Bead Brownian Motion Research Articles

RecBCD Fails to Bypass the 5′-To-3′ Single-Stranded DNA Gap After Translocating Along Individual Chi-Containing Duplex DNA

The E. coli RecBCD plays an important role of initiating the repair of double-stranded DNA breaks (DSB). Translocating and recognizing chi sequence (5'-GCTGGTGG-3') have been implicated with a conformational change that enables the enzyme to preserve the 3'-to-5' single-stranded DNA for RecA assembly. RecBCD is composed of three subunits, RecB (3'-to-5' helicase), RecD (5'-to-3' helicase), and RecC. Here we used a single-molecule tethered particle motion technique to directly monitor the translocation of RecBCD along chi-contained DNA molecules. Using bead-labeled enzymes, we monitored the RecBCD translocation along individual DNA by measuring the gradual decrease in the bead Brownian motion as the enzyme moves along the DNA towards the surface. DNA substrates were designed that RecBCD would encounter a single-stranded DNA gap after the recognition of the chi sequence. While translocating along chi-free DNA substrates, the time traces showed no apparent pause, which neither 3'-to-5' nor 5'-to-3' ssDNA gap influents the movement of the enzyme1. However, over 50% of RecBCD enzymes failed to pass through the 5'-to-3' ssDNA gap after translocating over chi-containing duplex DNA. Considering RecD as a major 5'-to-3' ssDNA translocase in the RecBCD complex, our observation is consistent with the model that the conformation change occurs after chi recognition and RecD is disengaged from the 5'-ssDNA.(1) Chung, C.; Li, H. W. J. Am. Chem. Soc. 2013, 135, 8920.

Effect of Low-Pass Filtering in Force Calibration of Magnetic Tweezers

In typical experiments where magnetic tweezers are involved, precise measurement of the magnetic forces is of crucial importance. To achieve this, a widely applied method is to track the bead's Brownian motion trajectory and to calculate the force from its mean-squared-displacement. However, this method does not take into account the fact that the bead-tracking device always has a finite bandwidth, acting effectively as a low-pass filter. The result could be subjected to significant system errors, which overestimates the magnetic force. We analyze the power spectrum of the bead's Brownian motion, and provide a corrected formula to calculate the magnetic force, which is free of system errors induced by limited detection bandwidth. A dsDNA force-extension curve is experimentally measured. The curve is consistent with the WLC model, exhibiting correctness of the new formula. On the other hand, the force given by the traditional method shows significant deviation from the WLC model, which is 3 times larger at most.

Direct Observation of RecBCD Helicase as Single-Stranded DNA Translocases

The heterotrimeric Escherichia coli RecBCD enzyme comprises two helicase motors with different polarities: RecB (3'-to-5') and RecD (5'-to-3'). This superfamily I helicase is responsible for initiating DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (>200 nt) with no apparent secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt-end DNA, and translocate along the DNA substrates containing an ssDNA gap, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over a long gap in either 3'-to-5' or 5'-to-3' ssDNA direction indicates that RecBCD helicase possesses ssDNA translocase activities in both polarities. Most RecBCD active tethers showed full translocation across the ssDNA to the dsDNA region, with about 19% of enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. In addition, we prepared DNA substrates containing two opposite polarities (5'-to-3' and 3'-to-5') of ssDNA regions intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with less than 40% of tethers dissociating when entering into the second ssDNA region. These results suggest a model that RecBCD is able to switch between ssDNA translocases and rethread the other strand of ssDNA.

Direct Observation of Recbcd Helicase as ssDNA Translocases

The E. coli RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB (3’-to-5’) and RecD (5’-to-3’). This Superfamily I helicase is responsible for the initiation of DNA double-strand-break (DSB) repair in the homologous recombination pathway. We used single-molecule tethered particle motion (TPM) experiments to visualize the RecBCD helicase translocation over long single-stranded (ss) DNA (> 200 nt) without secondary structure. The bead-labeled RecBCD helicases were found to bind to the surface-immobilized blunt duplex DNA, and translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to-5’ or 5’-to-3’ direction indicates that both RecB and RecD are ssDNA translocases. We also applied continuous force (∼ 0.2 pN) to stretch DNA substrates and to remove any potentially transient looped ssDNA structure, and observed continuous translocation of RecBCD. It confirms the ssDNA translocase activities of RecBCD helicase. About 78 % of active tethers showed full translocation across the ssDNA to the dsDNA region, and the other 22 % enzymes dissociated from the ss/dsDNA junction after translocating across the ssDNA region. We also prepared a double-gapped substrate containing two regions of ssDNA with opposite polarities (5’-to-3’ and 3’-to-5’) intermitted by duplex DNA. RecBCD was able to translocate across both ssDNA regions in either ssDNA orientation orders, with 20 - 40 % of tethers dissociating while entering the second ssDNA region. These results suggest a mechanism that RecBCD is able to switch motors and rethread into the other strand after translocating along an ssDNA gapped region.

