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Incorporación progresiva de análogos de trifosfato de desoxinucleósido por una sola molécula de ADN polimerasa I

Kaitlin M Pugliese¹, O Tolga Gul¹, Yongki Choi¹

¹Departments of †Chemistry, §Physics and Astronomy, and ⊥Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.

Journal of the American Chemical Society

|July 7, 2015

Resumen

Las polimerasas de ADN pueden incorporar trifosfatos de desoxirribonucleósidos modificados (dNTP), con estudios de una sola molécula que revelan la dinámica enzimática durante la incorporación de bases. Esto sugiere un mecanismo dinámico de verificación de estabilidad para los pares de bases nacientes.

Área de la Ciencia:

La bioquímica es la bioquímica.
Biología Molecular Biología Molecular
Nanotecnología La nanotecnología es la nanotecnología.

Sus antecedentes:

Las polimerasas de ADN poseen mecanismos sofisticados para el reconocimiento preciso del trifosfato de desoxirribonucleósido (dNTP).
A pesar de esto, muestran una notable tolerancia a varios análogos de dNTP.
Comprender esta tolerancia a nivel molecular es crucial para los estudios de replicación y reparación del ADN.

Objetivo del estudio:

Para investigar la dinámica de una sola molécula del fragmento de ADN polimerasa I Klenow (KF) que interactúa con los análogos de dNTP.
Para resolver la acomodación de la enzima de estos análogos utilizando transistores de efecto de campo de nanotubos de carbono de pared única (SWCNT-FET).
Aclarar los mecanismos subyacentes a la tolerancia de la ADN polimerasa para los dNTP modificados.

Principales métodos:

Atado de moléculas individuales de KF a SWCNT-FET para mediciones de corriente de una sola molécula en tiempo real.
Analizando los cambios actuales (τclosed y τopen) durante la incorporación de base de los dNTP nativos y analógicos.
Utilizando nanocircuitos para detectar conformaciones enzimáticas alternativas durante la polimerización.

Principales resultados:

La duración del estado cerrado (τclosed) no se vio afectada por la incorporación analógica de dNTP.
La tasa de incorporación analógica de dNTP fue influenciada principalmente por la duración del estado abierto (τopen), lo que refleja el reconocimiento molecular.
Se incorporaron análogos específicos (α-tio-dNTP, 6-Cl-2APTP, 2-tio-dTTP, 2-tio-dCTP) más lentamente y se indujeron conformaciones enzimáticas alternativas.

Conclusiones:

La ADN polimerasa KF exhibe tolerancia a los análogos de dNTP, siendo el reconocimiento (τopen) el paso que limita la velocidad.
La enzima utiliza un mecanismo dinámico de comprobación de la estabilidad que implica rotaciones de hélice O para evaluar la estabilidad de los pares de bases nacientes.
Este mecanismo, junto con los efectos alostéricos, permite la acomodación de pares de bases no nativos, que difieren de los modelos anteriores que atribuyen todos los eventos al cierre de la enzima.

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