Single-molecule characterization of Sen1 translocation properties provides insights into eukaryotic factor-dependent transcription termination.

Sen1 is an essential helicase for factor-dependent transcription termination in Saccharomyces cerevisiae, whose molecular-motor mechanism has not been well addressed. Here, we use single-molecule experimentation to better understand the molecular-motor determinants of its action on RNA polymerase II (Pol II) complex. We quantify Sen1 translocation activity on single-stranded DNA (ssDNA), finding elevated translocation rates, high levels of processivity and ATP affinities. Upon deleting the N- and C-terminal domains, or further deleting different parts of the prong subdomain, which is an essential element for transcription termination, Sen1 displays changes in its translocation properties, such as slightly reduced translocation processivities, enhanced translocation rates and statistically identical ATP affinities. Although these parameters fulfil the requirements for Sen1 translocating along the RNA transcript to catch up with a stalled Pol II complex, we observe significant reductions in the termination efficiencies as well as the factions of the formation of the previously described topological intermediate prior to termination, suggesting that the prong may preserve an interaction with Pol II complex during factor-dependent termination. Our results underscore a more detailed rho-like mechanism of Sen1 and a critical interaction between Sen1 and Pol II complex for factor-dependent transcription termination in eukaryotes.

On the stability of stalled RNA polymerase and its removal by RapA.

Stalling of the transcription elongation complex formed by DNA, RNA polymerase (RNAP) and RNA presents a serious obstacle to concurrent processes due to the extremely high stability of the DNA-bound polymerase. RapA, known to remove RNAP from DNA in an ATP-dependent fashion, was identified over 50 years ago as an abundant binding partner of RNAP; however, its mechanism of action remains unknown. Here, we use single-molecule magnetic trapping assays to characterize RapA activity and begin to specify its mechanism of action. We first show that stalled RNAP resides on DNA for times on the order of 106 seconds and that increasing positive torque on the DNA reduces this lifetime. Using stalled RNAP as a substrate we show that the RapA protein stimulates dissociation of stalled RNAP from positively supercoiled DNA but not negatively supercoiled DNA. We observe that RapA-dependent RNAP dissociation is torque-sensitive, is inhibited by GreB and depends on RNA length. We propose that stalled RNAP is dislodged from DNA by RapA via backtracking in a supercoiling- and torque-dependent manner, suggesting that RapA's activity on transcribing RNAP in vivo is responsible for resolving conflicts between converging polymerase molecular motors.

Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA.

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 real-time observations of cotranscriptional R-loop formation at single-molecule 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.

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds.

Single-molecule data are of great significance in biology, chemistry, and medicine. However, new experimental tools to characterize, in a multiplexed manner, protein bond rupture under force are still needed. Acoustic force spectroscopy is an emerging manipulation technique which generates acoustic waves to apply force in parallel on multiple microbeads tethered to a surface. We here exploit this configuration in combination with the recently developed modular junctured-DNA scaffold that has been designed to study protein-protein interactions at the single-molecule level. By applying repetitive constant force steps on the FKBP12-rapamycin-FRB complex, we measure its unbinding kinetics under force at the single-bond level. Special efforts are made in analyzing the data to identify potential pitfalls. We propose a calibration method allowing in situ force determination during the course of the unbinding measurement. We compare our results with well-established techniques, such as magnetic tweezers, to ensure their accuracy. We also apply our strategy to study the force-dependent rupture of a single-domain antibody with its antigen. Overall, we get a good agreement with the published parameters that have been obtained at zero force and population level. Thus, our technique offers single-molecule precision for multiplexed measurements of interactions of biotechnological and medical interest.

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis.

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku. These observations are in contrast to human Ku, which is unable to bridge either of the two DNA substrates. We also demonstrate that bacterial Ku binds the DNA ends in a cooperative manner for synapsis initiation and remains stably bound at DNA junctions for several hours after ligation is completed, indicating that a system for removal of the proteins is active in vivo. Together these experiments shed light on the dynamics of bacterial NHEJ in DNA end recognition and processing. We speculate on the evolutionary similarities between bacterial and eukaryotic NHEJ and discuss how an increased understanding of bacterial NHEJ can open the door for future antibiotic therapies targeting this mechanism.

