Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB.

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.

Understanding the paradoxical mechanical response of in-phase A-tracts at different force regimes.

A-tracts are A:T rich DNA sequences that exhibit unique structural and mechanical properties associated with several functions in vivo. The crystallographic structure of A-tracts has been well characterized. However, the mechanical properties of these sequences is controversial and their response to force remains unexplored. Here, we rationalize the mechanical properties of in-phase A-tracts present in the Caenorhabditis elegans genome over a wide range of external forces, using single-molecule experiments and theoretical polymer models. Atomic Force Microscopy imaging shows that A-tracts induce long-range (∼200 nm) bending, which originates from an intrinsically bent structure rather than from larger bending flexibility. These data are well described with a theoretical model based on the worm-like chain model that includes intrinsic bending. Magnetic tweezers experiments show that the mechanical response of A-tracts and arbitrary DNA sequences have a similar dependence with monovalent salt supporting that the observed A-tract bend is intrinsic to the sequence. Optical tweezers experiments reveal a high stretch modulus of the A-tract sequences in the enthalpic regime. Our work rationalizes the complex multiscale flexibility of A-tracts, providing a physical basis for the versatile character of these sequences inside the cell.

Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis.

Genome replication induces the generation of large stretches of single-stranded DNA (ssDNA) intermediates that are rapidly protected by single-stranded DNA-binding (SSB) proteins. To date, the mechanism by which tightly bound SSBs are removed from ssDNA by the lagging strand DNA polymerase without compromising the advance of the replication fork remains unresolved. Here, we aimed to address this question by measuring, with optical tweezers, the real-time replication kinetics of the human mitochondrial and bacteriophage T7 DNA polymerases on free-ssDNA, in comparison with ssDNA covered with homologous and non-homologous SSBs under mechanical tension. We find important differences between the force dependencies of the instantaneous replication rates of each polymerase on different substrates. Modeling of the data supports a mechanism in which strong, specific polymerase-SSB interactions, up to ∼12 kBT, are required for the polymerase to dislodge SSB from the template without compromising its instantaneous replication rate, even under stress conditions that may affect SSB-DNA organization and/or polymerase-SSB communication. Upon interaction, the elimination of template secondary structure by SSB binding facilitates the maximum replication rate of the lagging strand polymerase. In contrast, in the absence of polymerase-SSB interactions, SSB poses an effective barrier for the advance of the polymerase, slowing down DNA synthesis.

Direct observation of OAM correlations from spatially entangled bi-photon states.

We present spatially-resolved observations of orbital angular momentum (OAM) conservation, via a Laguerre-Gauss (LG) basis decomposition, of spatially-entangled photon pairs produced in type-I collinear spontaneous parametric downconversion (SPDC). These results were obtained with a novel detection system for OAM-entangled photon pairs that combines a projective measurement for the signal photon to a specific value of the azimuthal index ls, with a spatially-resolved measurement for the idler photon using an intensified charge coupled (ICCD) camera. In combination with far-field diffraction of the idler photon through a triangular aperture, we are able to obtain: i) the spatial structure of the heralded idler photon, as governed by the user-selected topological charge of the signal photon; ii) the OAM spectrum; and iii) the topological charge (both magnitude and sign) for the heralded idler photon.

DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein.

Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB-DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during 'in situ' DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance.

Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase.

During DNA replication replicative polymerases move in discrete mechanical steps along the DNA template. To address how the chemical cycle is coupled to mechanical motion of the enzyme, here we use optical tweezers to study the translocation mechanism of individual bacteriophage Phi29 DNA polymerases during processive DNA replication. We determine the main kinetic parameters of the nucleotide incorporation cycle and their dependence on external load and nucleotide (dNTP) concentration. The data is inconsistent with power stroke models for translocation, instead supports a loose-coupling mechanism between chemical catalysis and mechanical translocation during DNA replication. According to this mechanism the DNA polymerase works by alternating between a dNTP/PPi-free state, which diffuses thermally between pre- and post-translocated states, and a dNTP/PPi-bound state where dNTP binding stabilizes the post-translocated state. We show how this thermal ratchet mechanism is used by the polymerase to generate work against large opposing loads (∼50 pN).

