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Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes. Here, the authors report the kinetics of Sen1/RNA simultaneous release, Sen1-Pol II complex dissociation, and Pol II diffusion during eukaryotic Sen1-dependent transcription termination through single-molecule fluorescence assays. [ABSTRACT FROM AUTHOR]

We characterize in real time the composition and catalytic state of the initial Escherichia coli transcription-coupled repair (TCR) machinery by using correlative single-molecule methods. TCR initiates when RNA polymerase (RNAP) stalled by a lesion is displaced by the Mfd DNA translocase, thus giving repair components access to the damage. We previously used DNA nanomanipulation to obtain a nanomechanical readout of protein-DNA interactions during TCR initiation. Here we correlate this signal with simultaneous single-molecule fluorescence imaging of labeled components (RNAP, Mfd or RNA) to monitor the composition and localization of the complex. Displacement of stalled RNAP by Mfd results in loss of nascent RNA but not of RNAP, which remains associated with Mfd as a long-lived complex on the DNA. This complex translocates at ∼4 bp/s along the DNA, in a manner determined by the orientation of the stalled RNAP on the DNA. [ABSTRACT FROM AUTHOR]

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 transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution. [ABSTRACT FROM AUTHOR]

Reports that single-molecule nanomanipulation of supercoiled DNA permits measurement of spatial and temporal parameters of protein-DNA interactions that affect DNA topology. Methods involved in the process; Reagent materials needed in the process; Preparation procedure for the preparation of DNA constructs.

Focuses on a single-molecule analysis of DNA uncoiling induced by a type II topoisomerase. Use of a single Drosophila melanogaster topoisomerase II; Monitoring of DNA extension in the presence of adenosine triphosphate; Relaxation of two supercoils during a single catalytic turnover.

Recent additions to the helicase family include motor proteins that do not actually unwind DNA, but rather translocate it. By sensing short polar sequences that orient the bacterial chromosome, the FtsK helicase translocates DNA so as to align the termini of replicated chromosomes with each other, facilitating the late stages of chromosome segregation. [ABSTRACT FROM AUTHOR]

The article comments on the study on DNA replication by B. A. Berghuis, et al. within the issue which describes a high-throughput single-molecule approach to probe the nanoscale mechanical properties of the Tus-Ter protein-DNA complex. The authors cites and discuss the results of the study. They remark that the reconstitution of strand separation on a Ter site has allowed the study to reveal the intrinsic strength of the Tus-Ter lock.

A correction to the article on a model that explain the low degree of positive deoxyribonucleic acid (DNA) supercoiling that was published in the previous issue is presented.