, a transposable element isolated from the genome of the housefly Musca domestica
(Warren et al.
), belongs to the hAT
family (Kempken & Windhofer, 2001
; Rubin et al.
), one of ten superfamilies into which eukaryotic transposons can be classified (Kapitonov & Jurka, 2004
). In general, hAT
elements have short terminal inverted repeats (5–27 bp), generate 8 bp target-site duplications upon transposition and encode a single transposase protein that catalyzes the DNA breakage and rejoining reactions required for transposition (Kempken & Windhofer, 2001
). All hAT
transposases display significant amino-acid sequence similarity, with the highest primary structure conservation at their C-termini (Calvi et al.
; Feldmar & Kunze, 1991
Various functional and structural subdomains of the 612-residue Hermes
transposase (Hermes; 70.1 kDa) have been determined or predicted (Fig. 1). For example, the N-terminus of Hermes contains residues important for nuclear localization (Michel & Atkinson, 2003
) and has been proposed to contain a DNA-binding BED domain (residues 25–78; Aravind, 2000
), while the C-terminus contains a sequence (residues 551–569) demonstrated to be important for multimerization (Michel et al.
). It has been proposed that hAT
transposases may carry a DSE catalytic triad (Bigot et al.
), a variant of the DDE motif found in the active site of other transposases such as HIV-1 integrase and Mu transposase (Dyda et al.
; Rice & Mizuuchi, 1995
), and that two acidic residues in particular, Asp402 and Glu572, are necessary for transposition (Michel et al.
). However, more recent biochemical characterization of Hermes in vitro
, combined with secondary-structure prediction, indicates that Hermes may be a member of the retroviral integrase superfamily and that residues Asp180, Asp248 and Glu572 are likely to form a metal-ion-binding site (Zhou et al.
Figure 1 Schematic diagram of the Hermes transposase. Six conserved primary sequence blocks (designated A–F) are found in most hAT transposases (Rubin et al., 2001 ). The three blocks D–F that cluster at the C-terminus are found within (more ...)
Our current understanding of the mechanisms of DNA transposition is derived from well studied prokaryotic and eukaryotic systems (Steiniger-White et al.
; Chaconas & Harshey, 2002
; Rio, 2002
), although high-resolution structural data are currently limited to prokaryotic transposases. To date, only one full-length eukaryotic transposase, Mos1
mariner from Drosophila mauritiana
, has been crystallized (Richardson et al.
). However, the structure of this mariner/Tc1 superfamily member has not yet been reported. Structural data are also available for the bipartite DNA-binding domain (residues 1–135) of the Caenorhabditis elegans
Tc3 transposase in complex with transposon DNA (van Pouderoyen et al.
; Watkins et al.
). Although the structure provides insight into how the Tc3 transposase recognizes DNA, no structural information has been garnered about the remaining portion of this enzyme.
Given the limited structural information available on eukaryotic transposases, we have cloned, overexpressed and crystallized a portion of Hermes with the objective of obtaining a high-resolution crystal structure. We anticipate the structural analysis of this transposase will provide insight into the mechanism by which hAT transposases catalyze DNA transposition.