Sequence-specific DNA binding activity of the cross-brace zinc finger motif of the piggyBac transposase

Abstract

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure–function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5′-TGCGT-3′/3′-ACGCA-5′ motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

Structural basis of seamless excision and specific targeting by piggyBac transposase

“…3b). At the level of the CRD monomer, the observed mode of DNA binding is broadly consistent with the model derived from NMR including the identification of Y558, R567, and K569 as crucial residues for specific recognition 43 , but the NMR model did not predict the severe DNA bend induced by CRD dimerization.…”

“…4). The CRD monomers refined against the cryo-EM maps are consistent with the cross-brace Zn finger structure determined by NMR 43 , although not identical due to side chain rearrangements at the hydrophobic CRD dimer interface (Fig. 3c).…”

“…Present in both LE35 and RE63 is a palindromic-like 19-bp internal repeat (the purple box sequence in Fig. 1b) that is the binding site for the C-terminal cysteine-rich domain (CRD; residues 553-594) of PB 43 . The CRD is required for PB activity and has been proposed to be the driver of TIR binding.…”

“…3). As DNase I footprinting data suggested that PB interacts with 4-5 bp of flanking DNA beyond its TTAA target 43 , target DNA included 11 bp on either side of the central TTAA ( Supplementary Fig. 3a).…”

Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium

“…Biochemical and structural studies of DD(D/E) transposases and retroviral integrases bound to their cognate DNA substrates have revealed that assembly of a productive transpososome often involves more transposase subunits than those actually engaged in catalysis, the supernumerary subunits playing an architectural role within the transposition complex ( Montaño et al, 2012 ; Hickman et al, 2014 ); reviewed in Hickman and Dyda, 2015 ). In particular, recent studies have shown that the T. ni PB transposase dimerizes in solution ( Jin et al, 2017 ) and higher-order complexes were proposed to assemble during piggyBac transposition ( Morellet et al, 2018 ). Within a transpososome, all transposase subunits are generally identical because they are encoded by a single gene carried by the mobile element itself.…”