Francisco Javier Cao-Garcia

Single-stranded DNA-binding (SSB) proteins play a crucial role in DNA replication by binding to single-stranded DNA (ssDNA) in multiple binding modes, depending on conditions such as salt and protein concentrations. The coverage-dependent effects on the kinetics of these binding modes remain incompletely understood. In particular, the bimodal binding kinetics and the further SSB-ssDNA shortening observed when SSB is removed from the media. Here, we develop a kinetic model extending the Tonks-McGhee-von Hippel framework to incorporate ligand crowding and mode transformations, capturing the inhibition of SSB binding and transitions to higher binding modes as coverage increases. This model quantitatively reproduces experimental binding kinetics and coverage-dependent behaviors observed for human mitochondrial SSB (HmtSSB) and E. coli SSB (EcoSSB). Our findings elucidate the impact of ligand crowding on SSB-ssDNA interactions and provide a generalizable framework for studying multimode ligand binding to polymers, with implications for understanding genome maintenance mechanisms.

Pif1-family helicases are essential for proper nuclear and mitochondrial genome maintenance, yet the regulation of their activities remains incompletely understood. Here, we use single-molecule manipulation and visualization techniques to dissect the real-time mechanochemical behavior of Pfh1, the sole Pif1-family helicase in Schizosaccharomyces pombe. We systematically varied force, ATP concentration, fork composition, and the single-stranded DNA–binding protein spRim1, to quantify the unwinding and single-stranded DNA translocation properties of Pfh1. We find that Pfh1 operates through unwinding-rewinding cycles during which coordinated interactions with both DNA strands at the fork optimize ATP utilization. Contacts with the translocating strand modulate ATP affinity, while interactions with the displaced strand control maximum unwinding velocity. Binding of spRim1 to the displaced strand disrupts the latter interactions, increasing the unwinding velocity. Stable interactions of the helicase with both strands at the fork may limit unwinding processivity to ~20 bp, eventually triggering transition to rewinding. Rewinding proceeds through an ATP-dependent process that is incompatible with strand switching, in which ATP turnover modulates DNA contacts and rewinding rate. Binding of spRim1 to the displaced strand further accelerates rewinding, possibly by competing with helicase–DNA interactions, and facilitates recovery of the active unwinding conformation once the fork has rewound. Together, these findings suggest that Pfh1 balances unwinding and rewinding through coordinated ATP-dependent strand interactions, providing insight into how Pif1-family helicases are controlled at replication forks.

Introduction: Observational and modeling studies have examined theinteractions between El Niño-Southern Oscillation (ENSO) and the equatorialAtlantic variability as incorporated into the classical charge-recharge oscillatormodel of ENSO. These studies included the role of the Atlantic in thepredictability of ENSO but assumed stationarity in the relationships, i.e., thatmodels’ coefficients do not change over time. A recent work by the authors haschallenged the stationarity assumption in the ENSO framework but withoutconsidering the equatorial Atlantic influence on ENSO.Methods: The present paper addresses the changing relationship betweenENSO and the Atlantic El Niño using an extended version of the rechargeoscillator model. The classical two-variable model of ENSO is extended byadding a linear coupling on the SST anomalies in the equatorial Atlantic. Themodel’s coefficients are computed for different periods. This calculation isdone using two methods to fit the model to the data: (1) the traditional method(ReOsc), and (2) a novel method (ReOsc+) based on fitting the Fisher’s Ztransform of the auto and cross-correlation functions.Results: We show that, during the 20th century, the characteristic damping rateof the SST and thermocline depth anomalies in the Pacific have decreased intime by a factor of 2 and 3, respectively. Moreover, the damping time of theENSO fluctuations has doubled from 10 to 20 months, and the oscillationperiod of ENSO has decreased from 60-70 months before the 1960s to 50months afterward. These two changes have contributed to enhancing ENSOamplitude. The results also show that correlations between ENSO and theAtlantic SST strengthened after the 70s and the way in which the impact of theequatorial Atlantic is added to the internal ENSO variability.Conclusions: The remote effects of the equatorial Atlantic on ENSO must beconsidered in studies of ENSO dynamics and predictability during specifictime-periods. Our results provide further insight into the evolution of the ENSOdynamics and its coupling to the equatorial Atlantic, as well as an improved toolto study the coupling of climatic and ecological variables.