Active DNA unwinding dynamics during processive DNA replication.

Duplication of double-stranded DNA (dsDNA) requires a fine-tuned coordination between the DNA replication and unwinding reactions. Using optical tweezers, we probed the coupling dynamics between these two activities when they are simultaneously carried out by individual Phi29 DNA polymerase molecules replicating a dsDNA hairpin. We used the wild-type and an unwinding deficient polymerase variant and found that mechanical tension applied on the DNA and the DNA sequence modulate in different ways the replication, unwinding rates, and pause kinetics of each polymerase. However, incorporation of pause kinetics in a model to quantify the unwinding reaction reveals that both polymerases destabilize the fork with the same active mechanism and offers insights into the topological strategies that could be used by the Phi29 DNA polymerase and other DNA replication systems to couple unwinding and replication reactions.

Proofreading dynamics of a processive DNA polymerase.

Replicative DNA polymerases present an intrinsic proofreading activity during which the DNA primer chain is transferred between the polymerization and exonuclease sites of the protein. The dynamics of this primer transfer reaction during active polymerization remain poorly understood. Here we describe a single-molecule mechanical method to investigate the conformational dynamics of the intramolecular DNA primer transfer during the processive replicative activity of the Phi 29 DNA polymerase and two of its mutants. We find that mechanical tension applied to a single polymerase-DNA complex promotes the intramolecular transfer of the primer in a similar way to the incorporation of a mismatched nucleotide. The primer transfer is achieved through two novel intermediates, one a tension-sensitive and functional polymerization conformation and a second non-active state that may work as a fidelity check point for the proofreading reaction.

DNA replication machineries: Structural insights from crystallography and electron microscopy.

Since the discovery of DNA as the genetic material, scientists have been investigating how the information contained in this biological polymer is transmitted from generation to generation. X-ray crystallography, and more recently, cryo-electron microscopy techniques have been instrumental in providing essential information about the structure, functions and interactions of the DNA and the protein machinery (replisome) responsible for its replication. In this chapter, we highlight several works that describe the structure and structure-function relationships of the core components of the prokaryotic and eukaryotic replisomes. We also discuss the most recent studies on the structural organization of full replisomes.

Mechanical properties of high-G.C content DNA with a-type base-stacking.

The sequence of a DNA molecule is known to influence its secondary structure and flexibility. Using a combination of bulk and single-molecule techniques, we measure the structural and mechanical properties of two DNAs which differ in both sequence and base-stacking arrangement in aqueous buffer, as revealed by circular dichroism: one with 50% G·C content and B-form and the other with 70% G·C content and A-form. Atomic force microscopy measurements reveal that the local A-form structure of the high-G·C DNA does not lead to a global contour-length decrease with respect to that of the molecule in B-form although it affects its persistence length. In the presence of force, however, the stiffness of high-G·C content DNA is similar to that of balanced-G·C DNA as magnetic and optical tweezers measured typical values for the persistence length of both DNA substrates. This indicates that sequence-induced local distortions from the B-form are compromised under tension. Finally, high-G·C DNA is significantly harder to stretch than 50%-G·C DNA as manifested by a larger stretch modulus. Our results show that a local, basepair configuration of DNA induced by high-G·C content influences the stretching elasticity of the polymer but that it does not affect the global, double-helix arrangement.

Condensation prevails over B-A transition in the structure of DNA at low humidity.

B-A transition and DNA condensation are processes regulated by base sequence and water activity. The constraints imposed by interhelical interactions in condensation compromise the observation of the mechanism by which B and A base-stacking modes influence the global state of the molecule. We used a single-molecule approach to prevent aggregation and mechanical force to control the intramolecular chain association involved in condensation. Force-extension experiments with optical tweezers revealed that DNA stretches as B-DNA under ethanol and spermine concentrations that favor the A-form. Moreover, we found no contour-length change compatible with a cooperative transition between the A and B forms within the intrinsic-force regime. Experiments performed at constant force in the entropic-force regime with magnetic tweezers similarly did not show a bistable contraction of the molecules that could be attributed to the B-A transition when the physiological buffer was replaced by a water-ethanol mixture. A total, stepwise collapse was found instead, which is characteristic of DNA condensation. Therefore, a low-humidity-induced change from the B- to the A-form base-stacking alone does not lead to a contour-length shortening. These results support a mechanism for the B-A transition in which low-humidity conditions locally change the base-stacking arrangement and globally induce DNA condensation, an effect that may eventually stabilize a molecular contour-length reduction.

