Chemistry of the DDE enzymes

The DDE catalytic site

It has become clear that many of the enzymes involved in transposition reactions are related and, moreover, are part of a larger family of phosphoryltransferases which also includes RNaseH and the RuvC "Holliday junction resolvase" (Dyda, et al., Rice & Mizuuchi, 1995, Rice, et al., 1996, Hickman, et al., Montano & Rice, 2011, Dyda, et al., 2012, Montano, et al., 2012, Hickman & Dyda, 2015).

These transposases catalyse cleavage at the 3' ends of the element by an attacking nucleophile (generally H₂O) to expose a free 3'OH group (Fig 1.40.1). This hydroxyl in turn acts as a nucleophile to attack a 5' phosphate group in the target DNA (strand transfer) in a single-step transesterification reaction. Under certain conditions the enzyme is also capable of "disintegrating" the transposon end by catalysing the attack of the 3' target OH group on the new transposon-target junction (Chow, et al., 1992, Vos, et al., 1993, Polard, et al., 1996).

The reaction(s) do not require an external energy source and do not involve a covalently linked enzyme-substrate intermediate as do certain site-specific recombination reactions.

It is worth underlining that, since it is the donor strand itself which performs the cleavage-ligation step in the target DNA, no cleaved target molecule is detected in the absence of strand transfer.

An acidic amino acid triad (DDE: Asp, Asp, Glu) present in these enzymes is intimately involved in catalysis presumably by co-ordinating divalent metal cations (in particular Mg²⁺) which assist the various nucleophilic attacking- and leaving-groups during the course of the reaction (Fig 1.40.1). The reaction is an in-line nucleophilic attack resulting in chiral inversion of the target phosphate. Chiral inversion has been observed for retroviral integration (Engelman, et al., 1991, Gerton, et al., 1999) bacteriophage Mu (Mizuuchi & Adzuma, 1991), and Tn10 (Kennedy, et al., 2000) transposition and in a related reaction, V(D)J recombination (van Gent, et al., 1996) and was revealed by substituting a non-bridging oxygen for a sulphur group which fixes the normally achiral phosphate group in one or other of its alternative chiral forms (Fig 1.40.1).For many ISs and the retroviral integrases (IN) this triad is known as the DD(35)E motif and is highly conserved (Fayet, et al., 1990, Katzman, et al., 1991, Kulkosky, et al., 1992); (Yuan & Wessler, 2011). In addition, alignments of several Tpases (Rezsohazy, et al., 1993) revealed regions of amino acid conservation designated, N1, N2, and N3 and C1 (Rezsohazy, et al., 1993) which encompass the D (N2), D (N3) and E (C1) regions of typical DDE motifs respectively. The C1 region is probably the most defined structural element. It appears to be part of an a-helix with additional conserved amino acids including a K or R residue approximately 7 amino acids or two helical turns downstream from the E residue (Doak, et al., 1994, Polard & Chandler, 1995, Jenkins, et al., 1997). Less well-conserved residues often occur at approximately one helical turn (3 or 4 residues) upstream and downstream E (DDE motifs). For retroviruses this has been shown to interact with the terminal base pairs of the element presumably contributing to correct positioning of the transposon end in the active site (Jenkins, et al., 1997, Esposito & Craigie, 1998).

Although this conservation in the primary sequence is lower in certain of the other groups of elements and not all families have been explored in sufficient detail to assure that the alignments are biologically relevant, mutagenesis studies with some of these elements (e.g. the Mu, Tn7, IS10, and Tc1/3 Tpases and the retroviral integrases) clearly underline the importance of these residues. Moreover, structural analysis showed that these acidic amino acids are arranged close to each other in a similar, three-dimensional manner in other phosphoryltransferases such as RNaseH and RuvC (Rice & Mizuuchi, 1995, Rice, et al., 1996), otherwise unrelated to Tpases. These primary amino acid conservations are also reflected in conserved structural features. Major conserved features identified in the structures of the catalytic cores of retroviral integrases (Fig 1.40.1), and the Mu and IS50 Tpases include 2 b-sheets each harboring one of the D residues and the long a-helix including the E residue. The a-helix is designated a-4 in HIV and C1 in the Tpases (Rezsohazy et al., 1993, Goldgur, et al., 1998), see (Haren et al., 1999)). It is one of the most conserved regions in the catalytic core. Mutagenesis and crosslinking studies with IN_HIV suggested that it played a role in positioning both the nucleophile and viral DNA (Jenkins et al., 1997, Esposito & Craigie, 1998, Gerton et al., 1998). In particular K159 or K156, which cross-link to the terminal CA dinucleotide, are located on the same side of the a-helix and are strongly conserved. Q148 also lies on the same side and appears to interact with the terminal end of the non-processed strand (see (Engelman & Cherepanov, 2014)).

An amide or basic amino acid is highly conserved at this or the neighboring position and mutation results in severe impairment of catalysis (IN: (Gerton et al., 1998); IS10: (Bolland & Kleckner, 1996); IS50: see (Reznikoff, 2008); IS903: (Tavakoli, et al., 1997).

