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
UCM identifierORCIDScopus Author IDWeb of Science ResearcherIDDialnet IDGoogle Scholar ID

Search Results

Now showing 1 - 5 of 5
  • Publication
    Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis
    (Oxford University Press, 2019-06-20) 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.
  • Publication
    DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein
    (Oxford University Press, 2017-07-07) 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, Borja
    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), 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.
  • Publication
    Mechanics, thermodynamics, and kinetics of ligand binding to biopolymers
    (Public LibraryI Science, 2017-04-05) Jarillo Díaz, Javier; Morín, José A.; Beltrán de Heredia Rodríguez, Elena; García Villaluenga, Juan Pedro; Ibarra, Borja; Cao García, Francisco Javier
    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.
  • Publication
    Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase
    (Oxford University Press, 2015-04-20) Morin, José A.; Cao García, Francisco Javier; Lázaro, José M.; Arias González, J. Ricardo; Valpuesta, José M.; Carrascosa, José L.; Salas, Margarita; Ibarra, Borja
    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 (similar to 50 pN).
  • Publication
    Kinetic modeling of molecular motors: pause model and parameter determination from single-molecule experiments
    (IOP Publishing, 2016-05) Morin, José A.; Ibarra, Borja; Cao García, Francisco Javier
    Single-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 model