Transposition and site-specific recombination

cut-and-paste mechanisms to a variety of genetic rearrangements
Bernard Hallet *, David J. Sherratt
Received 28 May 1997 ; revised 1 July 1997 ; accepted 1 July 1997
In bacteria, two categories of specialised recombination promote a variety of DNA rearrangements. Transposition is the
process by which genetic elements move between different locations of the genome, whereas site-specific recombination is a
reaction in which DNA strands are broken and exchanged at precise positions of two target DNA loci to achieve determined
biological function. Both types of recombination are represented by diverse genetic systems which generally encode their own
recombination enzymes. These enzymes, generically called transposases and site-specific recombinases, can be grouped into
several families on the basis of amino acid sequence similarities, which, in some cases, are limited to a signature of a few
residues involved in catalysis. The well characterised site-specific recombinases are found to belong to two distinct groups,
whereas the transposases form a large super-family of enzymes encompassing recombinases from both prokaryotes and
eukaryotes. In spite of important differences in the catalytic mechanisms used by these three classes of enzymes to cut and
rejoin DNA molecules, similar strategies are used to coordinate the biochemical steps of the recombination reaction and to
control its outcome. This review summarises our current understanding of transposition and site-specific recombination,
attempting to illustrate how relatively conserved DNA cut-and-paste mechanisms can be used to bring about a variety of
complex DNA rearrangements.
; Site- speci¢c recombinatio n ; Recomb ination mechanism ; Nucleoprote in comple x
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2. Transpositional and site-speci¢c recombination : overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2.1. A plethora of transposable elements with common transposition motifs . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
2.2. Site-speci¢c recombination : integration, resolution, inversion and transposition . . . . . . . . . . . . . . . . . . . . . 160
3. Three mechanisms of DNA cut-and-paste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
3.1. The DDE transposases : a universal one-step transesteri¢cation reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3.2. Site-speci¢c recombinases : two-step transesteri¢cations by distinct mechanisms . . . . . . . . . . . . . . . . . . . . . . 165
3.2.1. The resolvase/invertase family : concerted breakage and rejoining of four DNA strands . . . . . . . . . . . 166
3.2.2. The V
family : sequential pairs of DNA strand exchange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
0168-6445 / 97 / $32.00 ß 1997 Federation of Europ ean Microbiolog ical Societies. Published by Elsevier Science B.V.
0 1 6 8 - 6 4 4 5 ( 9 7 ) 0 0 0 5 5 - 7
FEMS Microbiology Reviews 21 (1997) 157^178
* Corresponding author. Tel. : +44 (1865) 275-290 ; fax : +44 (1865) 275-297 ; e-mail : Hallet@bioch.ox.ac.uk
Transposition and site-speci¢c recombination : adapting DNA
Microbiology Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
Abstract
Keywords : Transposon
Contents
Int
PII S

158
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
4. Higher order nucleoprotein complexes : an additional level of control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
recombination by distinct biochemical mechanisms
1. Introduction
may show very similar outcomes.
The present review attempts to give a general survey
of the specialised recombination repertoire in
It is now well documented that genetic information
can be reshu¥ed by inversion, insertion, duplication,
deletion, or translocation of DNA segments.
bacteria, trying to outline the di¡erences, but also
discussing some aspects which in the light of recent
Whereas some rearrangements ful¢l speci¢c physiological
functions or are involved in programmed
studies, sound very much like `variations on a
processes, others occur `spontaneously' with respect
theme'. Although this review is focused on bacterial
to time and position in the genome and contribute to
recombination systems, those in eukaryotes show
the genetic diversity of a population. In bacteria, as
many similarities. Other discussion on site-speci¢c
in most organisms, DNA rearrangements can be
recombination and transposition can be found in
recent reviews [2^10].
mediated by general recombination between homologous
DNA sequences, but also by a variety of specialised
recombination mechanisms commonly
grouped into two categories : genetic transposition
2. Transpositional and site-speci¢c recombination:
and conservative site-speci¢c recombination.