Measurements of Real-Time Replication Kinetics of DNA Polymerases on ssDNA Templates Coated with Single-Stranded DNA-Binding Proteins.

Optical tweezers can monitor and control the activity of individual DNA polymerase molecules in real time, providing in this way unprecedented insight into the complex dynamics and mechanochemical processes that govern their operation. Here, we describe an optical tweezers-based assay to determine at the single-molecule level the effect of single-stranded DNA-binding proteins (SSB) on the real-time replication kinetics of the human mitochondrial DNA polymerase during the synthesis of the lagging strand.

In vitro single-molecule manipulation studies of viral DNA replication.

Faithfull replication of genomic information relies on the coordinated activity of the multi-protein machinery known as the replisome. Several constituents of the replisome operate as molecular motors that couple thermal and chemical energy to a mechanical task. Over the last few decades, in vitro single-molecule manipulation techniques have been used to monitor and manipulate mechanically the activities of individual molecular motors involved in DNA replication with nanometer, millisecond, and picoNewton resolutions. These studies have uncovered the real-time kinetics of operation of these biological systems, the nature of their transient intermediates, and the processes by which they convert energy to work (mechano-chemistry), ultimately providing new insights into their inner workings of operation not accessible by ensemble assays. In this chapter, we describe two of the most widely used single-molecule manipulation techniques for the study of DNA replication, optical and magnetic tweezers, and their application in the study of the activities of proteins involved in viral DNA replication.

DNA replication: In vitro single-molecule manipulation data analysis and models.

DNA replication is a key biochemical process of the cell cycle. In the last years, analysis of in vitro single-molecule DNA replication events has provided new information that cannot be obtained with ensembles studies. Here, we introduce crucial techniques for the proper analysis and modelling of DNA replication in vitro single-molecule manipulation data. Specifically, we review some of the main methods to analyze and model the real-time kinetics of the two main molecular motors of the replisome: DNA polymerase and DNA helicase. Our goal is to facilitate access to and understanding of these techniques to promotetheir use in the study of DNA replication at the single-molecule level. A proper analysis of single-molecule data is crucial to obtain a detailed picture of, among others, the kinetics rates, equilibrium contants and conformational changes of the system under study. The techniques presented here have been used or can be adapted to study the operation of other proteins involved in nucleic acids metabolism.

Optical Tweezers to Investigate the Structure and Energetics of Single-Stranded DNA-Binding Protein-DNA Complexes.

Optical tweezers enable the isolation and mechanical manipulation of individual nucleoprotein complexes. Here, we describe how to use this technique to interrogate the mechanical properties of individual protein-DNA complexes and extract information about their overall structural organization.

DNA replication machinery: Insights from in vitro single-molecule approaches.

The replisome is the multiprotein molecular machinery that replicates DNA. The replisome components work in precise coordination to unwind the double helix of the DNA and replicate the two strands simultaneously. The study of DNA replication using in vitro single-molecule approaches provides a novel quantitative understanding of the dynamics and mechanical principles that govern the operation of the replisome and its components. 'Classical' ensemble-averaging methods cannot obtain this information. Here we describe the main findings obtained with in vitro single-molecule methods on the performance of individual replisome components and reconstituted prokaryotic and eukaryotic replisomes. The emerging picture from these studies is that of stochastic, versatile and highly dynamic replisome machinery in which transient protein-protein and protein-DNA associations are responsible for robust DNA replication.

Dynamic constriction and fission of endoplasmic reticulum membranes by reticulon.

