aligned preferentially along the long axis of the phage.

DNA Supercoiling as a Regulator of Bacterial Gene Expression

Studies of gene expression have advanced to a stage at which we can ask quite detailed questions about the factors that influence transcription and the ways in which these factors interact with each other. Such factors include not only the specific proteins that bind to regulatory sites on DNA, but also more subtle aspects of DNA structure, such as local bending, Z-DNA regions, and palindromic sequences.