Characterization of human XPD helicase activity with single-molecule magnetic tweezers.

XPD helicase is a DNA-unwinding enzyme involved in DNA repair. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5' to 3' direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA unwinding and single-stranded DNA translocation activities of human XPD in isolation. In our experimental setup, hXPD exhibited low unwinding processivity of ∼14 bp and slow unwinding rate of ∼0.3 bp/s. Measurements of the ssDNA translocation activity demonstrated that hXPD translocated on ssDNA at a similar rate as unwinding, revealing that slow rate was an intrinsic property of the hXPD translocation. Individual unwinding and translocation events were composed of pauses and runs with a distribution of lengths and rates. Analysis of these events unveiled similar mean run lengths and rates for unwinding and translocation, indicating that the unwinding behavior was a direct reflection of the translocation activity. The analysis also revealed that hXPD spent similar time stalling and unwinding/translocating. The detailed basal activity of hXPD reported here provides a baseline for future studies on how hXPD activity is regulated by other TFIIH components.

Characterization of human XPD helicase activity with Single Molecule Magnetic Tweezers.

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5-prime to 3-prime direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD by itself. In our experimental setup, hXPD exhibits low unwinding processivity of ~14 bp and slow overall unwinding rate of ~0.3 bp/s. Individual unwinding and translocation events were composed of fast and slow runs and pauses. Analysis of these events gave similar mean run sizes and rates for unwinding and translocation, suggesting that unwinding is a reflection of translocation. The analysis also revealed that hXPD spent similar time stalling and unwinding. hXPD translocated on ssDNA at a similar overall rate as that of unwinding, pointing to an active helicase. However, we observed modest effects of DNA sequence on stalling and unwinding initiation position. Considering the slow unwinding rate, high probability of base pair separation at the ssDNA/dsDNA fork, and the observed DNA sequence dependences, we propose that hXPD is most likely a partially active helicase. Our results provide detailed information on the basal activity of hXPD which enhances our mechanistic understanding of hXPD activity.

Tropical forests post-logging are a persistent net carbon source to the atmosphere.

Logged and structurally degraded tropical forests are fast becoming one of the most prevalent land-use types throughout the tropics and are routinely assumed to be a net carbon sink because they experience rapid rates of tree regrowth. Yet this assumption is based on forest biomass inventories that record carbon stock recovery but fail to account for the simultaneous losses of carbon from soil and necromass. Here, we used forest plots and an eddy covariance tower to quantify and partition net ecosystem CO2 exchange in Malaysian Borneo, a region that is a hot spot for deforestation and forest degradation. Our data represent the complete carbon budget for tropical forests measured throughout a logging event and subsequent recovery and found that they constitute a substantial and persistent net carbon source. Consistent with existing literature, our study showed a significantly greater woody biomass gain across moderately and heavily logged forests compared with unlogged forests, but this was counteracted by much larger carbon losses from soil organic matter and deadwood in logged forests. We estimate an average carbon source of 1.75 ± 0.94 Mg C ha-1 yr-1 within moderately logged plots and 5.23 ± 1.23 Mg C ha-1 yr-1 in unsustainably logged and severely degraded plots, with emissions continuing at these rates for at least one-decade post-logging. Our data directly contradict the default assumption that recovering logged and degraded tropical forests are net carbon sinks, implying the amount of carbon being sequestered across the world's tropical forests may be considerably lower than currently estimated.

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion.

Helicases are essential for nearly all nucleic acid processes across the tree of life, yet detailed understanding of how they couple ATP hydrolysis to translocation and unwinding remains incomplete because their small (∼300 picometer), fast (∼1 ms) steps are difficult to resolve. Here, we use Nanopore Tweezers to observe single Escherichia coli RecQ helicases as they translocate on and unwind DNA at ultrahigh spatiotemporal resolution. Nanopore Tweezers simultaneously resolve individual steps of RecQ along the DNA and conformational changes of the helicase associated with stepping. Our data reveal the mechanochemical coupling between physical domain motions and chemical reactions that together produce directed motion of the helicase along DNA. Nanopore Tweezers measurements are performed under either assisting or opposing force applied directly on RecQ, shedding light on how RecQ responds to such forces in vivo. Determining the rates of translocation and physical conformational changes under a wide range of assisting and opposing forces reveals the underlying dynamic energy landscape that drives RecQ motion. We show that RecQ has a highly asymmetric energy landscape that enables RecQ to maintain velocity when encountering molecular roadblocks such as bound proteins and DNA secondary structures. This energy landscape also provides a mechanistic basis making RecQ an 'active helicase,' capable of unwinding dsDNA as fast as it translocates on ssDNA. Such an energy landscape may be a general strategy for molecular motors to maintain consistent velocity despite opposing loads or roadblocks.

Direct observation of topoisomerase IA gate dynamics.

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although type IA topoisomerases are assumed to accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the Escherichia coli type IA topoisomerases I and III. We found that after cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation, and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allowed us to develop a mechanistic model of interactions between type IA topoisomerases and single-stranded DNA.

