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Analysis of LINE-1 Retrotransposition at the Single Nucleus Level
Published on: April 23, 2016
El retrotransposón L1 humano codifica una endonucleasa conservada requerida para la retrotransposición
Q Feng1, J V Moran, H H Kazazian
1Department of Molecular Biology and Genetics, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Cell
|November 29, 1996
Resumen
Los investigadores descubrieron un dominio crítico de endonucleasa (EN) en los elementos L1 humanos. Esta proteína EN corta el ADN en los sitios objetivo, facilitando la retrotransposición de L1 y primando la transcripción inversa.
Área de la Ciencia:
- Biología Molecular Biología Molecular
- Genética La genética.
- La bioquímica es la bioquímica.
Sus antecedentes:
- Los elementos L1 humanos son retrotransposones no LTR abundantes.
- El marco de lectura abierto L1 2 (ORF2) codifica una transcriptasa inversa (RT).
- La función del L1 ORF2 N-terminal no se había caracterizado previamente.
Objetivo del estudio:
- Para identificar y caracterizar el dominio de la endonucleasa en L1 ORF2.2.
- Para investigar la actividad enzimática y la especificidad del objetivo de la endonucleasa L1.
- Para determinar el papel de la endonucleasa L1 en la retrotransposición L1.
Principales métodos:
- Purificación de las proteínas de la endonucleasa L1 (L1 ENp).
- En ensayos de escisión de ADN in vitro se utilizan plásmidos superenrollados.
- Mutagénesis dirigida al sitio de los residuos conservados en L1 EN.
- Prueba para medir la eficiencia de retrotransposición de L1.
Principales resultados:
- Se identificó un dominio de endonucleasa (EN) en el extremo N de L1 ORF2, conservado en poli (A) retrotransposones.
- Purificado L1 ENp crea 5'-fosfato, 3'-hidroxilo nicks en el ADN, independientemente de los sitios de AP.
- L1 ENp divide preferentemente secuencias similares a L1 en sitios objetivo in vivo.
- Las mutaciones en los residuos conservados de L1 EN eliminan la actividad de nicking y la retrotransposición de L1.
Conclusiones:
- El dominio L1 EN funciona como una endonucleasa esencial para la retrotransposición de L1.
- Es probable que L1 EN corte el sitio de ADN objetivo para la inserción de L1.
- Se propone que la actividad de L1 EN prime la transcripción inversa durante la retrotransposición.
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