Estudio a nivel de moléculas individuales de la actividades de la actividad de replicación del ADN de la polimerasa del bacteriófago Phi29

Resumen

Abstract The accurate replication of DNA is essential to keep genetic integrity. Replicative DNA polymerases are the enzymes responsible for DNA synthesis; they work as molecular motors that translocates along DNA through a series of conformational changes fuelled by dNTP binding and hydrolysis. This process requires overcoming the energetic barrier associated with the base pair melting of its double helix and a fine tuned coordination between the processes of DNA unwinding and replication. One intriguing question that remains poorly understood is the exact mechanism of the coupling of these two reactions. Moreover, little is known about the mechanochemistry of translocation: How thermal and chemical energies are converted to movement? Or more specifically, which step of the dNTP binding and incorporation cycle lead to translocation? In the bacteriophage Phi29 the processes of replication and unwinding are tightly coupled within the same protein: the Phi29 DNA polymerase. This polymerase also presents the basic architecture of the polymerase domain, common to all known polymerases, and the basic features of Family B DNA polymerases. In this thesis we use optical tweezers to characterize, at a single molecule level, the basis for the tight coupling between replication and unwinding and the mechanochemistry of translocation in the Phi29 DNA polymerase. We found, after including the pause kinetic in a model to quantify the unwinding mechanism, that the wild type and an unwinding deficient polymerase variant both destabilize the fork with the same active mechanism: the polymerase destabilize at least two base pairs in the junction with an energy per base pair of 4Gd = 2 kBT. We also found that this polymerase presents a robust mechanism, capable of processing DNA at high opposing forces. For all conditions tested (opposing and aiding forces (¿50 to 20 pN)) the velocity without pauses follows a Michaelis-Menten relation (for dNTP concentrations of 5, 10, 50, 100, 200, 500 ¿M). The force dependence of the Michaelis-Menten parameters (Vmax and Km) indicates that the force dependent step in the polymerization cycle is related to the binding step, suggesting a Brownian ratchet type of mechanism. Key words: DNA replication; Phi29 DNA polymerase; Molecular motors; Single Molecule; Optical tweezers