A high-throughput assay for Tn5 Tnp-induced DNA cleavage.
Nucleic Acids Res
; 32(10): e83, 2004 Jun 16.
Article
em En
| MEDLINE
| ID: mdl-15201394
ABSTRACT
Transposition causes genomic instability by mobilizing DNA elements. This phenomenon is mechanistically related to other DNA rearrangements, such as V(D)J recombination and retroviral DNA integration. A conserved active site architecture within the transposase/integrase superfamily catalyzes these distinct phenomena. The Tn5 transposase (Tnp) falls within this protein class, and many intermediates of the Tn5 transposition reaction have been characterized. Here, we describe a method for the rapid identification of Tn5 Tnp small molecule effectors. This high-throughput screening strategy will aid in the identification of compounds that perturb Tnp-induced DNA cleavage. This method is advantageous, since it identifies effectors that specifically inhibit catalysis without inhibiting Tnp-DNA binding interactions. Effectors identified using this method will serve as a valuable aid both in the isolation and characterization of metal-bound reaction intermediates and in co-crystallization studies involving the effector, Tnp and DNA, to identify the structural basis of the interaction. Furthermore, since Tn5 Tnp shares a similar active site architecture to other transposase/integrase superfamily members, this strategy and any effectors identified using this method will be readily applicable to these other systems.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA
/
Transposases
Idioma:
En
Ano de publicação:
2004
Tipo de documento:
Article