overview
Although transposition and site-speci¢c recombination
seem to be fundamentally distinct processes
2.1. A plethora of transposable elements with common
that often have very di¡erent biological outcomes,
motifs
transposition
increasing evidence indicates that they are related
Transposable elements or transposons can be described
as discrete DNA segments that are able to
move between di¡erent, non homologous, genomic
in many ways. In general recombination, genetic material
is exchanged through a cascade of events involving
multiple proteins assembled in di¡erent enzymatic
complexes [1]. In contrast, both categories of
specialised recombination systems utilise relatively
loci. Transposition of an element is thus a recombination
reaction involving three separate sites : the
simple recombination machineries in which one (or
two transposon ends and the new target locus. In
sometimes two) enzyme, generically referred to as the
autonomous transposable elements, the transposase
transposase and site-speci¢c recombinase, catalyses
is encoded by a gene located within the element.
the essential DNA breakage and joining reactions.
These proteins act at speci¢c DNA sequences which
Transposon ends often contain inverted repeated sequences
on which the transposase binds speci¢cally.
are characteristic for each genetic element.
Although most transposons can transpose into many
di¡erent places, insertion is never totally random.
For all systems analysed, coordination of the recombination
reactions requires the formation of an
While a speci¢c target consensus sequence has been
enzymatically competent nucleoprotein complex in
found for some elements, the insertion of many
which the recombinase and at least two distant
transposons is also in£uenced by regional features
of the target locus such as a local DNA structure,
the presence host protein binding sites, or DNA
DNA sites are brought together. Whereas the molecular
transactions controlling the assembly of this active
complex can vary from one system to another
supercoiling (reviewed in Ref. [10]). The transposon
Tn7 is distinguished by its ability to transpose either
within the same family, unrelated systems also appear
to use convergent strategies. As a consequence,
to a speci¢c site of the bacterial chromosome
a single recombination mechanism can be adapted to
(attTn7) at a high frequency or to many di¡erent
many di¡erent recombination functions. Conversely,
sites at a low frequency (see below) [11].

Fig. 1. Two major modes of transposition. In non-replicative
transposition, the element (rectangle) is excised from the donor
locus by double-strand breaks at both ends (solid triangles) and
transferred to the target site (cut-and-paste mechanism). The
product of intermolecular transposition by the replicative mechanism
is a cointegrate molecule in which the donor and target
backbones are fused. The element is duplicated during the process
and each copy remains attached to the donor backbone at
one end, whereas the other end is joined to the target. The cointegrate
may be resolved by homologous or site-speci¢c recombination
between the two transposon copies to restore the initial
donor molecule and yield a target copy in which the element is
inserted. Both modes of transposition usually generate short target
duplications £anking the element in the new target locus
(open
triangles).
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 159
transposition. In a non-replicative or `cut-and-paste'
mechanism, the element is excised from its original
location and inserted into the new target locus. This
mode of transposition results in the simple insertion
of the element in the target DNA (Fig. 1). The
gapped donor molecule resulting from the transposon
excision is either degraded or repaired by homologous
recombination using a second copy of the donor
as a template [12,13]. Rarely, the ends of the
donor molecule may be resealed. In replicative transposition,
the element is copied so that insertion of
the element into the same DNA molecule leads to
deletion and/or replicative inversion, while transposition
from one circular molecule to another generates
a cointegrate structure in which the donor and
target backbones are joined by directly repeated copies
of the transposon at each junction. This cointegrate
may be subsequently resolved by recombination
between the two copies of the element, giving
rise to a restored donor molecule and a target molecule
in which one copy of the transposon has inserted
(Fig. 1) [9]. Both modes of transposition generate
short target sequence duplications of a
characteristic length (2^14 bp) that form direct repeats
£anking the element in its new locus. These
duplications are created by repair of the gaps arising
from staggered cuts of the target DNA (see below).
Beyond this overall description hides a broad diversity
of transposable elements in bacteria. More
than 400 di¡erent elements have been identi¢ed
and characterised from a wide range of archaebacteria
and eubacteria (J. Mahillon and M. Chandler,
personal communication). The size and genetic organisation
of bacterial transposons is highly variable.
They range from relatively small and compact (0.8^2
kb) insertion sequences (IS), which in the simplest
form contain a single transposase gene [14], to larger
(and more complex) multi-drug resistance transposons
such as Tn7 (14 kb) [11], or the bacteriophage
Mu (37.5 kb) which uses transposition as part of its
life cycle strategy [6,15].
