hAT Transposable Elements

The mechanism of hAT element excision. The chemical mechanism of hAT element excision is shown based on studies undertaken with the Hermes element and transposase ( 9 ). The initial nick occurs on the nontransferred strand of the transposon that leads to the formation of an intermediate structure in which a hairpin loop is formed at the end of the flanking DNA with this second nick exposing the 3′OH of the terminal nucleotide on the transferred strand of the transposon. Therefore, it can undertake strand transfer to the target DNA molecule.

Conserved amino acids across hAT transposases. The four functional domains of hAT transposases are shown with conserved amino acids between 22 hAT transposases shown below. The DDE catalytic triad is shown in red and, based on the cocrystal structure of the Hermes transposase bound with the 16-mer L terminal inverted repeat (TIR), amino acids involved in DNA binding of the TIR, which are moderately conserved across the hAT transposases, are shown in blue. The relative positions of other amino acids that bind to the Hermes L TIR but are not conserved across the hAT transposases are shown by black bars. The position of the DNA binding cleft in the Hermes transposase is show by the blue bar located at the N-end of the insertion domain. Yellow-highlighted amino acids are very highly conserved across these transposases. The locations of the three conserved regions identified in the HFLI hobo transposase are shown underneath.

Comparison of hAT transposase sequences. (A) Consensus alignment of 22 hAT transposases. The alignment was obtained using the program M-Coffee, which uses multiple sequence alignment programs. The location of each sequence within its transposase is shown by the amino acid numbers. The location of the DDE motif is shown above the alignments, conserved resides are shown below the alignments along with the locations of the domains and the six conserved blocks originally identified by Rubin et al. ( 12 ). Amino acids identified as being critical for Hermes transposase activity and/or DNA binding from the Hermes transpososome cocrystal by Hickman et al. ( 6 ) are shown in red as are identical amino acids present in the same locations in the other transposases. The C and H amino acids proposed to constitute the BED domain are shown in blue. Accession numbers are: Tol2 (BAA87039), Tgf2 (AFC96942), Herves (AAS21248), Tam3 (CAA38906), Ac (CAA29005), nDart1 (BAI39457), TCUP (ABC59221.1), Hobo (A39652), Homer (AAD03082), Hermes (AAB60236), TcBuster (ABF20545), AeBuster1 (ABF20543), Tag1 (AAC25101), Restless (CAA93759), Tol1 (BAF64515), Crypt1 (AF283502), BuT2 (AF368884), and IpTip100 (BAA36225). (B) Relative location of the conserved regions identified in Fig. 3A on the Hermes transposase and Hermes terminal inverted repeat tetramer ( 6 ).

Comparison of hAT transposase sequences. (A) Consensus alignment of 22 hAT transposases. The alignment was obtained using the program M-Coffee, which uses multiple sequence alignment programs. The location of each sequence within its transposase is shown by the amino acid numbers. The location of the DDE motif is shown above the alignments, conserved resides are shown below the alignments along with the locations of the domains and the six conserved blocks originally identified by Rubin et al. ( 12 ). Amino acids identified as being critical for Hermes transposase activity and/or DNA binding from the Hermes transpososome cocrystal by Hickman et al. ( 6 ) are shown in red as are identical amino acids present in the same locations in the other transposases. The C and H amino acids proposed to constitute the BED domain are shown in blue. Accession numbers are: Tol2 (BAA87039), Tgf2 (AFC96942), Herves (AAS21248), Tam3 (CAA38906), Ac (CAA29005), nDart1 (BAI39457), TCUP (ABC59221.1), Hobo (A39652), Homer (AAD03082), Hermes (AAB60236), TcBuster (ABF20545), AeBuster1 (ABF20543), Tag1 (AAC25101), Restless (CAA93759), Tol1 (BAF64515), Crypt1 (AF283502), BuT2 (AF368884), and IpTip100 (BAA36225). (B) Relative location of the conserved regions identified in Fig. 3A on the Hermes transposase and Hermes terminal inverted repeat tetramer ( 6 ).

The organization of subterminal direct repeats in 17 active hAT transposon from the Ac and Buster families and five transposons from the Tip family. The relative positions of these repeats in the 300 bp at each end of the transposon is shown together with the sequences of these repeats that are located under the name of each transposon. Asterisks denote where biochemical studies using purified transposase have confirmed binding to these repeats. The orientation of the repeats on the top or bottom strand of the transposon is depicted by the position of the filled circle above or below the transposon ends. In some cases, the direct repeats overlap with the terminal inverted repeats.

