Single-stranded DNA Translocation Research Articles

Two residues in the DNA binding site of Pif1 helicase are essential for nuclear functions but dispensable for mitochondrial respiratory growth.

Pif1 helicase functions in both the nucleus and mitochondria. Pif1 tightly couples ATP hydrolysis, single-stranded DNA translocation, and duplex DNA unwinding. We investigated two Pif1 variants (F723A and T464A) that have each lost one site of interaction of the protein with the DNA substrate. Both variants exhibit minor reductions in affinity for DNA and ATP hydrolysis but have impaired DNA unwinding activity. However, these variants translocate on single-stranded DNA faster than the wildtype enzyme and can slide on the DNA substrate in an ATP-independent manner. This suggests they have lost their grip on the DNA, interfering with coupling ATP hydrolysis to translocation and unwinding. Yeast expressing these variants have increased gross chromosomal rearrangements, increased telomere length, and can overcome the lethality of dna2Δ, similar to phenotypes of yeast lacking Pif1. However, unlike pif1Δ mutants, they are viable on glycerol containing media and maintain similar mitochondrial DNA copy numbers as Pif1 wildtype. Overall, our data indicate that a tight grip of the trailing edge of the Pif1 enzyme on the DNA couples ATP hydrolysis to DNA translocation and DNA unwinding. This tight grip appears to be essential for the Pif1 nuclear functions tested but is dispensable for mitochondrial respiratory growth.

E. coli RecBCD nuclease domain regulates helicase activity but not single-stranded DNA translocation activity

E. coli RecBCD nuclease domain regulates helicase activity but not single-stranded DNA translocation activity

Exploring ssDNA translocation through α-hemolysin using coarse-grained steered molecular dynamics.

Protein nanopores have proven to be effective for single-molecule studies, particularly for single-stranded DNA (ssDNA) translocation. Previous experiments demonstrated their ability to distinguish differences in purine and pyrimidine bases and in the orientation of the ssDNA molecule inside nanopores. Unfortunately, the microscopic details of ssDNA translocation over experimental time scales, which are not accessible through all-atom molecular dynamics (MD), have yet to be examined. However, coarse-grained (CG) MD simulations enable systems to be simulated over longer characteristic times closer to experiments than all-atom MD. This paper studies ssDNA translocation through α-hemolysin nanopores exploiting steered MD using the MARTINI CG force field. The impacts of the sequence length, orientation inside the nanopore and DNA charges on translocation dynamics as well as the conformational dynamics of ssDNA during the translocation are explored. Our results highlight the efficacy of CG molecular dynamics in capturing the experimental properties of ssDNA translocation, including a wide distribution in translocation times per base. In particular, the phosphate charges of the DNA molecule are crucial in the translocation dynamics and impact the translocation rate. Additionally, the influence of the ssDNA molecule orientation on the translocation rate is explained by the conformational differences of ssDNA inside the nanopore during its translocation. Our study emphasizes the significance of obtaining sufficient statistics via CG MD, which can elucidate the great variety of translocation processes.

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.

Human XPD is an active helicase with low processivity and overall unwinding rate.

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. Of particular note is its role in the large multidomain complex transcription factor IIH (TFIIH). As part of TFIIH, XPD opens a bubble of ∼ 26 base pairs of DNA for access by proteins in the nucleotide excision repair pathway (NER). XPD uses the energy from ATP to translocate in the 5’ to 3’ direction on one strand of duplex DNA, displacing the opposite strand in the process. We have measured the double-stranded DNA (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD using magnetic tweezers assays. By following changes in the extension of tethered DNA molecules under force, we are able to measure the movement of individual helicase molecule on DNA in real time. Processivity, overall rate, pause position, step size, and step rates for both dsDNA unwinding and ssDNA translocation of XPD are measured and compared. The enzyme exhibits low processivity in helicase activity, with an average ∼10 bases unwound, while the translocation of the enzyme on ssDNA is more processive, with ∼30 bases translocated. This difference in processivity on dsDNA unwinding vs ssDNA translocation may be due to interactions between the displaced strand and XPD limiting the overall processivity. However, these interactions do not affect rates, as same average rate (∼0.3 nucleotides/s) for both unwinding and ssDNA translocation has been observed. This same rate also indicates that the enzyme works as an active helicase. There is no obvious correlation between pause positions and DNA sequence. The observed low unwinding processivity (lower than the expected NER repair bubble) and rate suggests that other proteins in the TFIIH complex enhance the helicase activity of XPD.

A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair.

Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5'-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5'-ssDNA, ensuring the correct ssDNA bubble size before cleavage.

