Separation of plasmid DNA topoisomers by multimodal chromatography

The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.     

A triangular loop of domain D1 of FlgE is essential for hook assembly but not for the mechanical function

The bacterial flagellar hook is a short, curved tubular structure made of FlgE. The hook connects the basal body as a rotary motor and the filament as a helical propeller and functions as a universal joint to smoothly transmit torque produced by the motor to the filament. Salmonella FlgE consists of D0, Dc, D1 and D2 domains. Axial interactions between a triangular loop of domain D1 (D1-loop) and domain D2 are postulated to be responsible for hook supercoiling. In contrast, Bacillus FlgE lacks the D1-loop and domain D2. Here, to clarify the roles of the D1-loop and domain D2 in the mechanical function, we carried out deletion analysis of Salmonella FlgE. A deletion of the D1-loop conferred a loss-of-function phenotype whereas that of domain D2 did not. The D1-loop deletion inhibited hook polymerization. Suppressor mutations of the D1-loop deletion was located within FlgD, which acts as the hook cap to promote hook assembly. This suggests a possible interaction between the D1-loop of FlgE and FlgD. Suppressor mutant cells produced straight hooks, but retained the ability to form a flagellar bundle behind a cell body, suggesting that the loop deletion does not affect the bending flexibility of the Salmonella hook.     

The Acidic C-terminal Tail of the GyrA Subunit Moderates the DNA Supercoiling Activity of Bacillus subtilis Gyrase

Gyrase is a type II DNA topoisomerase that introduces negative supercoils into DNA in an ATP-dependent reaction. It consists of a topoisomerase core, formed by the N-terminal domains of the two GyrA subunits and by the two GyrB subunits, that catalyzes double-stranded DNA cleavage and passage of a second double-stranded DNA through the gap in the first. The C-terminal domains (CTDs) of the GyrA subunits form a β-pinwheel and bind DNA around their positively charged perimeter. As a result, DNA is bound as a positive supercoil that is converted into a negative supercoil by strand passage. The CTDs contain a conserved 7-amino acid motif that connects blades 1 and 6 of the β-pinwheel and is a hallmark feature of gyrases. Deletion of this so-called GyrA-box abrogates DNA bending by the CTDs and DNA-induced narrowing of the N-gate, affects T-segment presentation, reduces the coupling of DNA binding to ATP hydrolysis, and leads to supercoiling deficiency. Recently, a severe loss of supercoiling activity of Escherichia coli gyrase upon deletion of the non-conserved acidic C-terminal tail (C-tail) of the CTDs has been reported. We show here that, in contrast to E. coli gyrase, the C-tail is a very moderate negative regulator of Bacillus subtilis gyrase activity. The C-tail reduces the degree of DNA bending by the CTDs but has no effect on DNA-induced conformational changes of gyrase that precede strand passage and reduces DNA-stimulated ATPase and DNA supercoiling activities only 2-fold. Our results are in agreement with species-specific, differential regulatory effects of the C-tail in gyrases from different organisms.     

The Escherichia cohi minB mutation resembles gyrB in defective nucleoid segregation and decreased negative supercoiling of plasmids

Summary Nucleoid segregation in the Escherichia coli minB mutant and in cells that over-produce minB gene products appeared defective as measured from fluorescence micrographs. Electrophoretic resolution of topoisomers of plasmid isolates from the minB strain revealed a decreased level of negative supercoiling; in addition, multimerization was observed. Over-production of the minB gene product also resulted in a decreased level of negative supercoiling. This phenotype is typical of the gyrB(ts) mutant, which is known to be affected in chromosome decatenation and supercoiling. We propose that the minB mutation and over-production of the minB gene products cause a defect in nucleoid segregation, which may be related to the decrease in negative supercoiling. As in the gyrB(ts) mutant, retardation of nucleoid segregation is proposed to inhibit constriction initiation in the cell centre and to give rise to nucleoid-free cell poles. As a consequence, these cells divide between nucleoid and cell pole, resulting in minicell and (sometimes) in anucleate cell formation.     

Separate and combined biochemical activities of the subunits of a naturally split reverse gyrase

Reverse gyrase reanneals denatured DNA and induces positive supercoils in DNA, an activity that is critical for life at very high temperatures. Positive supercoiling occurs by a poorly understood mechanism involving the coordination of a topoisomerase domain and a helicase-like domain. In the parasitic archaeon Nanoarchaeum equitans, these domains occur as separate subunits. We express the subunits, and characterize them both in isolation and as a heterodimer. Each subunit tightly associates and interacts with the other. The topoisomerase subunit enhances the catalytic specificity of the DNA-dependent ATPase activity of the helicase-like subunit, and the helicase-like subunit inhibits the relaxation activity of the topoisomerase subunit while promoting positive supercoiling. DNA binding preference for both single- and double-stranded DNA is partitioned between the subunits. Based on a sensitive topological shift assay, the binding preference of helicase-like subunit for underwound DNA is modulated by its binding with ATP cofactor. These results provide new insight into the mechanism of positive supercoil induction by reverse gyrase.     

Purified Saccharomyces cerevisiae RNA polymerase II interacts homologously with two different promoters as revealed by P1 endonuclease analysis

Summary The intergenic region of the Saccharomyces cerevisiae GAL1-GAL10 divergent promoters has been circularized in vitro in different topological states. In defined conditions, purified homologous RNA polymerase II forms two stable complexes (half-life -5 h) with this DNA in the presence of the four ribonucleotides, as determined by measurement (Gamper and Hearst 1983) of the amount and stability of the resulting unwinding. Each stable complex induces in the closed DNA domain a region of hypersensitivity to P1 endonuclease. The two induced hypersensitive regions are very similar: each maps on one promoter, spans over the 100 bp DNA sequence that encompasses the RNA Initiation Sites (RIS) and the TATA box, is composed by three subregions (one on the RIS, one proximal or overlapping the TATA sequence, one intermediate). We show that this promoter-localized interaction is supercoil-dependent.     

Gap compression/extension mechanism of bacterial flagellar hook as the molecular universal joint

Bacterial flagellar hook acts as a molecular universal joint, transmitting torque produced by the flagellar basal body, a rotary motor, to the flagellar filament. The hook forms polymorphic supercoil structures and can be considered as an assembly of 11 circularly arranged protofilaments. We investigated the molecular mechanism of the universal joint function of the hook by a approximately two-million-atom molecular dynamics simulation. On the inner side of the supercoil, protein subunits are highly packed along the protofilament and no gaps remain for further compression, whereas subunits are slightly separated and are hydrogen bonded through one layer of water molecules on the outer side. As for the intersubunit interactions between protofilaments, subunits are packed along the 6-start helix in a left-handed supercoil whereas they are highly packed along the 5-start helix in a right-handed supercoil. We conclude that the supercoiled structures of the hook in the left- and right-handed forms make maximal use of the gaps between subunits, which we call "gap compression/extension mechanism". Mutual sliding of subunits at the subunit interface accompanying rearrangements of intersubunit hydrogen bonds is interpreted as a mechanism to allow continuous structural change of the hook during flagellar rotation at low energy cost.