Bet-hedging is a phenotype diversification strategy that combines a fast-growing vulnerable phenotype with a slow-growing resistant phenotype. In environments switching between favorable and unfavorable conditions, bet-hedging optimizes growth and reduces fluctuations over a long time, which is expected to reduce extinction risk. Here, we address directly how bet-hedging can reduce extinction probability in an agent-based model. An agent-based model is required due to the small number of individuals close to extinction. We also show that the agent-based model converges to the collective model behavior for populations of 100 individuals or more. However, the collective model provides relevant qualitative insight even for small populations. The collective model provides expressions for extinction that stress the relevance of the initial population number, showing that a factor four increase in the total population has a greater effect than a change of strategy from maximum growth to minimal extinction. This work provides further insight into finite population effects on extinction risk of populations following a bet-hedging strategy.

Surface sensing initiates bacterial colonization of substrates. The protein PilY1 plays key roles during this process—surface detection, host adhesion, and motility—while experiencing mechanical perturbations of varying magnitudes. InPseudomonas aeruginosa, the adhesion and motility functions of PilY1 are associated with integrin and calcium ligand-binding sites; however, how mechanical forces influence PilY1’s dynamics and its interactions with these ligands remain unknown. Here, using single-molecule magnetic tweezers, we reveal that PilY1 is a mechanosensor protein that exhibits different behaviors depending on the force load. At high forces (>20 pN), PilY1 unfolds through a hierarchical sequence of intermediates, whose mechanical stability increases with calcium binding. This enhanced stability may help counteract type IV pilus retraction forces during motility. At low forces (<7 pN), we identify the dynamics of the integrin-binding domain, which is reminiscent of the behavior of mechanosensor proteins. Integrin binding induces a force-dependent conformational change in this domain, shortening its unfolded extension. Our findings suggest that PilY1 roles are force- and ligand-modulated, which could entail a mechanical-based compartmentalization of its functions.

ATP synthases are proteins that catalyse the formation of ATP through the rotatory movement of their membrane‐spanning subunit. In mitochondria, ATP synthases are found to arrange as dimers at the high‐curved edges of cristae. Here, a direct link is explored between the rotatory movement of ATP synthases and their preference for curved membranes. An active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles is found. Coarse‐grained simulations confirm the curvature‐seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein‐protein contacts. The formation of transient protein dimers relies on the membrane‐mediated attractive interaction of the order of 1.5 k_BT produced by a hydrophobic mismatch upon protein rotation. Transient dimers are sustained by a conic‐like arrangement characterized by a wedge angle of θ ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. The results suggest a new role of the rotational movement of ATP synthases for their dynamic self‐assembly in biological membranes.

Feedback control uses the state information of the system to actuate on it. The information used implies an effective entropy reduction of the controlled system, potentially increasing its performance. How to compute this entropy reduction has been formally shown for a general system and has been explicitly computed for spatially discrete systems. Here, we address a relevant example of how to compute the entropy reduction by information in a spatially continuous feedback-controlled system. Specifically, we consider a feedback flashing ratchet, which constitutes a paradigmatic example for the role of information and feedback in the dynamics and thermodynamics of transport induced by the rectification of Brownian motion. A Brownian particle moves in a periodic potential that is switched on and off by a controller. The controller measures the position of the particle at regular intervals and performs the switching depending on the result of the measurement. This system reaches a long-time dynamical regime with a nonzero mean particle velocity, even for a symmetric potential. Here, we calculate the efficiency at maximum power in this long-time regime, computing all the required contributions. We show how the entropy reduction can be evaluated from the entropy of the non-Markovian sequence of control actions, and we also discuss the required sampling effort for its accurate computation. Moreover, the output power developed by the particle against an external force is investigated, which—for some values of the system parameters—is shown to become larger than the input power provided by the switching of the potential. The apparent efficiency of the ratchet thus becomes higher than one, if the entropy reduction contribution is not considered. This result highlights the relevance of including the entropy reduction by information in the thermodynamic balance of feedback-controlled devices, specifically when writing the second principle. The inclusion of the entropy reduction by information leads to a well-behaved efficiency over all the range of parameters investigated.

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.

Different types of ligands compete in binding to polymers with different consequences for the physical and chemical properties of the resulting complex. Here, we derive a general kinetic model for the competitive binding kinetics of different types of ligands to a linear polymer, using the McGhee and von Hippel detailed binding-site counting procedure. The derived model allows the description of the competitive binding process in terms of the size of the ligand, binding, and release rates, and cooperativity parameters. We illustrate the implications of the general theory showing the equations for the competitive binding of two ligands. The size of the ligand, given by the number of monomers occluded, is shown to have a great impact on competitive binding. Ligands requiring a large available gap for binding are strongly inhibited by smaller ligands. Ligand size then has a leading role compared to binding affinity or cooperativity. For ligands that can bind in different modes (i.e., different number of monomers), this implies that they are more effective in covering or passivating the polymer in lower modes, if the different modes have similar binding energies.