The Pore-Forming Properties of SsoHel308 Helicase from Saccharolobus solfataricus

The pore-forming activity of SsoHel308 helicase from extreme thermophilic archaea Saccharolobus solfataricus has been demonstrated for the first time. This protein embedded in rabbit erythrocyte membranes may cause erythrocyte hemolysis. It has been shown that this enzyme forms pores in a planar artificial bilayer membrane and acts as a transformer. After embedding this enzyme into biolayer lipid membranes, the membrane conductivity is altered. Taken together, our results show that SsoHel308 helicase is able to form pores in artificial bilayer membranes and, in some cases, the current that flows across the membranes shares features typical of ion channels. The short lifetime of the pores in the membrane significantly reduces the toxicity of helicase for a living cell. The possibility of directed translocation of single-stranded DNA in the presence of ATP will enable the use of this enzyme as a molecular syringe for injecting single-stranded DNA into living cells.

Spontaneous Translocation of Single-Stranded DNA in Graphene-MoS2 Heterostructure Nanopores: Shape Effect.

The appropriate translocation speed of a single-stranded DNA (ssDNA) through a solid-state nanopore is crucial for DNA sequencing technologies. By studying the geometry effect of graphene-MoS2 hetero-nanopores with molecular dynamics simulations, we have found that the shape of these nanopores (circular, square, or triangular, with similar size) may have a significant effect on the spontaneous translocation of ssDNA, with the triangular nanopore showing the slowest translocation and the circular one the fastest. Further analyses reveal that such differences in the spontaneous ssDNA translocation arise from different electrostatic attractions between the positively charged Mo atoms exposed in the pore and the negatively charged phosphate groups (PO4-) in nucleotides; the "sharpness" and the total number of the exposed Mo atoms of the nanopores are responsible for different electrostatic attractions between ssDNA and the nanopore. Our findings suggest that graphene-MoS2 heterostructure nanopores with lower symmetries (i.e., having sharper corners) are capable of slowing down the ssDNA translocation, which might help better facilitate the nucleotide sensing and DNA sequencing. The conclusion from these findings might also extend to other solid-state nanopores in designing appropriate shapes for better controlling of the translocation speed.

A dual-constriction biological nanopore resolves homonucleotide sequences with high fidelity.

Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35-residue N-terminal region of its extracellular interaction partner CsgF to produce a dual-constriction pore with improved signal and basecalling accuracy for homopolymer regions. The electron cryo-microscopy structure of CsgG in complex with full-length CsgF shows that the 33 N-terminal residues of CsgF bind inside the β-barrel of the pore, forming a defined second constriction. In complexes of CsgG bound to a 35-residue CsgF constriction peptide, the second constriction is separated from the primary constriction by ~25 Å. We find that both constrictions contribute to electrical signal modulation upon ssDNA translocation. DNA sequencing using a prototype CsgG:CsgF protein pore with two constrictions improved single-read accuracy by 25 to 70 % in homopolymers up to 9 nucleotides long.

Mechanism and Kinetics of Lipid Bilayer Formation in Solid-State Nanopores.

Solid-state nanopores provide a highly versatile platform for rapid electrical detection and analysis of single molecules. Lipid bilayer coating of the nanopores can reduce nonspecific analyte adsorption to the nanopore sidewalls and increase the sensing selectivity by providing possibilities for tethering specific ligands in a cell-membrane mimicking environment. However, the mechanism and kinetics of lipid bilayer formation from vesicles remain unclear in the presence of nanopores. In this work, we used a silicon-based, truncated pyramidal nanopore array as the support for lipid bilayer formation. Lipid bilayer formation in the nanopores was monitored in real time by the change in ionic current through the nanopores. Statistical analysis revealed that a lipid bilayer is formed from the instantaneous rupture of individual vesicle upon adsorption in the nanopores, differing from the generally agreed mechanism that lipid bilayer forms at a high vesicle surface coverage on a planar support. The dependence of the lipid bilayer formation process on the applied bias, vesicle size, and concentration was systematically studied. In addition, the nonfouling properties of the lipid bilayer coated nanopores were demonstrated during long single-stranded DNA translocation through the nanopore array. The findings indicate that the lipid bilayer formation process can be modulated by introducing nanocavities intentionally on the planar surface to create active sites or changing the vesicle size and concentration.

Molecular Dynamics Simulation Study of Transverse and Longitudinal Ionic Currents in Solid-State Nanopore DNA Sequencing

We report all-atom molecular dynamics (MD) simulations of single-stranded DNA (ssDNA) translocation in 1 M KCl solution through a silicon nitride solid-state nanopore with one or two nanochannels p...

