Joseph J. Loparo

Real-time single-molecule observation of rolling-circle DNA replication

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

Local hydrogen bonding dynamics and collective reorganization in water: Ultrafast infrared spectroscopy of HOD/D2O

We present an investigation into hydrogen bonding dynamics and kinetics in water using femtosecond infrared spectroscopy of the OH stretching vibration of HOD in D(2)O. Infrared vibrational echo peak shift and polarization-selective pump-probe experiments were performed with mid-IR pulses short enough to capture all relevant dynamical processes. The experiments are self-consistently analyzed with a nonlinear response function expressed in terms of three dynamical parameters for the OH stretching vibration: the frequency correlation function, the lifetime, and the second Legendre polynomial dipole reorientation correlation function. It also accounts for vibrational-relaxation-induced excitation of intermolecular motion that appears as heating. The long time, picosecond behavior is consistent with previous work, but new dynamics are revealed on the sub-200 fs time scale. The frequency correlation function is characterized by a 50 fs decay and 180 fs beat associated with underdamped intermolecular vibrations of hydrogen bonding partners prior to 1.4 ps exponential relaxation. The reorientational correlation function observes a 50 fs librational decay prior to 3 ps diffusive reorientation. Both of these correlation functions compare favorably with the predictions from classical molecular dynamics simulations. The time-dependent behavior can be separated into short and long time scales by the 340 fs correlation time for OH frequency shifts. The fast time scales arise from dynamics that are mainly local: fluctuations in hydrogen bond distances and angles within relatively fixed intermolecular configurations. On time scales longer than the correlation time, dephasing and reorientations reflect collective reorganization of the liquid structure. Since the OH transition frequency and dipole are only weakly sensitive to these collective coordinates, this is a kinetic regime which gives an effective rate for exchange of intermolecular structures.

Characterization of spectral diffusion from two-dimensional line shapes

The analysis of line shapes in two-dimensional optical and infrared spectroscopies is a powerful approach to characterizing the dynamics of molecules in the condensed phase. Changes in line shape from diagonally elongated to symmetric as a function of waiting time arise from evolution of the transition frequency. We describe a number of quantitative measures of frequency fluctuations and spectral diffusion through the analysis of two-dimensional (2D) line shapes. These metrics are identical to the systems frequency correlation function and independent of population relaxation in the limit of a short time approximation for the 2D response. We also test the broader applicability of these expressions for analyzing three-level vibrational systems and experiments with finite pulses.

Multidimensional infrared spectroscopy of water. I. Vibrational dynamics in two-dimensional IR line shapes

In this and the following paper, we describe the ultrafast structural fluctuations and rearrangements of the hydrogen bonding network of water using two-dimensional (2D) infrared spectroscopy. 2D IR spectra covering all the relevant time scales of molecular dynamics of the hydrogen bonding network of water were studied for the OH stretching absorption of HOD in D2O. Time-dependent evolution of the 2D IR line shape serves as a spectroscopic observable that tracks how different hydrogen bonding environments interconvert while changes in spectral intensity result from vibrational relaxation and molecular reorientation of the OH dipole. For waiting times up to the vibrational lifetime of 700 fs, changes in the 2D line shape reflect the spectral evolution of OH oscillators induced by hydrogen bond dynamics. These dynamics, characterized through a set of 2D line shape analysis metrics, show a rapid 60 fs decay, an underdamped oscillation on a 130 fs time scale induced by hydrogen bond stretching, and a long time decay constant of 1.4 ps. 2D surfaces for waiting times larger than 700 fs are dominated by the effects of vibrational relaxation and the thermalization of this excess energy by the solvent bath. Our modeling based on fluctuations with Gaussian statistics is able to reproduce the changes in dispersed pump-probe and 2D IR spectra induced by these relaxation processes, but misses the asymmetry resulting from frequency-dependent spectral diffusion. The dynamical origin of this asymmetry is discussed in the companion paper.

