Search

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]

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report realtime observations of cotranscriptional R-loop formation at singlemolecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation. [ABSTRACT FROM AUTHOR]

Reverse gyrases (RGs) are the only topoisomerases capable of generating positive supercoils in DNA. Members of the type IA family, they do so by generating a single-strand break in substrate DNA and then manipulating the two single strands to generate positive topology. Here, we use single-molecule experimentation to reveal the obligatory succession of steps that make up the catalytic cycle of RG. In the initial state, RG binds to DNA and unwinds ∼2 turns of the double helix in an ATP-independent fashion. Upon nucleotide binding, RG then rewinds ∼1 turn of DNA. Nucleotide hydrolysis and/or product release leads to an increase of 2 units of DNA writhe and resetting of the enzyme, for a net change of topology of +1 turn per cycle. Final dissociation of RG from DNA results in rewinding of the 2 turns of DNA that were initially disrupted. These results show how tight coupling of the helicase and topoisomerase activities allows for induction of positive supercoiling despite opposing torque. [ABSTRACT FROM AUTHOR]

Initiation is a highly regulated, rate-limiting step in transcription. We used a series of approaches to examine the kinetics of RNA polymerase (RNAP) transcription initiation in greater detail. Quenched kinetics assays, in combination with gel-based assays, showed that RNAP exit kinetics from complexes stalled at later stages of initiation (e.g., from a 7-base transcript) were markedly slower than from earlier stages (e.g., from a 2- or 4-base transcript). In addition, the RNAP-GreA endonuclease accelerated transcription kinetics from otherwise delayed initiation states. Further examination with magnetic tweezers transcription experiments showed that RNAP adopted a long-lived backtracked state during initiation and that the paused-backtracked initiation intermediate was populated abundantly at physiologically relevant nucleoside triphosphate (NTP) concentrations. The paused intermediate population was further increased when the NTP concentration was decreased and/or when an imbalance in NTP concentration was introduced (situations that mimic stress). Our results confirm the existence of a previously hypothesized paused and backtracked RNAP initiation intermediate and suggest it is biologically relevant; furthermore, such intermediates could be exploited for therapeutic purposes and may reflect a conserved state among paused, initiating eukaryotic RNA polymerase II enzymes. [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]

The article offers information related to genome sequencing which has revealed patterns of mutation demonstrating that DNA repair proteins fight damage more efficiently in some regions than others. Topics discussed include techniques that monitor DNA repair directly at a genome-wide scale, genome-wide picture of nucleotide excision repair (NER) in bacteria and patterns of NER in Escherichia coli.

Transcription-coupled repair (TCR) is one of the key nucleotide excision repair (NER) pathways required to preserve genome integrity. Although understanding TCR is still a major challenge, recent single-molecule experiments have brought new insights into the initial steps of TCR leading to new perspectives. [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]

Molecular motors have been extensively studied over the past 25 years using a range of optical microscopy methods. The specific example of RNA polymerase, responsible for transcribing the information encoded in DNA into RNA, illustrates the strength of the convergence between these recent approaches and the vast store of data collected over the past half century using the more 'classical' approaches of genetics, biochemistry, and structure elucidation. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [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.

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 KNAP-dependent DNA unwinding (with ≈1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with ≈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. [ABSTRACT FROM AUTHOR]

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.