ПОРООБРАЗУЮЩИЕ СВОЙСТВА ХЕЛИКАЗЫ SSOHEL308 ИЗ SACCHAROLOBUS SOLFATARICUS

The pore-forming activity of SsoHel308 helicase from extreme thermophilic archaea Saccharolobus solfataricus has been demonstrated for the first time. This protein, embedded into rabbit erythrocyte membranes, may cause erythrocyte hemolysis. It has been shown that this enzyme forms pores in a flat artificial bilayer membrane and acts as the transformer. After embedding this enzyme into bilayer lipid membranes, the membrane conductivity alters. Taken together, our results show that SsoHel308 helicase is able to form pores in artificial bilayer membranes and, in some cases, the current that flows across the membranes shares features typical of ion channels. The short lifetime of the pores in the membrane significantly reduces the toxicity of helicase for a living cell, and the possibility of directed translocation of single-stranded DNA in the presence of ATP will enable the use of this enzyme as a molecular syringe for injecting single-stranded DNA into living cells.

Single-Stranded DNA Translocation Recordings through Solid-State Nanopores on Glass Chips at 10 MHz Measurement Bandwidth.

Accurate and low-cost analysis of biomolecules is important for many applications. This work seeks to further improve the measurement bandwidths achievable with solid-state nanopores, which have emerged as an important platform for this analysis. We report single-stranded DNA translocation recordings at a bandwidth of 10 MHz copolymers of 80 (C20A20C20A20), 90 (C30A30C30), and 200 (C50A50C50A50) nucleotides through Si nanopores with effective diameters of 1.4-2.1 nm and effective membrane thicknesses 0.5-8.9 nm. By optimizing glass chips with thin nanopores and by integrating them with custom-designed amplifiers based on complementary metal-oxide-semiconductor technology, this work demonstrates detection of translocation events as brief as 100 ns with a signal-to-noise ratio exceeding seven at a measurement bandwidth of 10 MHz. We also report data robustness and variability across 13 pores of similar size and thickness, yielding a current blockade between 30 and 60% with a mean ionic current blockade (ΔI) of ∼3-9 nA and a characteristic dwell time of ∼2-21 ns per nucleotide. These measurements show that characteristic translocation rates are at least 10 times faster than previously recorded. We detect transient intraevent fluctuations, multiple current levels within translocation events, and variability of DNA translocation event signatures and durations.

Revealing the mechanism of DNA passing through graphene and boron nitride nanopores.

Nanopores on 2D materials have great potential for DNA sequencing, which is attributed to their high sequencing speed and reduced cost. However, identifying DNA bases at such a high speed with nanometer precision has remained a big challenge. Here, we implemented theoretical calculations to show the translocation of single-stranded DNA (ssDNA) through solid-state nanopores on a 2D hexagonal boron nitride (h-BN) and graphene sheet. A base-specific ssDNA sequencing technique was devised, based on the individual differences in the ion current responses for the (polyA)16, (polyG)16, (polyC)16, and (polyT)16 bases of ssDNA. Our sequential procedure for sequencing is built on a comparative approach between the current signals obtained from the nanopores to achieve base-specific detection. Our results indicate that at higher voltages (1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 V nm-1), DNA translocation is tracked though the 1.5 and 2.0 nm nanopores, and at the 1.5 nm pore size, folded ssDNA close to the nanopore accounts for 93% and 81% of events for graphene and h-BN. Our calculations indicate charge transfer from the graphene to ssDNA, while the reverse happens in the case of the h-BN membrane. These results provide critical insights into our understanding of single molecule sequencing through solid-state nanopore research.

Generation and Single-Molecule Characterization of a Sequence-Selective Covalent Cross-Link Mediated by Mechlorethamine at a C–C Mismatch in Duplex DNA for Discrimination of a Disease-Relevant Single Nucleotide Polymorphism

Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C-C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target complex. Cross-linked DNA generates an unmistakable deep and persistent current block (≥5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.

Active Transport by a Membrane Embedded Biomotor Nanopore

Helicase plays a vital role in all living organisms to unpackage genes. Its structure and function have been studied by various methodology. Molecular transport through nanoscale channels including membrane channels, biological and artificial nanopores are of great importance in several key biological processes. Both biological and artificial nanopores have been constructed and reconstituted into planar lipid bilayer for biosensing and DNA sequencing. However, most of the DNA and molecule translocation through those pores are passive transport aided by external electrical field force. Here we report that a helicase from single-stranded DNA bacteriophage with ring shape can be reengineered and inserted into planar lipid bilayers. We designed this novel biological nanopore with the capability of active DNA transport fueled by the chemical energy from ATP hydrolysis. The membrane-embedded helicase nanopore acts as a conductive channel to allow the translocation of single-stranded DNA, while still retain the helicase activity to unwind double-stranded DNA on membrane. The helicase activity could be inhibited by removal of Mg2+ or ATP in vitro. DNA with abasic furans can be distinguished during transmembrane-unwinding process. This 1.3 nm-diameter channel can also form pores in live cell membranes. This helicase nanopore is expected to provide an interesting tool to study motor activity at single-molecule level, and have potential applications in nanotechnology and nanomedicine.

