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Fig. 2 The characterized components of ResT. A The hairpin-binding module (HBM) and the tyrosine recombinase-like catalytic region overlaid on a domain representation of ResT as defined by partial protease digestion (Tourand et al. 2007). The numbers represent amino acid numbers. B Sequence alignment of ResT with the transposases of Tn5 and Tn10 highlighting the HBM of ResT. The YREK residue conserved in IS4 elements (Rezsohazy et al. 1993) is noted above the alignment. The hydrophobic pocket residues are located to the left of the loops indicating insertions in the transposases, and those of the YREK segment to the right. Light shading indicates sequence identity and similarity is shown by dark shading. The numbering below the schema indicates ResT amino acid numbers. Adapted from Bankhead and Chaconas (2004) with permission from the National Academy of Sciences USA

Fig. 2 The characterized components of ResT. A The hairpin-binding module (HBM) and the tyrosine recombinase-like catalytic region overlaid on a domain representation of ResT as defined by partial protease digestion (Tourand et al. 2007). The numbers represent amino acid numbers. B Sequence alignment of ResT with the transposases of Tn5 and Tn10 highlighting the HBM of ResT. The YREK residue conserved in IS4 elements (Rezsohazy et al. 1993) is noted above the alignment. The hydrophobic pocket residues are located to the left of the loops indicating insertions in the transposases, and those of the YREK segment to the right. Light shading indicates sequence identity and similarity is shown by dark shading. The numbering below the schema indicates ResT amino acid numbers. Adapted from Bankhead and Chaconas (2004) with permission from the National Academy of Sciences USA

chemical mechanisms). The reactions of both enzyme families are characterized by formation of a 3'-phosphotyrosyl enzyme-DNA intermediate and 5'-OH ends on the cleaved DNA (Gopaul and Duyne 1999; Grainge and Ja-yaram 1999; Hallet and Sherratt 1997).

To investigate whether the sequence relationship of part of ResT to the catalytic domains of the tyrosine recombinases and topoisomerases indicated that telomere resolution proceeds through a similar mechanism, evidence of a covalent ResT-DNA intermediate was sought. Such a complex is formed and has the expected 3' polarity. The overall reaction and formation of the covalent ResT-DNA complex is dependent upon the presence of the OH group of the tyrosine residue predicted from the sequence alignment to be the active site nucleophile (Y335). Mutation of this residue to phenylalanine abolishes both. The position of the cleavage sites on the rTel junction map to positions 3 nt from the axis of symmetry. The resulting 6-bp 5' stagger between the cleavage sites in the rTel falls within the 6-8-bp range found for all currently characterized tyrosine recombinases. One can infer from the simple buffer requirements of the reaction, along with the observation of a 3'-ResT-DNA complex that has the kinetic and reactive properties of a reaction intermediate, that telomere resolution catalysed by ResT also proceeds via a two-step transesterification (Kobryn and Chaconas 2002).

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