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Dinámica del cambio de sitio en la ADN polimerasa

Rajan Lamichhane¹, Svitlana Y Berezhna, Joshua P Gill

¹Department of Integrative Structural and Computational Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Journal of the American Chemical Society

|February 16, 2013

Resumen

Las polimerasas de ADN utilizan sitios distintos para la síntesis y corrección de pruebas de ADN. Este estudio revela que los sustratos de ADN se mueven entre estos sitios a través de la transferencia intramolecular e intermolecular, siendo la transferencia intramolecular más rápida, lo que mejora la eficiencia de la reparación del ADN.

Área de la Ciencia:

Biología Molecular Biología Molecular
La bioquímica es la bioquímica.
La biofísica es la biofísica.

Sus antecedentes:

Las polimerasas de ADN sintetizan ADN y poseen actividad de 3'-5' exonucleasa para la corrección de pruebas.
Los dominios enzimáticos separados para las actividades de la polimerasa (pol) y la exonucleasa (exo) requieren un mecanismo de transferencia para el extremo inicial del ADN.

Objetivo del estudio:

Para monitorear directamente el movimiento de sustratos de ADN entre los sitios pol y exo del fragmento de ADN polimerasa I Klenow (KF) utilizando la transferencia de energía de resonancia Förster de una sola molécula (smFRET).
Aclarar los mecanismos y la cinética de la transferencia de ADN entre los sitios activos de la polimerasa y la exonucleasa.

Principales métodos:

Desarrollo y aplicación de un sistema de transferencia de energía por resonancia Förster (smFRET) de una sola molécula.
Observación directa del movimiento del sustrato de ADN entre los sitios de la KF polimerasa y la exonucleasa.
Análisis de tiempo de permanencia para determinar las constantes de velocidad para las vías de transferencia.

Principales resultados:

Los sustratos de ADN pueden transferirse entre los sitios pol y exo a través de vías intramoleculares (canalización) e intermoleculares (disociación-reasociación).
La transferencia intramolecular es cinéticamente favorecida por encima de la transferencia intermolecular.
Los terminales de primeros mispirados acceden al sitio exo con más frecuencia en presencia de sustratos de trifosfato de desoxinucleósido (dNTP), lo que mejora la revisión.

Conclusiones:

El estudio proporciona evidencia directa de movimiento físico coordinado de ADN entre los sitios de la KF polimerasa y la exonucleasa.
Tanto los mecanismos de transferencia intramolecular e intermolecular contribuyen a la función de corrección de pruebas de la enzima.
La presencia de dNTPs mejora la corrección de fallos al promover el acceso de terminales mal emparejados al sitio de la exonucleasa.

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