The organization of subterminal direct repeats in 17 active hAT transposon from the Ac and Buster families and five transposons from the Tip family. The relative positions of these repeats in the 300 bp at each end of the transposon is shown together with the sequences of these repeats that are located under the name of each transposon. Asterisks denote where biochemical studies using purified transposase have confirmed binding to these repeats. The orientation of the repeats on the top or bottom strand of the transposon is depicted by the position of the filled circle above or below the transposon ends. In some cases, the direct repeats overlap with the terminal inverted repeats.

Alignment of the left terminal inverted repeats (TIRs) of the 17 active transposons from the Ac and Buster families and the five from the Tip family. (A) Alignment showing the conservation of A2 and G5 amongst these transposons with a lower degree of conservation of C11 when the TIRs exceed 10 bp in length. (B) The weblogo generated from the first 8 bp of each of the TIRs in (A) with the amino acids that interact with these in the Hermes transpoase shown below ( 6 ). These are grouped according to their location in the DNA binding domain (DNA), the first catalytic domain (CAT 1), the insertion domain (INS), or the second catalytic domain (CAT 2) of the transposase. *Denotes that the interaction with the TIR is with the main chain of the amino acid. Amino acids in red interact with the nontransferred strand, those in black with the transferred strand. The nontransferred strand is shown in the weblogo.

Amino acid sequence comparison of domesticated hAT transposases. (A) Consensus alignment of 11 domesticated genes derived from hAT transposases and the Hermes transposase. The location of each sequence within its transposase is shown by the amino acid numbers. The location of conserved resides are shown below the alignments along with the locations of the domains. Amino acids identified as being critical for Hermes transposase activity and/or DNA binding from the Hermes transpososome cocrystal by Hickman et al. ( 6 ) are shown in red as are identical amino acids present in the same locations in the other proteins. The C and H amino acids proposed to constitute the BED domain are shown in blue. Accession numbers are: ZBED1 (AAH15030), ZBED4 (NP_055653), ZBED5 (Q49AG3), ZBED6 (NP_001167579), DAYSLEEPER (Q9M2N5), KIAA0543_ZnF862 (060290), P52rIPK (O43422), DREF (BAA24727), GON-14a (CCD71205), GTF21RD2 (AAP14955), and b-Gary (CAJ32531). (B) Conserved key amino acids in domesticated genes derived from hAT transposases and the Hermes transposase. The amino acids are listed along the top of the table. Underneath is a diagram showing the domain they reside in. Black boxes indicate that the amino acid is present in this location in the domesticated gene. Gray boxes denote an amino acid is present that is chemically similar to the amino acid listed at the top of the table and this is shown in the box. Empty boxes indicate no conservation. The number of these amino acids missing from each protein is listed in the final column. The number missing at each location is listed in the final row.

Amino acid sequence comparison of domesticated hAT transposases. (A) Consensus alignment of 11 domesticated genes derived from hAT transposases and the Hermes transposase. The location of each sequence within its transposase is shown by the amino acid numbers. The location of conserved resides are shown below the alignments along with the locations of the domains. Amino acids identified as being critical for Hermes transposase activity and/or DNA binding from the Hermes transpososome cocrystal by Hickman et al. ( 6 ) are shown in red as are identical amino acids present in the same locations in the other proteins. The C and H amino acids proposed to constitute the BED domain are shown in blue. Accession numbers are: ZBED1 (AAH15030), ZBED4 (NP_055653), ZBED5 (Q49AG3), ZBED6 (NP_001167579), DAYSLEEPER (Q9M2N5), KIAA0543_ZnF862 (060290), P52rIPK (O43422), DREF (BAA24727), GON-14a (CCD71205), GTF21RD2 (AAP14955), and b-Gary (CAJ32531). (B) Conserved key amino acids in domesticated genes derived from hAT transposases and the Hermes transposase. The amino acids are listed along the top of the table. Underneath is a diagram showing the domain they reside in. Black boxes indicate that the amino acid is present in this location in the domesticated gene. Gray boxes denote an amino acid is present that is chemically similar to the amino acid listed at the top of the table and this is shown in the box. Empty boxes indicate no conservation. The number of these amino acids missing from each protein is listed in the final column. The number missing at each location is listed in the final row.