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Substrate Generation for Endonucleases of CRISPR/Cas Systems
Published on: September 8, 2012
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Bases estructurales y mecánicas de la adquisición de espaciadores dependientes de PAM en los sistemas CRISPR-Cas
Jiuyu Wang1, Jiazhi Li2, Hongtu Zhao2
1Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Beijing Key Laboratory of Noncoding RNA, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Cell
|October 20, 2015
Resumen
Las bacterias usan sistemas CRISPR-Cas para recordar el ADN viral. Este estudio revela la estructura de los complejos Cas1-Cas2, mostrando cómo seleccionan e insertan fragmentos de ADN viral (espaciadores) en la matriz CRISPR para la memoria inmune.
Área de la Ciencia:
- Microbiología
- Biología molecular
- Biología estructural
Sus antecedentes:
- Las bacterias poseen inmunidad adaptativa contra los virus a través del sistema CRISPR-Cas.
- Las matrices CRISPR almacenan secuencias de ADN de fagos y plásmidos invasores como espaciadores.
- La adquisición de espaciadores es un paso crucial para establecer la inmunidad adaptativa.
Objetivo del estudio:
- Para aclarar los mecanismos estructurales de la selección de protospacer y el procesamiento de ADN por el complejo Cas1-Cas2.
- Comprender el papel del móvil adyacente del protospacer (PAM) en la inmunidad adaptativa CRISPR-Cas.
Principales métodos:
- Se utilizó la cristalografía de rayos X para determinar las estructuras de los complejos Cas1-Cas2-ADN.
- Se realizaron ensayos bioquímicos para analizar las actividades de unión y escisión del ADN.
Principales resultados:
- El estudio reveló la estructura de un complejo Cas1-Cas2 unido a un ADN protospacer bifurcado con voladizos 3' flanqueantes.
- Las subunidades Cas1a reconocen la secuencia PAM en el voladizo de 3' de una manera específica de la base.
- La hendidura se produce a 5 nt del límite del dúplex, generando un intermedio de 33 nt para la inserción.
- La unión del protospacer induce un cambio conformacional en Cas1-Cas2, lo que facilita el reconocimiento.
Conclusiones:
- Los hallazgos proporcionan información mecanicista basada en la estructura sobre el proceso de adquisición de protospacer dependiente de PAM.
- Este trabajo aclara cómo los sistemas CRISPR-Cas muestran y procesan el ADN extraño para la inmunidad adaptativa.
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