Modulation of Escherichia coli UvrD Single-Stranded DNA Translocation by DNA Base Composition

Escherichia coli UvrD is an SF1A DNA helicase/translocase that functions in chromosomal DNA repair and replication of some plasmids. UvrD can also displace proteins such as RecA from DNA in its capacity as an anti-recombinase. Central to all of these activities is its ATP-driven 3′–5′ single-stranded (ss) DNA translocation activity. Previous ensemble transient kinetic studies have estimated the average translocation rate of a UvrD monomer on ssDNA composed solely of deoxythymidylates. Here we show that the rate of UvrD monomer translocation along ssDNA is influenced by DNA base composition, with UvrD having the fastest rate along polypyrimidines although decreasing nearly twofold on ssDNA containing equal amounts of the four bases. Experiments with DNA containing abasic sites and polyethylene glycol spacers show that the ssDNA base also influences translocation processivity. These results indicate that changes in base composition and backbone insertions influence the translocation rates, with increased ssDNA base stacking correlated with decreased translocation rates, supporting the proposal that base-stacking interactions are involved in the translocation mechanism.

Processivity of nucleic acid unwinding and translocation by helicases.

Helicases are a class of enzymes that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid (NA) strand separation. Besides the NA unwinding speed, another important factor for the helicase activity is the NA unwinding processivity. Here, we study the NA unwinding processivity with an analytical model that captures the phenomenology of the NA unwinding process. First, we study the processivity of the non-hexameric helicase that can unwind NA efficiently in the form of a monomer and the processivity of the hexameric helicase that can unwind DNA effectively, providing quantitative explanations of the available single-molecule experimental data. Then, we study the processivity of the non-hexameric helicases, in particular UvrD, in the form of a dimer and compare with that in the form of a monomer. The available single-molecule and some biochemical data showing that while UvrD monomer is a highly processive single-stranded DNA translocase it is inactive in DNA unwinding, whereas other biochemical data showing that UvrD is active in both single-stranded DNA translocation and DNA unwinding in the form of a monomer can be explained quantitatively and consistently. In addition, the recent single-molecule data are also explained quantitatively showing that constraining the 2B subdomain in closed conformation by intramolecular cross-linking can convert Rep monomer with a very poor DNA unwinding activity into a superhelicase that can unwind more than thousands of DNA base pairs processively, even against a large opposing force. Proteins 2016; 84:1590-1605. © 2016 Wiley Periodicals, Inc.

Processive DNA Unwinding by RecBCD Helicase in the Absence of Canonical Motor Translocation

Escherichia coli RecBCD is a DNA helicase/nuclease that functions in double-stranded DNA break repair. RecBCD possesses two motors (RecB, a 3′ to 5′ translocase, and RecD, a 5′ to 3′ translocase). Current DNA unwinding models propose that motor translocation is tightly coupled to base pair melting. However, some biochemical evidence suggests that DNA melting of multiple base pairs may occur separately from single-stranded DNA translocation. To test this hypothesis, we designed DNA substrates containing reverse backbone polarity linkages that prevent ssDNA translocation of the canonical RecB and RecD motors. Surprisingly, we find that RecBCD can processively unwind DNA for at least 80bp beyond the reverse polarity linkages. This ability requires an ATPase active RecB motor, the RecB “arm” domain, and also the RecB nuclease domain, but not its nuclease activity. These results indicate that RecBCD can unwind duplex DNA processively in the absence of ssDNA translocation by the canonical motors and that the nuclease domain regulates the helicase activity of RecBCD.

Stepwise Transport of Stretched ssDNA Through Graphene Nanopores

Among various biological and solid-state nanopores employed for DNA sensing, graphene nanopores have the greatest potential to be successful for DNA sequencing because graphene's single-atom-thin layer may offer single-base resolution recognition. However, the identification of individual bases could not be achieved yet by means of graphene nanopores, mainly because DNA translocation through the pore is too fast and thermal motion of bases is too strong to permit individual bases to be resolved. Here we demonstrate by means of molecular dynamics simulations that stretch-induced stepwise translocation of single-stranded DNA (ssDNA) through graphene nanopores can permit single-base resolution detection. The intrinsic stepwise DNA motion is brought about through alternating conformational changes between spontaneous adhesion of DNA bases to the rim of the graphene nanopore and unbinding due to mechanical force or electric field. A graphene membrane shaped as a quantum point contact permits, by means of transverse electronic conductance measurement, detection of the stepwise translocation of the DNA as predicted through quantum mechanical Green's function-based transport calculations. The measurement scheme described opens a route to enhance the signal-to-noise ratio by not only slowing down DNA translocation to provide sufficient time for base recognition, but also by stabilizing single DNA bases and, thereby, reducing thermal noise.