RecQ helicase triggers a binding mode change in the SSB-DNA complex to efficiently initiate DNA unwinding.

The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.

Functional mechanics of beetle mandibles: Honest signaling in a sexually selected system.

Male stag beetles possess colossal mandibles, which they wield in combat to obtain access to females. As with many other sexually selected weapons, males with longer mandibles win more fights. However, variation in the functional morphology of these structures, used in male-male combat, is less well understood. In this study, mandible bite force, gape, structural strength, and potential tradeoffs are examined across a wide size range for one species of stag beetle, Cyclommatus metallifer. We found that not only does male mandible size demonstrate steep positive allometry, but the shape, relative bite force, relative gape, and safety factor of the mandibles also change with male size. Allometry in these functionally important mandibular traits suggests that larger males with larger mandibles should be better fighters, and that the mandibles can be considered an honest signal of male fighting ability. However, negative allometry in mandible structural safety factor, wing size, and flight muscle mass suggest significant costs and a possible limit on the size of the mandibles. J. Exp. Zool. 325A:3-12, 2016. © 2015 Wiley Periodicals, Inc.

A heterotrimer model of the complete Microprocessor complex revealed by single-molecule subunit counting.

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stem-loop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

Attractive hydration forces in DNA-dendrimer interactions on the nanometer scale.

The energetic contribution of attractive hydration forces arising from water ordering is an interesting but often neglected aspect of macromolecular interactions. Ordering effects of water can bring about cooperativity in many intermolecular transactions, in both the short and long range. Given its high charge density, this is of particular importance for DNA. For instance, in nanotechnology, highly charged dendrimers are used for DNA compaction and transfection. Hypothesizing that water ordering and hydration forces should be maximal for DNA complexes that show charge complementarity (positive-negative), we present here an analysis of water ordering from molecular dynamics simulations and free energy calculations of the interaction between DNA and a nanoparticle with a high positive charge density. Our results indicate not only that complexation of the dendrimer with DNA affects the local water structure but also that ordered water molecules facilitate long-range interactions between the molecules. This contributes significantly to the free energy of binding of dendrimers to DNA and extends the interaction well beyond the electrostatic range of the DNA. Such water effects are of potentially substantial importance in cases when molecules appear to recognize each other across sizable distances, or for which kinetic rates are too fast to be due to pure diffusion. Our results are in good agreement with experiments on the role of solvent in DNA condensation by multivalent cations and exemplify a microscopic realization of mean-field phenomenological theories for hydration forces between mesoscopic surfaces.

Microscopic basis for the mesoscopic extensibility of dendrimer-compacted DNA.

The mechanism of DNA compaction by dendrimers is key to the design of nanotechnologies that can deliver genetic material into cells. We present atomistic simulations, mesoscopic modeling and single-molecule pulling experiments describing DNA dendrimer interactions. All-atom molecular dynamics were used to characterize pulling-force-dependent interactions between DNA and generation-3 PAMAM amine-terminated dendrimers, and a free energy profile and mean forces along the interaction coordinate are calculated. The energy, force, and geometry parameters computed at the atomic level are input for a Monte Carlo model yielding mesoscopic force-extension curves. Actual experimental single-molecule curves obtained with optical tweezers are also presented, and they show remarkable agreement with the virtual curves from our model. The calculations reveal the microscopic origin of the hysteresis observed in the phase transition underlying compaction. A broad range of ionic and pulling parameters is sampled, and suggestions for windows of conditions to probe new single-molecule behavior are made.

An experimentally guided umbrella sampling protocol for biomolecules.

We present a simple method for utilizing experimental data to improve the efficiency of numerical calculations of free energy profiles from molecular dynamics simulations. The method involves umbrella sampling simulations with restraining potentials based on a known approximate estimate of the free energy profile derived solely from experimental data. The use of the experimental data results in optimal restraining potentials, guides the simulation along relevant pathways, and decreases overall computational time. In demonstration of the method, two systems are showcased. First, guided, unguided (regular) umbrella sampling simulations and exhaustive sampling simulations are compared to each other in the calculation of the free energy profile for the distance between the ends of a pentapeptide. The guided simulation use restraints based on a simulated "experimental" potential of mean force of the end-to-end distance that would be measured by fluorescence resonance energy transfer (obtained from exhaustive sampling). Statistical analysis shows a dramatic improvement in efficiency for a 5 window guided umbrella sampling over 5 and 17 window unguided umbrella sampling simulations. Moreover, the form of the potential of mean force for the guided simulations evolves, as one approaches convergence, along the same milestones as the extensive simulations, but exponentially faster. Second, the method is further validated by replicating the forced unfolding pathway of the titin I27 domain using guiding umbrella sampling potentials determined from actual single molecule pulling data. Comparison with unguided umbrella sampling reveals that the use of guided sampling encourages unfolding simulations to converge faster to a forced unfolding pathway that agrees with previous results and produces a more accurate potential of mean force.