Using a Single-Molecule Method to Visualize RecBCD Helicase Translocation along Single-Stranded DNA

The E. coli RecBCD helicase initiates the repair of double strand DNA break in the homologous recombination pathway. RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB is a 3’-to- 5’ helicase and RecD is a 5’-to-3’ helicase. How RecBCD unwinds and translocates along duplex DNA is not clearly defined. Here we used a single-molecule tethered particle motion (TPM) experiment to visualize the RecBCD helicase translocation over long distance single-stranded (ss) DNA. We first prepared DNA substrates containing a > 200 nt long, unstructured ssDNA gap flanked by double-stranded DNA for RecBCD loading. In the TPM experiments, the bead-labeled, biotinylated RecBCD helicases are found to recognize and bind to the blunt, double-stranded DNA end, and successfully translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion amplitude. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to- 5’ or 5’-to-3’ direction indicates that wild-type RecBCD functions an ssDNA translocase.

Developing Single-Molecule TPM Experiments for Direct Observation of Successful RecA-Mediated Strand Exchange Reaction

RecA recombinases play a central role in homologous recombination. Once assembled on single-stranded (ss) DNA, RecA nucleoprotein filaments mediate the pairing of homologous DNA sequences and strand exchange processes. We have designed two experiments based on tethered particle motion (TPM) to investigate the fates of the invading and the outgoing strands during E. coli RecA-mediated pairing and strand exchange at the single-molecule level in the absence of force. TPM experiments measure the tethered bead Brownian motion indicative of the DNA tether length change resulting from RecA binding and dissociation. Experiments with beads labeled on either the invading strand or the outgoing strand showed that DNA pairing and strand exchange occurs successfully in the presence of either ATP or its non-hydrolyzable analog, ATPγS. The strand exchange rates and efficiencies are similar under both ATP and ATPγS conditions. In addition, the Brownian motion time-courses suggest that the strand exchange process progresses uni-directionally in the 5′-to-3′ fashion, using a synapse segment with a wide and continuous size distribution.

Actively Controlled Manipulation of a Magnetic Microbead Using Quadrupole Magnetic Tweezers

This paper presents the theoretical analysis and experimental investigation of actively controlled manipulation of a magnetic microbead using quadrupole magnetic tweezers. Bead dynamics, magnetic actuation, and visual measurement are analyzed. A feedback control law is developed and implemented to stabilize and steer the motion of the magnetic microbead. It is developed in two steps. First, an inverse model, which is associated with a lumped-parameter analytical force model, is derived to enable feedback linearization. Second, linear controllers are designed to achieve motion stabilization and manipulation of the magnetic microbead. A proportional-gain controller along with feedback linearization is implemented to establish a stable trapping of the magnetic bead to facilitate system calibration. Experiments are then performed to validate the derived inverse force model and theoretical analysis. In addition, a minimum-variance controller is designed and employed to reduce the variance of the bead's Brownian motion. The control performance in terms of variance reduction, nanostepping, and large-range steering is then experimentally demonstrated.

Investigating the Nucleation and Extension Rates of E.coli and Deinococcus RecA Along Duplex DNA

RecA is a protein which promotes the exchange between two homologous DNA molecules in homologous recombination process. When individual RecA molecules assemble on DNA, the DNA is stretched and underwound to form a nucleoprotein filament with its rigidity and end-to-end length increased. We have developed single-molecule tethered particle motion (TPM) experiments to study the assembly dynamics of RecA proteins on individual duplex DNA molecules. The TPM method is capable of measuring the changes in DNA length by observing the bead's Brownian motion, thus allowing us to monitor RecA nucleation and extension in real-time. Using much shorter DNA (a few hundreds basepairs), TPM experiments offer improved sensitivity, since the DNA length change can be readily detected as soon as a few RecA bounded to duplex DNA molecules. Our experiments indicated a faster nucleation rate compared to the previous reports (Galletto et al., 2006). Moreover, we have compared the nucleation and extension rates of E. coli RecA with the RecA from Deinococcus radiodurans, UV-resistant bacteria, under different nucleotide states, ATP and ATPγS. Deinococcus radiodurans RecA are found to nucleate faster (∼1.6×10-2 bp-1min-1) but extend slower (∼0.3-1.5 RecA/sec) under ATP.This difference reflects the physiological role of Dr. RecA when extensive UV-damaged DNA molecules are present.

Studying RecBCD Helicase Translocation Along χ-DNA Using Tethered Particle Motion with a Stretching Force

Escherichia coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments showed that RecBCD recognizes an 8 nt DNA sequence, χ, and lowers its unwinding rate afterward under saturating ATP condition. We have developed a single-molecule force-tethered particle motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in detail. In the FTPM experiment, a stretching force is applied to the DNA-bead complex that suppresses the bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean-square displacement of the bead's Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties of conventional TPM experiments, such as nonlinearity and low resolution of long DNA substrates. The FTPM method offers the best resolution in the presence of only a small stretching force (0.20 pN). We used the FTPM method to investigate RecBCD helicase motion along 4.1 kb long χ-containing duplex DNA molecules, and observed that the translocation rate of RecBCD changes after the χ sequence under limited ATP concentrations. This suggests that χ recognition by RecBCD does not require saturating ATP conditions, contrary to what was previously reported.