Direct observation of helicase-topoisomerase coupling within reverse gyrase.

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.

Reconstruction of bacterial transcription-coupled repair at single-molecule resolution.

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 site. 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 attached. 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 Mfd­RNAP than to DNA damage. These observations provide a quantitative framework for comparing complementary DNA repair pathways in vivo.

Mechanism of efficient double-strand break repair by a long non-coding RNA.

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair.

DNA mismatch repair (MMR) maintains genome stability through repair of DNA replication errors. In Escherichia coli, initiation of MMR involves recognition of the mismatch by MutS, recruitment of MutL, activation of endonuclease MutH and DNA strand incision at a hemimethylated GATC site. Here, we studied the mechanism of communication that couples mismatch recognition to daughter strand incision. We investigated the effect of catalytically-deficient Cas9 as well as stalled RNA polymerase as roadblocks placed on DNA in between the mismatch and GATC site in ensemble and single molecule nanomanipulation incision assays. The MMR proteins were observed to incise GATC sites beyond a roadblock, albeit with reduced efficiency. This residual incision is completely abolished upon shortening the disordered linker regions of MutL. These results indicate that roadblock bypass can be fully attributed to the long, disordered linker regions in MutL and establish that communication during MMR initiation occurs along the DNA backbone.

TopA, the Sulfolobus solfataricus topoisomerase III, is a decatenase.

DNA topoisomerases are essential enzymes involved in all the DNA processes and among them, type IA topoisomerases emerged as a key actor in the maintenance of genome stability. The hyperthermophilic archaeon, Sulfolobus solfataricus, contains three topoisomerases IA including one classical named TopA. SsoTopA is very efficient at unlinking DNA catenanes, grouping SsoTopA into the topoisomerase III family. SsoTopA is active over a wide range of temperatures and at temperatures of up to 85°C it produces highly unwound DNA. At higher temperatures, SsoTopA unlinks the two DNA strands. Thus depending on the temperature, SsoTopA is able to either prevent or favor DNA melting. While canonical topoisomerases III require a single-stranded DNA region or a nick in one of the circles to decatenate them, we show for the first time that a type I topoisomerase, SsoTopA, is able to efficiently unlink covalently closed catenanes, with no additional partners. By using single molecule experiments we demonstrate that SsoTopA requires the presence of a short single-stranded DNA region to be efficient. The unexpected decatenation property of SsoTopA probably comes from its high ability to capture this unwound region. This points out a possible role of TopA in S. solfataricus as a decatenase in Sulfolobus.

Eeny meeny miny moe, catch a transcript by the toe, or how to enumerate eukaryotic transcripts.

In this issue of Genes & Development, Revyakin and colleagues (pp. 1691-1702) measure the relation between individual RNA polymerase II transcription events and transcription factor assembly by counting RNA transcripts retained on the template DNA using single-molecule fluorescence.

C-terminal lysine repeats in Streptomyces topoisomerase I stabilize the enzyme-DNA complex and confer high enzyme processivity.

Streptomyces topoisomerase I (TopA) exhibits exceptionally high processivity. The enzyme, as other actinobacterial topoisomerases I, differs from its bacterial homologs in its C-terminal domain (CTD). Here, bioinformatics analyses established that the presence of lysine repeats is a characteristic feature of actinobacterial TopA CTDs. Streptomyces TopA contains the longest stretch of lysine repeats, which terminate with acidic amino acids. DNA-binding studies revealed that the lysine repeats stabilized the TopA-DNA complex, while single-molecule experiments showed that their elimination impaired enzyme processivity. Streptomyces coelicolor TopA processivity could not be restored by fusion of its N-terminal domain (NTD) with the Escherichia coli TopA CTD. The hybrid protein could not re-establish the distribution of multiple chromosomal copies in Streptomyces hyphae impaired by TopA depletion. We expected that the highest TopA processivity would be required during the growth of multigenomic sporogenic hyphae, and indeed, the elimination of lysine repeats from TopA disturbed sporulation. We speculate that the interaction of the lysine repeats with DNA allows the stabilization of the enzyme-DNA complex, which is additionally enhanced by acidic C-terminal amino acids. The complex stabilization, which may be particularly important for GC-rich chromosomes, enables high enzyme processivity. The high processivity of TopA allows rapid topological changes in multiple chromosomal copies during Streptomyces sporulation.

