IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings

Abstract

Transposases are ubiquitous enzymes that catalyze DNA rearrangement events with broad impacts on gene expression, genome evolution, and the spread of drug-resistance in bacteria. Here, we use biochemical and structural approaches to define the molecular determinants by which IstA, a transposase present in the widespread IS21 family of mobile elements, catalyzes efficient DNA transposition. Solution studies show that IstA engages the transposon terminal sequences to form a high-molecular weight complex and promote DNA integration. A 3.4 Å resolution structure of the transposase bound to transposon ends corroborates our biochemical findings and reveals that IstA self-assembles into a highly intertwined tetramer that synapses two supercoiled terminal inverted repeats. The three-dimensional organization of the IstA•DNA cleaved donor complex reveals remarkable similarities with retroviral integrases and classic transposase systems, such as Tn7 and bacteriophage Mu, and provides insights into IS21 transposition.

Molecular basis for transposase activation by a dedicated AAA+ ATPase

“…1a ). Note that it has previously been shown that IstA exhibits similar levels of in vitro activity using either the isolated right TIR or an equimolar mixture of left and right TIRs 21 . The donor molecule comprised both a transferred strand, pre-cleaved at a characteristic CA dinucleotide that is typically attacked by transposases and retroviral integrases, and a non-transferred strand bearing a short (5 nucleotide (nt)) 5′ overhang.…”

“…The IS 21 family is particularly widespread, has been found in clinically important multidrug-resistant strains and has shaped the evolution of human pathogens 18 , 19 . Owing to their simplicity and robust activity, IS 21 elements are a particularly appealing model for mechanistic studies 20 , 21 . The archetypal IS 21 sequence contains two terminal inverted repeats (TIRs) that are typically composed of at least two repeated elements (L1 and L2 for the left ends, and R1 and R2 for the right ends) 20 (Fig.…”

“…The IstA transposase possess two helix-turn-helix (HTH) DNA-binding domains, a DDE catalytic motif and a β-barrel followed by a flexible carboxy-terminal region 21 (Fig. 1b ).…”

“…It has been reported that IstA, the domain organization of which is similar to that of the MuA and TnsB transposases, can oligomerize into a highly intertwined tetramer that synapses two transposon ends into a supercoiled configuration 21 . However, this tetramer state is inactive and requires the regulatory IstB factor to perform transposition 21 , 23 . IstB, similar to the MuB and TnsC helper proteins, is a member of the AAA+ family and a close paralogue of the DnaC/I helicase loader.…”

“…Previous studies have established that the IstB ATPase, similar to TnsC and MuB 25 , 26 , 28 , is essential for stimulating DNA transposition by its cognate transposase 21 , 23 . Although ATP is required to facilitate proper IstB assembly 27 , the precise role of nucleotide binding and hydrolysis in IstA recruitment, activation and DNA transposition has been unclear.…”