Dynamic coupling between conformations and nucleotide states in DNA gyrase.

Gyrase is an essential bacterial molecular motor that supercoils DNA using a conformational cycle in which chiral wrapping of > 100 base pairs confers directionality on topoisomerization. To understand the mechanism of this nucleoprotein machine, global structural transitions must be mapped onto the nucleotide cycle of ATP binding, hydrolysis and product release. Here we investigate coupling mechanisms using single-molecule tracking of DNA rotation and contraction during Escherichia coli gyrase activity under varying nucleotide conditions.

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension.

Single-molecule measurements of DNA twist and extension have been used to reveal physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields >100× improvement in time resolution over previous techniques.

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity.

DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit.

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: II. The shape of the GyrA subunit C-terminal domain (CTD) is not a sole determinant for controlling supercoiling efficiency.

DNA topoisomerases are essential enzymes that can overwind, underwind, and disentangle double-helical DNA segments to maintain the topological state of chromosomes. Nearly all bacteria utilize a unique type II topoisomerase, gyrase, which actively adds negative supercoils to chromosomes using an ATP-dependent DNA strand passage mechanism; however, the specific activities of these enzymes can vary markedly from species to species. Escherichia coli gyrase is known to favor supercoiling over decatenation (Zechiedrich, E.

All tangled up: how cells direct, manage and exploit topoisomerase function.

Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands.

A naturally chimeric type IIA topoisomerase in Aquifex aeolicus highlights an evolutionary path for the emergence of functional paralogs.

Bacteria frequently possess two type IIA DNA topoisomerases, gyrase and topo IV, which maintain chromosome topology by variously supercoiling, relaxing, and disentangling DNA. DNA recognition and functional output is thought to be controlled by the C-terminal domain (CTD) of the topoisomerase DNA binding subunit (GyrA/ParC). The deeply rooted organism Aquifex aeolicus encodes one type IIA topoisomerase conflictingly categorized as either DNA gyrase or topo IV.