Backtracked and paused transcription initiation intermediate of Escherichia coli RNA polymerase.

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.

Initiation of transcription-coupled repair characterized at single-molecule resolution.

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.

Simple calibration of TIR field depth using the supercoiling response of DNA.

The combination of single-molecule fluorescence and nanomanipulation techniques into a single experimental platform enables one to carry out correlative analysis of the composition and the activity of complex, multicomponent molecular systems. Here we describe implementation and calibration of such a combined system allowing simultaneous single-molecule force spectroscopy and fluorescence imaging of proteins acting on the DNA using magnetic trapping coupled with fluorescence excitation based on a Total Internal Reflection (TIR), or evanescent, field. We propose a simple and robust in situ method for calibration of the TIR field depth against the mechanical properties of nanomanipulated DNA, and which is made possible by the fact that the magnetic bead used to trap and nanomanipulate DNA and measure its conformation also exhibits autofluorescence in the TIR field. Indeed, the fact that the bead size is on the 1-micron scale does not preclude sensitive probing of an intensity field which decays exponentially on the 0.1micron-scale. We demonstrate the usefulness of this approach by mapping out TIR field depth as a function of the angle of incidence of the illuminating laser at the glass-water interface and showing that one recovers the expected theoretical relationship between field depth and angle of incidence.

A highly processive topoisomerase I: studies at the single-molecule level.

Amongst enzymes which relieve torsional strain and maintain chromosome supercoiling, type IA topoisomerases share a strand-passage mechanism that involves transient nicking and re-joining of a single deoxyribonucleic acid (DNA) strand. In contrast to many bacterial species that possess two type IA topoisomerases (TopA and TopB), Actinobacteria possess only TopA, and unlike its homologues this topoisomerase has a unique C-terminal domain that lacks the Zn-finger motifs characteristic of type IA enzymes. To better understand how this unique C-terminal domain affects the enzyme's activity, we have examined DNA relaxation by actinobacterial TopA from Streptomyces coelicolor (ScTopA) using real-time single-molecule experiments. These studies reveal extremely high processivity of ScTopA not described previously for any other topoisomerase of type I. Moreover, we also demonstrate that enzyme processivity varies in a torque-dependent manner. Based on the analysis of the C-terminally truncated ScTopA mutants, we propose that high processivity of the enzyme is associated with the presence of a stretch of positively charged amino acids in its C-terminal region.

Biology, one molecule at a time.

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.

Use of DNA forceps to measure receptor-ligand dissociation equilibrium constants in a single-molecule competition assay.

The ability of biophysicists to decipher the behavior of individual biomolecules has steadily improved over the past thirty years. However, it still remains unclear how an ensemble of data acquired at the single-molecule level compares with the data acquired on an ensemble of the same molecules. We here propose an assay to tackle this question in the context of dissociation equilibrium constant measurements. A sensor is built by engrafting a receptor and a ligand onto a flexible dsDNA scaffold and mounting this assembly on magnetic tweezers. This way, looking at the position of the magnetic bead enables one to determine in real-time if the two molecular partners are associated or not. Next, to quantify the affinity of the scrutinized single-receptor for a given competitor, various amounts of the latter molecule are introduced in solution and the equilibrium response of the sensor is monitored throughout the titration protocol. Proofs of concept are established for the binding of three rapamycin analogs to the FKBP12 cis-trans prolyl isomerase. For each of these drugs the mean affinity constant obtained on a ten of individual receptors agrees with the one previously determined in a bulk assay. Furthermore, experimental contingencies are sufficient to explain the dispersion observed over the single-molecule values.