DNA gyrase genes in mycobacterium tuberculosis: a single operon driven by multiple promoters

The two genes encoding DNA gyrase in Mycobacterium tuberculosis are present next to each other in the genome, with gyrB upstream of gyrA. We show that the primary transcript is dicistronic. However, in addition to the principal promoter, there are multiple weaker promoters that appear to fine-tune transcription. With these and other mycobacterial promoters, we propose consensus promoter sequences for two distinct sigma factors. In addition to this, the gyr genes in M. tuberculosis, as in other species, are subject to autoregulation, albeit with slower kinetics, probably reflecting the slower metabolism of the organism

Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents

DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival. There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations. However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive. Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB. Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme. Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl-N-nitro-N-nitrosoguanidine, that act independently of DNA gyrase. Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources. In contrast, GyrI is not effective against lesions induced by ultraviolet radiation. Furthermore, the expression of GyrI does not significantly alter the topology of DNA. Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged

A hairpin near the 5′ end stabilises the DNA gyrase mRNA in Mycobacterium smegmatis

RNA is amongst the most labile macromolecules present in the cells. The steady-state levels of mRNA are regulated both at the stages of synthesis and degradation. Recent work in Escherichia coli suggests that controlling the rate of degradation is as important as the process of synthesis. The stability of mRNA is probably more important in slow- growing organisms like mycobacteria. Here, we present our analysis of the cis elements that determine the stability of the DNA gyrase message in Mycobacterium smegmatis. The message appears to be stabilised by a structure close to its 5′ end. The effect is especially pronounced in a nutrient-depleted state. These results largely parallel the model proposed in E.coli for mRNA degradation/ stability with subtle differences. Furthermore, these results suggest that the slow-growing organisms might use stable mRNAs as a method to reduce the load of transcription on the cell

DNA Gyrase Genes in Mycobacterium tuberculosis: a Single Operon Driven by Multiple Promoters

The two genes encoding DNA gyrase in Mycobacterium tuberculosis are present next to each other in the genome, with gyrB upstream of gyrA. We show that the primary transcript is dicistronic. However, in addition to the principal promoter, there are multiple weaker promoters that appear to fine-tune transcription. With these and other mycobacterial promoters, we propose consensus promoter sequences for two distinct sigma factors. In addition to this, the gyr genes in M. tuberculosis, as in other species, are subject to auto regulation, albeit with slower kinetics, probably reflecting the slower metabolism of the organism

GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase

DNA gyrase is the target of two plasmid-encoded toxins CcdB and microcin B17, which ensure plasmid maintenance. These proteins stabilize gyrase–DNA covalent complexes leading to double-strand breaks in the genome. In contrast, the physiological role of chromosomally encoded inhibitor of DNA gyrase (GyrI) in Escherichia coli is unclear and its mechanism of inhibition has not been established. We demonstrate that the mode of inhibition of GyrI is distinct from all other gyrase inhibitors. It inhibits DNA gyrase prior to, or at the step of, binding of DNA by the enzyme. GyrI reduces intrinsic as well as toxin-stabilized gyrase–DNA covalent complexes. Furthermore, GyrI reduces microcin B17-mediated double-strand breaks in vivo, imparting protection to the cells against the toxin, substantiating the in vitro results. Thus, GyrI is an antidote to DNA gyrase-specific proteinaceous poisons encoded by plasmid addiction systems

Mobilization of RAG-Generated Signal Ends by Transposition and Insertion In Vivo

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated “core” RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability

DNA mismatches and GC-rich motifs target transposition by the RAG1/RAG2 transposase

In addition to their essential role in V(D)J recombination, the RAG proteins function as a transposase capable of inserting the V(D)J recombination intermediate, the signal end DNA fragment, into target DNA. RAG-mediated transposition has been suggested to contribute to genome instability and the development of lymphoid malignancies. Previous studies suggested that the RAG transposase exhibits a target site preference for GC rich sequences and hairpin structures. Here we demonstrate that a transposition hot spot (5′-GCCGCCGGGCC-3′), smaller portions of this hot spot and other GC rich motifs are able to target RAG-mediated transposition. Tracks of GC base pairs have been shown to have an unusually high rate of base pair breathing. Intriguingly, we find that DNA mismatches can efficiently target RAG-mediated transposition and suppress the use of other target sites. Hairpins, however, are not generally preferred targets. Our results indicate that target DNA melting may be a crucial step during RAG-mediated transposition, and that target site selection by the RAG transposase may be intimately linked to mutagenic and metabolic processes that transiently present favorable DNA structures to the transposition machinery

The Additional 165 Amino Acids in the B Protein of Escherichia coli DNA Gyrase Have an Important Role in DNA Binding

DNA gyrase is the only enzyme known to negatively super coil DNA. The enzyme is a heterotetramer of A2B2 subunit composition. Alignment of the primary sequence of gyrase B (GyrB) from various species shows that they can be grouped into two classes. The GyrB of Gram-negative eubacteria has a stretch of about 165 amino acids in the C-terminal half, which is lacking in other GyrB subunits and type II topoisomerases. In Escherichia coli, no function has so far been attributed to this stretch. In this study, we have tried to assess the function of this region both in vivo and in vitro. A deletant (GyrBD160) lacking this region is non-functional in vivo. The holoenzyme reconstituted from gyrase A (GyrA) and GyrBD160 shows reduced but detectable supercoiling and quinolone-induced cleavage activity in vitro. GyrBD160 retains its ability to bind to GyrA and novobiocin. However, when reconstituted with GyrA, the deletant shows greatly impaired DNA binding. The intrinsic ATPase activity of the GyrBD160 is comparable to that of wild type GyrB, but this activity is not stimulated by DNA. These studies indicate that the additional stretch present in GyrB is essential for the DNA binding ability of E.coli gyrase