Person: Cao García, Francisco Javier
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First Name
Francisco Javier
Last Name
Cao García
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Físicas
Department
Estructura de la Materia, Física Térmica y Electrónica
Area
Física Aplicada
Identifiers
11 results
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Now showing 1 - 10 of 11
Item Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase(Nucleic acids research, 2015) Morin, José A.; Cao García, Francisco Javier; Lázaro, José M.; Arias González, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, BorjaDuring 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 (similar to 50 pN).Item Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis(Nucleic acids research, 2019) Cerrón, Fernando; Lorenzo, Sara de; Lemishko, Kateryna M.; Ciesielski, Grzegorz L.; Kaguni, Laurie S.; Cao García, Francisco Javier; Ibarra, Borja; otros, ...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 similar to 12 k(B) T, 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.Item Domain wall dynamics in expanding spaces(Physical review E (statistical, nonlinear, biological, and soft matter physics), 2006) Cao García, Francisco Javier; Zamora-Sillero, Elias; Quintero, Niurka R.We study the effects on the dynamics of kinks due to expansions and contractions of the space. We show that the propagation velocity of the kink can be adiabatically tuned through slow expansions and/or contractions, while its width is given as a function of the velocity. We also analyze the case of fast expansions and/or contractions, where we are no longer on the adiabatic regime. In this case the kink moves more slowly after an expansion-contraction cycle as a consequence of the loss of energy through radiation. All these effects are numerically studied in the nonlinear Klein-Gordon equations (both for the sine-Gordon and for the 𝜙4 potential), and they are also studied within the framework of the collective coordinate evolution equations for the width and the center of mass of the kink. These collective coordinate evolution equations are obtained with a procedure that allows us to consider even the case of large expansions and/or contractions.Item Kinetic modeling of molecular motors: pause model and parameter determination from single-molecule experiments(Journal of statistical mechanics: theory and experiment, 2016) Morin, José A.; Ibarra, Borja; Cao García, Francisco JavierSingle-molecule manipulation experiments of molecular motors provide essential information about the rate and conformational changes of the steps of the reaction located along the manipulation coordinate. This information is not always sufficient to define a particular kinetic cycle. Recent single-molecule experiments with optical tweezers showed that the DNA unwinding activity of a Phi29 DNA polymerase mutant presents a complex pause behavior, which includes short and long pauses. Here we show that different kinetic models, considering different connections between the active and the pause states, can explain the experimental pause behavior. Both the two independent pause model and the two connected pause model are able to describe the pause behavior of a mutated Phi29 DNA polymerase observed in an optical tweezers single-molecule experiment. For the two independent pause model all parameters are fixed by the observed data, while for the more general two connected pause model there is a range of values of the parameters compatible with the observed data (which can be expressed in terms of two of the rates and their force dependencies). This general model includes models with indirect entry and exit to the long-pause state, and also models with cycling in both directions. Additionally, assuming that detailed balance is verified, which forbids cycling, this reduces the ranges of the values of the parameters (which can then be expressed in terms of one rate and its force dependency). The resulting model interpolates between the independent pause model and the indirect entry and exit to the long-pause state modelItem Reliability of rectified transport: Coherence and reproducibility of transport by open-loop and feedback-controlled Brownian ratchets(Physical review E, 2018) Jarillo, Javier; García Villaluenga, Juan Pedro; Cao García, Francisco JavierBrownian ratchets are small-scale systems which rectify thermal fluctuations to produce a net current of particles. They have inspired many models of molecular motors that perform transport in the noisy environment of living cells. For the most common ratchet systems, this rectification is achieved by means of the switching of a periodic and spatially asymmetric potential (flashing ratchets) or by means of a rocking force (rocking ratchets). The rectification mechanism can be applied without information on the state of the system (open-loop ratchets) or using information on the state of the system (feedback or closed-loop ratchets). In order to characterize the transport, the most used quantity is the mean velocity of the center of mass of the system. However, another important transport attribute that has not received much attention is its quality. Here we analyze the quality of transport by studying the coherence and reproducibility of the transport induced by several representative open-and closed-loop rectification protocols under the maximum mean velocity conditions. We find that for few-particle systems, the best protocol is the rocked feedback protocol, producing the transport of particles with the highest coherence and reproducibility per distance traveled at the maximum mean velocity, while for larger systems it is overtaken by its open-loop counterpart. Our results also show that protocols with similar maximum mean velocities can have quite different coherences and reproducibilities. This highlights the importance of studying the reliability of rectified transport to develop performant synthetic rectification devices. These contributions to the emerging field of reliable transport in noisy environments are expected also to provide insight into the performance of natural molecular motors.Item Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB(NUCLEIC ACIDS RESEARCH, 2023) Plaza-G.A., Ismael; Lemishko, Kateryna M.; Crespo, Rodrigo; Truong, Thinh Q.; Kaguni, Laurie S.; Cao García, Francisco Javier; Ciesielski, Grzegorz L.; Ibarra, BorjaMany 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.Item Fluctuation dynamics of bilayer vesicles with intermonolayer sliding: Experiment and theory(Chemistry and Physics of Lipids, 2015) Mell, Michael; Moleiro, Lara H.; Hertle, Yvonne; López-Montero, Iván; Cao García, Francisco Javier; Fouquet, Peter; Hellweg, Thomas; Monroy, FranciscoThe presence of coupled modes of membrane motion in