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Escherichia coli Mfd translocase enables transcription-coupled repair by displacing RNA polymerase (RNAP) stalled on a DNA lesion and then coordinating assembly of the UvrAB(C) components at the damage site1-4. Recent studies have shown that after binding to and dislodging stalled RNAP, Mfd remains on the DNA in the form of a stable, slowly translocating complex with evicted RNAP attached5,6. Here we find, using a series of single-molecule assays, that recruitment of UvrA and UvrAB to Mfd-RNAP arrests the translocating complex and causes its dissolution. Correlative single-molecule nanomanipulation and fluorescence measurements show that dissolution of the complex leads to loss of both RNAP and Mfd. Subsequent DNA incision by UvrC is faster than when only UvrAB(C) are available, in part because UvrAB binds 20-200 times more strongly to Mfd-RNAP than to DNA damage. These observations provide a quantitative framework for comparing complementary DNA repair pathways in vivo., The conformational changes that take place in Mfd upon docking to, and activation by, stalled RNAP (7-9) enable it to bind to DNA upstream of RNAP and translocate along DNA [...]

Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2009.01.008 Byline: Achillefs N. Kapanidis (1), Terence Strick (2) Abstract: Single-molecule techniques have moved from being a fascinating curiosity to a highlight of life science research. The single-molecule approach to biology offers distinct advantages over the conventional approach of taking bulk measurements; this additional information content usually comes at the cost of the additional complexity. Popular single-molecule methods include optical and magnetic tweezers, atomic force microscopy, tethered particle motion and single-molecule fluorescence spectroscopy; the complement of these methods offers a wide range of spatial and temporal capabilities. These approaches have been instrumental in addressing important biological questions in diverse areas such as protein-DNA interactions, protein folding and the function(s) of membrane proteins. Author Affiliation: (1) Department of Physics and Interdisciplinary Research Collaboration in Bionanotechnology, Clarendon Laboratory, University of Oxford, Parks Road, Oxford, OX1 3PU, UK (2) Institut Jacques Monod, Centre National de la Recherche Scientifique, Unite Mixte de Recherche 7592 and University of Paris 7 - Paris Diderot, 4 rue M.-A. Lagroua Weill-Halle, 75205 Paris Cedex 13, France

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with [approximately equal to]1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with [approximately equal to]5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.

Research describing the structure and interactions of single biomolecules is presented. In particular the manipulation of protein and DNA molecules by pulling and twisting is investigated.

Preferential relaxation of positively supercoiled DNA by Escherichia coli topoisomerase IV (topo IV) has been studied using single-molecule and ensemble measurements. The rate of supercoil relaxation by topo IV is orders of magnitude faster than had been thought. One enzyme at each replication fork may be enough. How topo IV can quickly remove (+) supercoils to support DNA replication without relaxing the essential (-) supercoils of the chromosome is explained.

Author(s): Terence R. Strick (corresponding author); Vincent Croquette; David Bensimon Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a [...]