A high-throughput assay for Tn5 Tnp-induced DNA cleavage

AUTOR(ES)

Ason, Brandon

FONTE

Oxford University Press

RESUMO

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.

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