From parasites to partners: exploring the intricacies of host-transposon dynamics and coevolution.

Transposable elements, often referred to as "jumping genes," have long been recognized as genomic parasites due to their ability to integrate and disrupt normal gene function and induce extensive genomic alterations, thereby compromising the host's fitness. To counteract this, the host has evolved a plethora of mechanisms to suppress the activity of the transposons. Recent research has unveiled the host-transposon relationships to be nuanced and complex phenomena, resulting in the coevolution of both entities. Transposition increases the mutational rate in the host genome, often triggering physiological pathways such as immune and stress responses. Current gene transfer technologies utilizing transposable elements have potential drawbacks, including off-target integration, induction of mutations, and modifications of cellular machinery, which makes an in-depth understanding of the host-transposon relationship imperative. This review highlights the dynamic interplay between the host and transposable elements, encompassing various factors and components of the cellular machinery. We provide a comprehensive discussion of the strategies employed by transposable elements for their propagation, as well as the mechanisms utilized by the host to mitigate their parasitic effects. Additionally, we present an overview of recent research identifying host proteins that act as facilitators or inhibitors of transposition. We further discuss the evolutionary outcomes resulting from the genetic interactions between the host and the transposable elements. Finally, we pose open questions in this field and suggest potential avenues for future research.

Evolution of YacG to safeguard DNA gyrase from external perturbation.

Cells have evolved strategies to safeguard their genome integrity. We describe a mechanism to counter double strand breaks in the chromosome that involves the protection of an essential housekeeping enzyme from external agents. YacG is a DNA gyrase inhibitory protein from Escherichia coli that protects the bacterium from the cytotoxic effects of catalytic inhibitors as well as cleavage-complex stabilizers of DNA gyrase. By virtue of blocking the primary DNA binding site of the enzyme, YacG prevents the accumulation of double strand breaks induced by gyrase poisons. It also enables the bacterium to resist the growth-inhibitory property of novobiocin. Gyrase poison-induced oxidative stress upregulates YacG production, probably as a cellular response to counter DNA damage. YacG-mediated protection of the genome is specific for gyrase targeting agents as the protection is not observed from the action of general DNA damaging agents. YacG also intensifies the transcription stress induced by rifampicin substantiating the importance of gyrase activity during transcription. Although essential for bacterial survival, DNA gyrase often gets entrapped by external inhibitors and poisons, resulting in cell death. The existence of YacG to specifically protect an essential housekeeping enzyme might be a strategy adopted by bacteria for competitive fitness advantage.

Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein.

Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coli gyrase, a type IIA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holoenzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state.

Dpb2 integrates the leading-strand DNA polymerase into the eukaryotic replisome.

BACKGROUND: The eukaryotic replisome is a critical determinant of genome integrity with a complex structure that remains poorly characterized. A central unresolved issue is how the Cdc45-MCM-GINS helicase is linked to DNA polymerase epsilon, which synthesizes the leading strand at replication forks and is an important focus of regulation. RESULTS: Here, we use budding yeast to show that a conserved amino-terminal domain of the Dpb2 subunit of Pol ε (Dpb2NT) interacts with the Psf1 component of GINS, via the unique "B domain" of the latter that is dispensable for assembly of the GINS complex but is essential for replication initiation. We show that Dpb2NT is required during initiation for assembly of the Cdc45-MCM-GINS helicase. Moreover, overexpressed Dpb2NT is sufficient to support assembly of the Cdc45-MCM-GINS helicase during initiation, upon depletion of endogenous Dpb2. This produces a replisome that lacks DNA polymerase epsilon, and although cells are viable, they grow extremely poorly. Finally, we use a novel in vitro assay to show that Dpb2NT is essential for Pol ε to interact with the replisome after initiation. CONCLUSIONS: These findings indicate that the association of Dpb2 with the B domain of Psf1 plays two critical roles during chromosome replication in budding yeast. First, it is required for initiation, because it facilitates the incorporation of GINS into the Cdc45-MCM-GINS helicase at nascent forks. Second, it plays an equally important role after initiation, because it links the leading strand DNA polymerase to the Cdc45-MCM-GINS helicase within the replisome.

Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.