Transposition reactions and different types of gene rearrangement.

If DDE transposases are capable of catalysing only single strand cleavage to generate a 3'OH at the end of the transposon, how do IS elements with move from one place to another While initiation of a transposition reaction catalysed by the DDE transposases proceeds via transfer of the 3' end of the transposon (transferred strand), the outcome of the reaction is governed by cleavage of the 5' (non-transferred) end of the element as discussed in Groups with DDE Transposases (Fig 1.8.2).

Cleavage of the transferred strand alone.

5' strand cleavage does not occur concomitantly with 3' strand transfer (Fig 1.8.2), the donor and target molecules become covalently linked. Subsequent 5' strand cleavage will separate the element from the donor backbone and will also result in direct insertion (Fig 1.8.2). In the case of retroviruses, only the 3' cleavage occurs, removing 2 bp from the end of the double strand DNA viral copy. However, since no donor backbone is attached to the viral DNA, direct insertion can ensue. In cases where the 5' transposon end remains attached to the donor backbone DNA the result of 3' strand transfer is to join transposon and target leaving a 3'OH in the target DNA at the junction. This can act as a primer for replication of the element and generate cointegrates where donor and target molecules are separated by a single transposon copy at each junction. During its lytic cycle bacteriophage Mu similarly undergoes only 3' cleavage of the transferred strand, the donor backbone remains attached and cointegrate molecules result if replication occurs. Members of the Tn3 family appear to transpose in this way and certain Tn7 and IS903 mutants can be induced to undergo cointegrate formation.

Cointegrates from donor dimer plasmids

It is important to note that cointegrates identical to those produced by replicative transposition can also be produced by a non-replicative process either from a plasmid dimer (Fig 1.40.2) (Berg, 1983, Lichens-Park & Syvanen, 1988) or from tandemly repeated copies of an IS element (Reimmann & Haas, 1987, Reimmann, et al., 1988, Spielmann-Ryser, et al., 1991, Olasz, et al., 1997, Kiss & Olasz, 1999, Turlan, et al., 2000). For copy out - paste in transposition, the two outcomes lead to either simple insertion (Fig 1.40.2, left) or cointegrates (Fig 1.40.2, middle). For cut and paste transposition, a dimer plasmid donor gives rise directly to a cointegrate structure (Fig 1.40.2, right).

Transposase structures and the Transpososome.

Only a single prokaryotic IS DDE transposase, that of IS50, has yielded structural information (Davies, et al., 1999, Davies, et al., 2000) and this has provided important insights into transposition of its cognate IS (e.g (Vaezeslami, et al., 2007, Gradman & Reznikoff, 2008, Gradman & Reznikoff, 2008, Klenchin, et al., 2008)). The only other prokaryotic DDE whose structure is available is that of the transpososome of bacteriophage Mu (Montano, et al., 2012). On the other hand, a limited number of transposase and DDE transposase-DNA co-complex structures are available and have yielded important insights into the details of the transposition reaction (Figs 1.8.3 and 1.40.3). They all tend to support the detailed reaction mechanism. These include several retroviral integrases (IN) such as HIV, ASV and PFV (Hare, et al., 2009, Hare, et al., 2009, Valkov, et al., 2009, Cherepanov, 2010, Cherepanov, et al., 2010, Li, et al., 2010, Engelman & Cherepanov, 2014, Maskell, et al., 2015); the mariner transposon Mos1 (Richardson, et al., 2004, Richardson, et al., 2007, Richardson, et al., 2009, Cuypers, et al., 2013); and the hAT transposon HERMES (revealing a complex set of interactions between neighboring monomers in the octomeric transposase complex (Hickman, et al., 2005, Perez, et al., 2005, Hickman, et al., 2014).

These studies underline a large degree of diversity in the different transposase/integrase and in their architecture and the detailed interaction with their cognate DNA sequences even though they share a well conserved catalytic domain topology. A recurring theme in all these structures is that the complexes tend to be stabilised by a network of inter- and intra-protein contacts and contacts with the DNA. Moreover, the cleavages appear to occur "in trans": cleavages on one end are catalysed by a transposase molecule bound to the other DNA end (Montano & Rice, 2011)(Hickman, et al., 2010, Dyda, et al., 2012). This phenomenon includes both prokaryotic transposable elements Tn5 (Naumann & Reznikoff, 2000) and Mu (Savilahti & Mizuuchi, 1996) (Aldaz, et al., 1996) as well as the eukaryotic Mos1 transposon (Dornan, et al., 2015) and retroviruses (Hare, et al., 2010, Maertens, et al., 2010). This arrangement represents an important regulatory mechanism since no chemistry can occur on a single isolated transposon end which would result in non-productive transposition and would have a negative impact on the host replicon. Cleavage can only be initiated in the context of an architecturally defined and properly assembled "transpososome" in which both DNA ends are synapsed and which can give rise to productive transposition.

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