Site-directed mutagenesis studies of tn5 transposase residues involved in synaptic complex formation

Abstract

Transposition (the movement of discrete segments of DNA, resulting in rearrangement of genomic DNA) initiates when transposase forms a dimeric DNA-protein synaptic complex with transposon DNA end sequences. The synaptic complex is a prerequisite for catalytic reactions that occur during the transposition process. The transposase-DNA interactions involved in the synaptic complex have been of great interest. Here we undertook a study to verify the protein-DNA interactions that lead to synapsis in the Tn5 system. Specifically, we studied (i) Arg342, Glu344, and Asn348 and (ii) Ser438, Lys439, and Ser445, which, based on the previously published cocrystal structure of Tn5 transposase bound to a precleaved transposon end sequence, make cis and trans contacts with transposon end sequence DNA, respectively. By using genetic and biochemical assays, we showed that in all cases except one, each of these residues plays an important role in synaptic complex formation, as predicted by the cocrystal structure.

FIG. 1.

(A) Tn5 transposon structure. Tn5 consists of two nearly identical insertion sequences (IS50L and IS50R). IS50L carries the genes encoding kanamycin (kan), bleomycin (ble), and streptomycin (str) resistances. IS50R encodes Tnp and its inhibitor (Inh). Each IS50 is bracketed by two 19-bp ESs, called the OE and IE, that define the ends of the transposable element at which Tnp acts. The OE (in vivo assays) and ME (in vitro assays) were used in our studies. The base pairs of the ME that are shown in bold are from the OE sequence, and the others are from the IE sequence. (B) The 60-bp transposon substrate consists of 20 bp of DBB, 19 bp of ES, and 21 bp of transposon.

FIG. 2.

Schematic model of the Tn5 “cut-and-paste” transposition mechanism. Two Tnps bind to transposon ESs to form a synaptic complex (1, 24). Each monomer makes contacts with both its own ES (cis contacts) and the transposon ES bound to the other monomer (trans contacts) (9, 18). The catalytic domain of each Tnp is positioned in such a way as to cleave the transposon end of the other monomer (trans cleavage) (9, 18). DNA cleavage to excise the transposon proceeds through a number of steps. Tnp, in the presence of Mg²⁺, cleaves the DBB via a mechanism that involves a hairpin intermediate and results in a DEB complex (a synaptic complex with two cleaved ends) (4, 5). The chemical steps of the cleavage process are shown for one of the strands only. Following binding of the DEB complex to a nonspecific target DNA, the activated 3′-OH groups at the ends of the transposon perform nucleophilic attacks on the target DNA, accomplishing strand transfer with a 9-bp spacing (23). The Tnps leave the target DNA and the gaps are repaired, producing a 9-bp duplication (for reviews, see references , , and 21).

FIG. 3.

(A) Structure of Tn5 Tnp dimer bound to two OE sequences (PDB ID 1MUH) (9). Transferred and nontransferred strands are shown in green and purple, respectively. (B) Arg342, Glu344, and Asn348 and Ser438, Lys439, and Ser445 make cis and trans contacts with OE DNA, respectively. Mn²⁺ in the active site coordinates to Asp97, Asp188, Glu326 (DDE motif) and 3′-OH of the transferred strand. The DDE motif is a common catalytic triad of acidic residues among transposases and retroviral integrases, which provides a binding site for Mg²⁺ ions (9). Arg342 and Asn348 have water-mediated interactions with backbone phosphates of transferred and nontransferred strands, respectively. Glu344 has proposed base-specific contacts with G7 of the transferred strand and T8 and T9 of the nontransferred strand. Ser438 has a base-specific contact with A8 of the transferred strand and C7 of the nontransferred strand. Lys439 has a base-specific contact with G7 of the transferred strand. Ser445 has a water-mediated interaction with a backbone phosphate of the nontransferred strand. All these contacts are with the base pairs located in the major groove of ES DNA.

FIG. 4.

Results of in vivo papillation assay for the mutant and the control (EK/LP) Tnps. R342A, S438A, and K439A have no transposition activity. S445A and N348A show impaired activity, and E344A shows enhanced hyperactivity compared to EK/LP.

FIG. 5.

(A) PEC formation with the control (EK/LP) and the mutant Tnps. PECs and the unbound 60-bp substrates are marked. (B) Percentage of PECs formed by each protein, which is measured as the amount of ES-containing oligonucleotide in PECs divided by the total amount of ES-containing oligonucleotide in each reaction. These results show that R342A, S438A, and K439A form insignificant amounts of PECs. N348A and S445A show impaired activities, and E344A demonstrates enhanced hyperactivity in PEC formation compared to EK/LP. Error bars indicate standard deviations.

FIG. 6.

Oɛ¹ of Glu344 is located within 3.32 and 2.97 Å of C⁴ and C⁵ of T9 located in the major groove of nontransferred strand, respectively, which will cause steric interactions between Glu344 and the ES.