RESUMEN
The adaptive evolution of large asexual populations is generally characterized by competition between clones carrying different beneficial mutations. Interference slows down the adaptation speed and makes the theoretical description of the dynamics more complex with respect to the successional occurrence and fixation of beneficial mutations typical of small populations. A simplified modeling framework considering multiple beneficial mutations with equal and constant fitness advantage is known to capture some of the essential features of laboratory evolution experiments. However, in these experiments the relative advantage of a beneficial mutation is generally dependent on the genetic background. In particular, the general pattern is that, as mutations in different loci accumulate, the relative advantage of new mutations decreases, a trend often referred to as "diminishing return" epistasis. Here, we propose a phenomenological model that generalizes the fixed-advantage framework to include this negative epistasis in a simple way. We evaluate analytically as well as with direct simulations the quantitative consequences of diminishing returns on the evolutionary dynamics. The speed of adaptation decreases in time and reaches a limit value corresponding to neutral evolution in the long time limit. This corresponds to an increase of the diversity in terms of "classes of mutation" in the population. Finally, we show how the model can be compared with dynamic data on fitness and number of beneficial mutations from laboratory evolution experiments.
Asunto(s)
Epistasis Genética/fisiología , Evolución Molecular , Genética de Población , Modelos Genéticos , MutaciónRESUMEN
OBJECTIVE: The aim of this work is to demonstrate a novel single-molecule DNA sequence comparison assay that is purely based on DNA mechanics. METHODS: A molecular construct that contained the two homologous but non-identical DNA sequences that were to be compared was prepared such that a four-way (Holliday) junction could be formed by the formation of heteroduplexes through the inter-recombination of the strands. Magnetic tweezers were used to manipulate the force and the winding applied to this construct for inducing both the formation and the migration of a Holliday junction. The end-to-end distance of the construct was measured as a function of the winding and was used to monitor the behavior of the Holliday junction in different regions of the intra-molecular recombination. MAIN RESULTS: In the appropriate buffer, the magnet rotation induces the migration of the Holliday junction in the regions where there is no sequence difference between the recombining sequences. In contrast, even a single-base difference between the recombining sequences leads to a long-lasting blockage of the migration in the same buffer; this effect was obtained when the junction was positioned near this locus (the site of the single-base difference) and forced toward the formation of heteroduplexes that comprise the locus. The migration blockages were detected through the identification of the formation of plectonemes. The detection of the presence of sequence differences and their respective mappings were obtained from the series of blockages that were detected. SIGNIFICANCE: This work presents a novel single-molecule sequence comparison assay that is based on the use of a Holliday junction as an ultra-sensitive nanomechanism; the mismatches act as blocking grains of sand in the Holliday "DNA gearbox". This approach will potentially have future applications in biotechnology.
Asunto(s)
ADN Cruciforme/química , ADN/química , ADN/genética , ADN Cruciforme/genética , Modelos Genéticos , Conformación de Ácido Nucleico , Recombinación GenéticaRESUMEN
Laboratory-based evolution experiments on microorganisms that do not recombine frequently show two distinct phases: an initial rapid increase in fitness followed by a slower regime. To explore the population structure and the evolutionary tree in the later stages of adaptation, we evolved a very large population (~3 × 10(10)) of Acinetobacter baylyi bacteria for approximately 2,800 generations from a single clone. The population was maintained in a chemostat at a high dilution rate. Nitrate in limiting amount and as the sole nitrogen source was used as a selection pressure. Analysis via resequencing of genomes extracted from populations at different generations provides evidence that long-term diversity can be established in the chemostat in a very simple medium. To find out which biological parameters were targeted by adaptation, we measured the maximum growth rate, the nitrate uptake, and the resistance to starvation. Overall, we find that maximum growth rate could be a reasonably good proxy for fitness. The late slow adaptation is compatible with selection coefficients spanning a typical range of 10(-3)-10(-2) per generation as estimated by resequencing, pointing to a possible subpopulations structuring.
Asunto(s)
Actinobacteria/genética , Adaptación Fisiológica/genética , Variación Genética , Genoma Bacteriano , Actinobacteria/fisiología , Proliferación Celular , Medios de Cultivo , Evolución Molecular , Aptitud Genética , Técnicas Microbiológicas , Nitratos/metabolismoRESUMEN
Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.
Asunto(s)
ADN Helicasas/metabolismo , ADN Cruciforme/metabolismo , Micromanipulación/métodos , ADN/metabolismo , MecánicaRESUMEN
A magnetic tweezers setup is used to control both the stretching force and the relative linking number DeltaLk of a palindromic DNA molecule. We show here, in absence of divalent ions, that twisting negatively the molecule while stretching it at approximately 1 pN induces the formation of a cruciform DNA structure. Furthermore, once the cruciform DNA structure is formed, the extrusion of several kilo-base pairs of palindromic DNA sequence is directly and reversibly controlled by varying DeltaLk. Indeed the branch point behaves as a nanomechanical gear that links rotation with translation, a feature related to the helicity of DNA. We obtain experimentally a very good linear relationship between the extension of the molecule and DeltaLk. We use then this experiment to obtain a precise measurement of the pitch of B-DNA in solution: 3.61 +/- 0.03 nm/turn.
Asunto(s)
ADN Superhelicoidal/química , Iones/química , Conformación de Ácido Nucleico , Fenómenos Biomecánicos , MicromanipulaciónRESUMEN
Polymerases form a class of enzymes that act as molecular motors as they move along their nucleic acid substrate during catalysis, incorporating nucleotide triphosphates at the end of the growing chain and consuming chemical energy. A debated issue is how the enzyme converts chemical energy into motion [J. Gelles and R. Landick, Cell 93, 13 (1998)]. In a single molecule assay, we studied how an opposing mechanical force affects the translocation rate of T7 RNA polymerase. Our measurements show that force acts as a competitive inhibitor of nucleotide binding. This result is interpreted in the context of possible models, and with respect to published crystal structures of T7 RNA polymerase. The transcribing complex appears to utilize only a small fraction of the energy of hydrolysis to perform mechanical work, with the remainder being converted to heat.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Biotina/química , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , ADN Viral/química , ADN Viral/metabolismo , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Cinética , Conformación Proteica , Dióxido de Silicio/química , Estreptavidina/química , Termodinámica , Uridina Trifosfato/química , Uridina Trifosfato/metabolismoRESUMEN
Within a single-molecule configuration, we have studied rotational drag on double stranded linear DNA by measuring the force during mechanical opening and closing of the double helix at different rates. The molecule is cranked at one end by the effect of unzipping and is free to rotate at the other end. In this configuration the rotational friction torque tau on double-stranded DNA leads to an additional contribution to the opening force. It is shown that the effect of rotational drag increases with the length of the molecule, is approximately proportional to the angular velocity of cranking, and we estimate that the torque tau is of the order of 1k(B)T for 10 000 base pairs of DNA cranked at 2000 turns per second.