Two-metal active site binding of a Tn5 transposase synaptic complex

Abstract

A synaptic complex of Tn5 transposase with an extended outside end DNA duplex was prepared and crystallized, and its crystal structure was determined in an effort to reveal the role of metal ions in catalysis. Two Mn2+ ions bound to the active site when a single nucleotide of donor DNA was added to the 3' end of the transferred strand. Marked conformational changes were observed in the DNA bases closest to the active site. The position of the metal ions and the conformational changes of the DNA provide insight into the mechanism of hairpin formation and cleavage, and is consistent with a two-metal model for catalysis.

Mixing active-site components: A recipe for the unique enzymatic activity of a telomere resolvase

“…The size of the linker or bridge region, which corresponds to the loop in Tn5 and Tn10 transposases, appears to vary from protein to protein, with only three amino acids in ResT and 16 residues in TelN and TelK. Secondary structure predictions in this region show a striking similarity between the above telomere resolvases and the known structure of the hairpin binding domain in the Tn5 transposase cocrystal structure (27,28). Moreover, the residues corresponding to the YREK motif are predicted to reside on the same face of an ␣-helix in all these enzymes, similarly displaced for interaction of these or neighboring residues with DNA.…”

“…(B) Amino acid sequence alignment of ResT (4, 13) with the cut-and-paste transposases of Tn5 and Tn10. Sequence analysis reveals that ResT contains a putative hairpin binding region similar to that found in the Tn5 (27,28,32) …”

“…This motif contains a hydrophobic binding pocket that stacks with the flippedout thymine base (Fig. 1A) at the penultimate position on the nontransferred strand (27,28,32). The hairpin binding domain also contains the conserved Y-(2)-R-(3)-E-(6)-K signature found in the transposases of IS4 family members (33).…”

“…This region of the transposase is involved in a series of interactions with the ends of the transposon DNA that distort the DNA and facilitate hairpin formation (Fig. 1A) (27,28,32). Some of the mutations in this region result in a transposase that cannot form the hairpin intermediate and therefore results in the accumulation of nicks at the transposon ends (29)(30)(31).…”

“…Structural analysis of the Tn5 transposase (27,28) coupled with biochemical and genetic experiments on both Tn5 (29,30) and Tn10 (31) has defined a hairpin binding module in these transposases (32). Because telomere resolution by ResT also involves the formation of a DNA hairpin, it seemed reasonable to examine the ResT sequence for the hairpin binding region found in the Tn5 and Tn10 transposases.…”