Mcm10 is essential for chromosome replication in eukaryotic cells and was previously thought to link the Mcm2-7 DNA helicase at replication forks to DNA polymerase alpha. Here, we show that yeast Mcm10 interacts preferentially with the fraction of the Mcm2-7 helicase that is loaded in an inactive form at origins of DNA replication, suggesting a role for Mcm10 during the initiation of chromosome replication, but Mcm10 is not a stable component of the replisome subsequently. Studies with budding yeast and human cells indicated that Mcm10 chaperones the catalytic subunit of polymerase alpha and preserves its stability. We used a novel degron allele to inactivate Mcm10 efficiently and this blocked the initiation of chromosome replication without causing degradation of DNA polymerase alpha. Strikingly, the other essential helicase subunits Cdc45 and GINS were still recruited to Mcm2-7 when cells entered S-phase without Mcm10, but origin unwinding was blocked. These findings indicate that Mcm10 is required for a novel step during activation of the Cdc45-MCM-GINS helicase at DNA replication origins.

Moonlighting function of glutamate racemase from Mycobacterium tuberculosis: racemization and DNA gyrase inhibition are two independent activities of the enzyme.

Glutamate racemase (MurI) provides d-glutamate, a key building block in the peptidoglycan of the bacterial cell wall. Besides having a crucial role in cell wall biosynthesis, MurI proteins from some bacteria have been shown to act as an inhibitor of DNA gyrase. Mycobacterium tuberculosis and Mycobacterium smegmatis MurI exhibit these dual characteristics. Here, we show that the two activities of M. tuberculosis MurI are unlinked and independent of each other. The racemization function of MurI is not essential for its gyrase-inhibitory property. MurI-DNA gyrase interaction influences gyrase activity but has no effect on the racemization activity of MurI. Overexpression of MurI in vivo provides resistance to the action of ciprofloxacin, suggesting the importance of the interaction in gyrase modulation. We propose that the moonlighting activity of MurI has evolved more recently than its racemase function, to play a transient yet important role in gyrase modulation.

YacG from Escherichia coli is a specific endogenous inhibitor of DNA gyrase.

We assign a function for a small protein, YacG encoded by Escherichia coli genome. The NMR structure of YacG shows the presence of an unusual zinc-finger motif. YacG was predicted to be a part of DNA gyrase interactome based on protein-protein interaction network. We demonstrate that YacG inhibits all the catalytic activities of DNA gyrase by preventing its DNA binding. Topoisomerase I and IV activities remain unaltered in the presence of YacG and its action appears to be restricted only to DNA gyrase. The inhibition of the enzyme activity is due to the binding of YacG to carboxyl terminal domain of GyrB. Overexpression of YacG results in growth inhibition and alteration in DNA topology due to uncontrolled inhibition of gyrase.

Inhibition of DNA gyrase activity by Mycobacterium smegmatis MurI.

Glutamate racemase (MurI) catalyzes the interconversion of l-glutamate to d-glutamate, one of the essential amino acids present in the peptidoglycan. In addition to this essential enzymatic function, MurI from Escherichia coli, Bacillus subtilis and Mycobacterium tuberculosis inhibit DNA gyrase activity. A single gene for murI found in the Mycobacterium smegmatis genome was cloned and overexpressed in a homologous expression system to obtain a highly soluble enzyme. In addition to the racemization activity, M. smegmatis MurI inhibits DNA gyrase activity by preventing DNA binding of gyrase. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. More importantly, MurI overexpression in vivo in mycobacterial cells provides protection against the action of ciprofloxacin. The DNA gyrase-inhibitory property thus appears to be a typical characteristic of MurI and would have probably evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double-strand breaks in the genome.

Glutamate racemase from Mycobacterium tuberculosis inhibits DNA gyrase by affecting its DNA-binding.

Glutamate racemase (MurI) catalyses the conversion of l-glutamate to d-glutamate, an important component of the bacterial cell wall. MurI from Escherichia coli inhibits DNA gyrase in presence of the peptidoglycan precursor. Amongst the two-glutamate racemases found in Bacillus subtilis, only one inhibits gyrase, in absence of the precursor. Mycobacterium tuberculosis has a single gene encoding glutamate racemase. Action of M.tuberculosis MurI on DNA gyrase activity has been examined and its mode of action elucidated. We demonstrate that mycobacterial MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species-specific as E.coli gyrase is also inhibited but is enzyme-specific as topoisomerase I activity remains unaltered. The mechanism of inhibition is different from other well-known gyrase inhibitors. MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA-binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalysed by DNA gyrase. MurI is thus not a typical potent inhibitor of DNA gyrase and instead its role could be in modulation of the gyrase activity.

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.