Multidimensional infrared spectroscopy of water. II. Hydrogen bond switching dynamics

We use multidimensional infrared spectroscopy of the OH stretch of HOD in D2O to measure the interconversion of different hydrogen bonding environments. The OH stretching frequency distinguishes hydrogen bonded (HB) and non-hydrogen-bonded (NHB) configurations by their absorption on the low (red) and high (blue) sides of the line shape. Measured asymmetries in the two dimensional infrared OH line shapes are manifestations of the fundamentally different spectral relaxations of HB and NHB. HB oscillators exhibit coherent oscillations within the hydrogen-bonded free energy well before undergoing activated barrier crossing, resulting in the exchange of hydrogen bonded partners. Conversely, NHB oscillators rapidly return to HB frequencies within 150 fs. These results support a picture where NHB configurations are only visited transiently during large fluctuations about a hydrogen bond or during the switching of hydrogen bonding partners. The results are not consistent with the presence of entropically stabilized dangling hydrogen bonds or a conceptual picture of water as a mixture of environments with varying hydrogen bond strength separated by barriers >kT.

Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki fragment synthesis. Here we use single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.

Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch

Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the Notch1 regulatory switch results in sensitivity to metalloprotease cleavage, and bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction.

Proliferating Cell Nuclear Antigen Uses Two Distinct Modes to Move along DNA

Proliferating cell nuclear antigen (PCNA) plays an important role in eukaryotic genomic maintenance by topologically binding DNA and recruiting replication and repair proteins. The ring-shaped protein forms a closed circle around double-stranded DNA and is able to move along the DNA in a random walk. The molecular nature of this diffusion process is poorly understood. We use single-molecule imaging to visualize the movement of individual, fluorescently labeled PCNA molecules along stretched DNA. Measurements of diffusional properties as a function of viscosity and protein size suggest that PCNA moves along DNA using two different sliding modes. Most of the time, the clamp moves while rotationally tracking the helical pitch of the DNA duplex. In a less frequently used second mode of diffusion, the movement of the protein is uncoupled from the helical pitch, and the clamp diffuses at much higher rates.

ParB spreading requires DNA bridging

The parABS system is a widely employed mechanism for plasmid partitioning and chromosome segregation in bacteria. ParB binds to parS sites on plasmids and chromosomes and associates with broad regions of adjacent DNA, a phenomenon known as spreading. Although essential for ParB function, the mechanism of spreading remains poorly understood. Using single-molecule approaches, we discovered that Bacillus subtilis ParB (Spo0J) is able to trap DNA loops. Point mutants in Spo0J that disrupt DNA bridging are defective in spreading and recruitment of structural maintenance of chromosomes (SMC) condensin complexes in vivo. DNA bridging helps to explain how a limited number of Spo0J molecules per parS site (~20) can spread over many kilobases and suggests a mechanism by which ParB proteins could facilitate the loading of SMC complexes. We show that DNA bridging is a property of diverse ParB homologs, suggesting broad evolutionary conservation.

Simultaneous single-molecule measurements of phage T7 replisome composition and function reveal the mechanism of polymerase exchange

A complete understanding of the molecular mechanisms underlying the functioning of large, multiprotein complexes requires experimental tools capable of simultaneously visualizing molecular architecture and enzymatic activity in real time. We developed a novel single-molecule assay that combines the flow-stretching of individual DNA molecules to measure the activity of the DNA-replication machinery with the visualization of fluorescently labeled DNA polymerases at the replication fork. By correlating polymerase stoichiometry with DNA synthesis of T7 bacteriophage replisomes, we are able to quantitatively describe the mechanism of polymerase exchange. We find that even at relatively modest polymerase concentration (∼2 nM), soluble polymerases are recruited to an actively synthesizing replisome, dramatically increasing local polymerase concentration. These excess polymerases remain passively associated with the replisome through electrostatic interactions with the T7 helicase for ∼50 s until a stochastic and transient dissociation of the synthesizing polymerase from the primer-template allows for a polymerase exchange event to occur.