closed shells is extensively predicted by theory. The bilayer structure inherent to lipid vesicles is suitable to support hybrid modes of ctirvature motion coupling membrane bending with the local reorganization of the bilayer material through relaxation of the dilatational stresses. Previous experiments evidenced the existence of such hybrid modes facilitating membrane bending at high curvatures in lipid vesicles [Rodriguez-Garcia, R., Arriaga, L.R., Mell, M., Moleiro, L.H., Lopez-Montero, I., Monroy, F., 2009. Phys. Rev. Lett. 102, 1282011. For lipid bilayers that are able to undergo intermonolayer sliding, the experimental fluctuation spectra are found compatible with a bimodal schema. The usual tension/bending fluctuations couple with the hybrid modes in a mechanical interplay, which becomes progressively efficient with increasing vesicle radius, to saturate at infinity radius into the behavior expected for a flat membrane. Grounded on the theory of closed shells, we propose an approximated expression of the bimodal spectrum, which predicts the observed dependencies on the vesicle radius. The dynamical features obtained from the autocorrelation functions of the vesicle fluctuations are found in quantitative agreement with the proposed theory. (C) 2014 Elsevier Ireland Ltd. All rights reserved.Item Modeling the Mechanics of Cell Division: Influence of Spontaneous Membrane Curvature, Surface Tension, and Osmotic Pressure(Frontiers in psychology, 2017) Beltrán de Heredia Rodríguez, Elena; Almendro Vedia, Víctor Galileo; Monroy, Francisco; Cao García, Francisco JavierMany cell division processes have been conserved throughout evolution and are being revealed by studies on model organisms such as bacteria, yeasts, and protozoa. Cellular membrane constriction is one of these processes, observed almost universally during cell division. It happens similarly in all organisms through a mechanical pathway synchronized with the sequence of cytokinetic events in the cell interior. Arguably, such a mechanical process is mastered by the coordinated action of a constriction machinery fueled by biochemical energy in conjunction with the passive mechanics of the cellular membrane. Independently of the details of the constriction engine, the membrane component responds against deformation by minimizing the elastic energy at every constriction state following a pathway still unknown. In this paper, we address a theoretical study of the mechanics of membrane constriction in a simplified model that describes a homogeneous membrane vesicle in the regime where mechanical work due to osmotic pressure, surface tension, and bending energy are comparable. We develop a general method to find approximate analytical expressions for the main descriptors of a symmetrically constricted vesicle. Analytical solutions are obtained by combining a perturbative expansion for small deformations with a variational approach that was previously demonstrated valid at the reference state of an initially spherical vesicle at isotonic conditions. The analytic approximate results are compared with the exact solution obtained from numerical computations, getting a good agreement for all the computed quantities (energy, area, volume, constriction force). We analyze the effects of the spontaneous curvature, the surface tension and the osmotic pressure in these quantities, focusing especially on the constriction force. The more favorable conditions for vesicle constriction are determined, obtaining that smaller constriction forces are required for positive spontaneous curvatures, low or negative membrane tension and hypertonic media. Conditions for spontaneous constriction at a given constriction force are also determined. The implications of these results for biological cell division are discussed. This work contributes to a better quantitative understanding of the mechanical pathway of cellular division, and could assist the design of artificial divisomes in vesicle-based self-actuated microsystems obtained from synthetic biology approaches.Item Efficiency at maximum power of a discrete feedback ratchet(Physical Review E, 2016) Jarillo Díaz, Javier; Tangarife, Tomás; Cao García, Francisco JavierEfficiency at maximum power is found to be of the same order for a feedback ratchet and for its open-loop counterpart. However, feedback increases the output power up to a factor of five. This increase in output power is due to the increase in energy input and the effective entropy reduction obtained as a consequence of feedback. Optimal efficiency at maximum power is reached for time intervals between feedback actions two orders of magnitude smaller than the characteristic time of diffusion over a ratchet period length. The efficiency is computed consistently taking into account the correlation between the control actions. We consider a feedback control protocol for a discrete feedback flashing ratchet, which works against an external load. We maximize the power output optimizing the parameters of the ratchet, the controller, and the external load. The maximum power output is found to be upper bounded, so the attainable extracted power is limited. After, we compute an upper bound for the efficiency of this isothermal feedback ratchet at maximum power output. We make this computation applying recent developments of the thermodynamics of feedback-controlled systems, which give an equation to compute the entropy reduction due to information. However, this equation requires the computation of the probability of each of the possible sequences of the controller's actions. This computation becomes involved when the sequence of the controller's actions is non-Markovian, as is the case in most feedback ratchets. We here introduce an alternative procedure to set strong bounds to the entropy reduction in order to compute its value. In this procedure the bounds are evaluated in a quasi-Markovian limit, which emerge when there are big differences between the stationary probabilities of the system states. These big differences are an effect of the potential strength, which minimizes the departures from the Markovianicity of the sequence of control actions, allowing also to minimize the departures from the optimal performance of the system. This procedure can be applied to other feedback ratchets and, more in general, to other control systems.Item DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein(Nucleic acids research, 2017) Morin, José A.; Cerron Campoo, Fernando; Jarillo Díaz, Javier; Beltrán de Heredia Rodríguez, Elena; Ciesielski, Grzegorz L.; Arias González, J. Ricardo; Kaguni, Laurie S.; Cao García, Francisco Javier; Ibarra, BorjaSingle-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), presentmultiple 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.