Studying RecBCD Helicase Translocation along Chi-DNA Using Tethered Particle Motion with a Stretching Force

E. coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments show that RecBCD recognizes an 8 nt DNA sequence, chi, and lowers its unwinding rate afterwards under saturating ATP condition. We have developed a single-molecule Force Tethered Particle Motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in details. In the FTPM experiment, a stretching force is applied to the DNA-bead complex, and suppressed bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean square displacement (MSD) of the bead Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties, such as non-linearality and low resolution of long DNA substrates in conventional TPM experiments. The FTPM method offers the best resolution (56 bp at 433 bp long DNA) in the presence of only a small stretching force (0.20 pN). We have used the FTPM method to investigate the RecBCD helicase motion along 4.1 kb long chi-containing duplex DNA molecules, and observed that translocation rate of RecBCD changes after chi sequence under limited ATP concentrations. This suggests that chi recognition by RecBCD does not require saturating ATP conditions, contrary to what have been previously reported.

Diversity in the Rates of Transcript Elongation by Single RNA Polymerase Molecules

Single-molecule measurements of the activities of a variety of enzymes show that rates of catalysis may vary markedly between different molecules in putatively homogeneous enzyme preparations. We measured the rate at which purified Escherichia coli RNA polymerase moves along a approximately 2650-bp DNA during transcript elongation in vitro at 0.5 mm nucleoside triphosphates. Individual molecules of a specifically biotinated RNA polymerase derivative were tagged with 199-nm diameter avidin-coated polystyrene beads; enzyme movement along a surface-linked DNA molecule was monitored by observing changes in bead Brownian motion by light microscopy. The DNA was derived from a naturally occurring transcription unit and was selected for the absence of regulatory sequences that induce lengthy pausing or termination of transcription. With rare exceptions, individual enzyme molecules moved at a constant velocity throughout the transcription reaction; the distribution of velocities across a population of 140 molecules was unimodal and was well fit by a Gaussian. However, the width of the Gaussian, sigma = 6.7 bp/s, was considerably larger than the precision of the velocity measurement (1 bp/s). The observations show that different transcription complexes have differences in catalytic rate (and thus differences in structure) that persist for thousands of catalytic turnovers. These differences may provide a parsimonious explanation for the complex transcription kinetics observed in bulk solution.

Tethered particle motion method for studying transcript elongation by a single RNA polymerase molecule

Schafer et al. (Nature 352:444-448 (1991)) devised the tethered particle motion (TPM) method to detect directly the movement of single, isolated molecules of a processive nucleic acid polymerase along a template DNA molecule. In TPM studies, the polymerase molecule is immobilized on a glass surface, and a particle (e.g., a 0.23 microns diameter polystyrene bead) is attached to one end of the enzyme-bound DNA molecule. Time-resolved measurements of the DNA contour length between the particle and the immobilized enzyme (the "tether length") are made by determining the magnitude of the Brownian motion of the DNA-tethered particle using light microscopy and digital image processing. We report here improved sample preparation methods that permit TPM data collection on transcript elongation by the Escherichia coli RNA polymerase at rates (approximately 10(2)-fold higher than those previously obtained) sufficient for practical use of microscopic kinetics techniques to analyze polymerase reaction mechanisms. In earlier TPM experiments, calculation of tether length from the observed Brownian motion was based on an untested numerical simulation of tethered bead Brownian motion. Using the improved methods, we have now empirically validated the TPM technique for tether lengths of 308-1915 base pairs (bp) using calibration specimens containing particles tethered by individual DNA molecules of known lengths. TPM analysis of such specimens yielded a linear calibration curve relating observed Brownian motion to tether length and allowed determination of the accuracy of the technique and measurement of how temporal bandwidth, tether length, and other experimental variables affect measurement precision. Under a standard set of experimental conditions (0.23 microns diameter bead, 0.23 Hz bandwidth, 23 degrees), accuracy is 108 and 258 bp r.m.s. at tether lengths of 308 and 1915 bp, respectively. Precision improves linearly with decreasing tether length to an extrapolated instrumentation limit of 10 bp r.m.s. and improves proportionally to the inverse square root of measurement bandwidth (1.9 x 10(2) bp Hz-1/2 for 1090-bp tethers). Measurements on large numbers of individual polymerase molecules reveal that time-averaged single-molecule elongation rates are more variable than is predicted from the random error in TPM measurements, demonstrating that the surface-immobilized RNA polymerase molecules are kinetically heterogeneous.