The endoplasmic reticulum (ER) is a continuous cell-wide membrane network. Network formation has been associated with proteins producing membrane curvature and fusion, such as reticulons and atlastin. Regulated network fragmentation, occurring in different physiological contexts, is less understood. Here we find that the ER has an embedded fragmentation mechanism based upon the ability of reticulon to produce fission of elongating network branches. In Drosophila, Rtnl1-facilitated fission is counterbalanced by atlastin-driven fusion, with the prevalence of Rtnl1 leading to ER fragmentation. Ectopic expression of Drosophila reticulon in COS-7 cells reveals individual fission events in dynamic ER tubules. Consistently, in vitro analyses show that reticulon produces velocity-dependent constriction of lipid nanotubes leading to stochastic fission via a hemifission mechanism. Fission occurs at elongation rates and pulling force ranges intrinsic to the ER, thus suggesting a principle whereby the dynamic balance between fusion and fission controlling organelle morphology depends on membrane motility.

Dynamics of individual molecular shuttles under mechanical force.

Molecular shuttles are the basis of some of the most advanced synthetic molecular machines. In these devices a macrocycle threaded onto a linear component shuttles between different portions of the thread in response to external stimuli. Here, we use optical tweezers to measure the mechanics and dynamics of individual molecular shuttles in aqueous conditions. Using DNA as a handle and as a single molecule reporter, we measure thousands of individual shuttling events and determine the force-dependent kinetic rates of the macrocycle motion and the main parameters governing the energy landscape of the system. Our findings could open avenues for the real-time characterization of synthetic devices at the single molecule level, and provide crucial information for designing molecular machinery able to operate under physiological conditions.

Mechanical measurement of hydrogen bonded host-guest systems under non-equilibrium, near-physiological conditions.

Decades after the birth of supramolecular chemistry, there are many techniques to measure noncovalent interactions, such as hydrogen bonding, under equilibrium conditions. As ensembles of molecules rapidly lose coherence, we cannot extrapolate bulk data to single-molecule events under non-equilibrium conditions, more relevant to the dynamics of biological systems. We present a new method that exploits the high force resolution of optical tweezers to measure at the single molecule level the mechanical strength of a hydrogen bonded host-guest pair out of equilibrium and under near-physiological conditions. We utilize a DNA reporter to unambiguously isolate single binding events. The Hamilton receptor-cyanuric acid host-guest system is used as a test bed. The force required to dissociate the host-guest system is ∼17 pN and increases with the pulling rate as expected for a system under non-equilibrium conditions. Blocking one of the hydrogen bonding sites results in a significant decrease of the force-to-break by 1-2 pN, pointing out the ability of the method to resolve subtle changes in the mechanical strength of the binding due to the individual H-bonding components. We believe the method will prove to be a versatile tool to address important questions in supramolecular chemistry.

Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers.

Ligands binding to polymers regulate polymer functions by changing their physical and chemical properties. This ligand regulation plays a key role in many biological processes. We propose here a model to explain the mechanical, thermodynamic, and kinetic properties of the process of binding of small ligands to long biopolymers. These properties can now be measured at the single molecule level using force spectroscopy techniques. Our model performs an effective decomposition of the ligand-polymer system on its covered and uncovered regions, showing that the elastic properties of the ligand-polymer depend explicitly on the ligand coverage of the polymer (i.e., the fraction of the polymer covered by the ligand). The equilibrium coverage that minimizes the free energy of the ligand-polymer system is computed as a function of the applied force. We show how ligands tune the mechanical properties of a polymer, in particular its length and stiffness, in a force dependent manner. In addition, it is shown how ligand binding can be regulated applying mechanical tension on the polymer. Moreover, the binding kinetics study shows that, in the case where the ligand binds and organizes the polymer in different modes, the binding process can present transient shortening or lengthening of the polymer, caused by changes in the relative coverage by the different ligand modes. Our model will be useful to understand ligand-binding regulation of biological processes, such as the metabolism of nucleic acid. In particular, this model allows estimating the coverage fraction and the ligand mode characteristics from the force extension curves of a ligand-polymer system.