Historically, bacterial transposable elements have
been grouped into several classes on the basis of
di¡erent criteria. However, it is quite clear that the
largest class, the IS elements, de¢ned by the sole
feature that they only encode functions required
for transposition, actually represents a very heterogeneous
group of genetically and functionally distinct
transposable elements. The identi¢cation and
comparison of an increased number of transposons
has made an alternative classi¢cation possible based
on sequence similarities ([14] ; J. Mahillon and M.
Chandler, personal communication). These studies
not only revealed the existence of several bacterial
families of related elements, but more importantly,
they also identi¢ed conserved transposase domains
amongst a broad diversity of transposons from prokaryotes
and eukaryotes. This was ¢rst shown for
the IS3 family of transposases which exhibit striking
similarities with the integrase proteins of retroviruses
and retrotransposons, notably by the presence of a
invariant triad of acidic residues termed the DDE
motif [16^18]. Subsequently, a DDE-like signature
has been identi¢ed within the transposase of many
di¡erent transposons including IS10, Tn7 and the
bacteriophage Mu, which are amongst the best characterised
bacterial elements [19^22]. The widespread
Bacterial transposons utilise two major modes of

160
Fig. 2. Outcomes from site-speci¢c recombination. Triangles
show the orientation of the recombination sites. a and b indicate
the position of distinct genetic markers around the recombination
loci. `Excision' and `integration' refer to recombination events involving
genetic entities of di¡erent size and/or function (e.g., the
bacterial chromosome and a phage genome), whereas `resolution'
and `fusion' apply to equivalent DNA molecules, (e.g., two plasmids).
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
occurrence of this signature indicates that transposases
and retroviral integrases from very di¡erent
sources form a super-family of recombination enzymes
which share a common catalytic mechanism.
It is worth noting that not all the proteins involved
in transposition contain a DDE motif, and that some
of them clearly belong to completely distinct classes
of enzymes. For example, the recombinases involved
in the translocation of di¡erent kinds of conjugative
transposons and members of the IS110/IS492 family,
belong to three di¡erent groups of site-speci¢c recombinases
[23^25]. IS1 transposase also shares similarities
with the restriction endonuclease EcoRII
[26]. In contrast, the IS91 family transposase is related
to proteins involved in replication by rollingcircle
mechanism [27,28]. Therefore, it is not surprising
that these elements appear to use quite di¡erent
recombination
mechanisms.
Transposable elements are often regarded as selfish
genetic entities that have evolved a set of mechanisms
to replicate faster than their host [29]. An
extreme case is bacteriophage Mu which transposes
into the host chromosome for the establishment of
segment. Recombination between sites on separate
lysogeny and, during lytic growth, uses the host machinery
to replicate its own DNA by undergoing
DNA molecules will integrate one molecule into
multiple rounds of replicative transposition [6,15].
By promoting various DNA rearrangements such
as deletions, inversions, and replicon fusions, transposable
elements can also be viewed as naturally
the other (Fig. 2). Although these DNA rearrangements
are reminiscent of those promoted by transposable
elements, one main di¡erence is that sitespeci¢c
recombination occurs between predetermined
occurring `genetic engineers' which by modifying
loci, whereas rearrangements mediated by transposons
largely rely on their target-site speci¢city. A
the genome of their host may contribute to the genetic
diversity of a population [30]. Through their
second major di¡erence lies in the conservative nature
of site-speci¢c recombination, i.e., DNA strand
exchange is completed without involving any DNA
ability to translocate genes, transposons are also potent
agents for horizontal transfer and in that respect,
they play a crucial role in the spread of antibiotic
resistance factors amongst bacteria.
synthesis or degradation. That allows site-speci¢c recombination
to be reciprocal, i.e., a second round of
2.2. Site-speci¢c recombination : integration,
recombination can restore the initial DNA con¢guration.
resolution, inversion and transposition
As a consequence of these distinguishing features,
site-speci¢c recombination is exploited for a range of
In DNA rearrangements mediated by site-speci¢c
biological functions [5]. In a number of examples,
site-speci¢c inversion is used to switch the expression
recombination, four DNA strands are broken, exchanged
and resealed at speci¢c positions of two
of genes between alternative patterns [32]. As the
separate recombination sites [3^5,31]. The outcome
of a recombination event depends on the relative
genes controlled by these systems often code for surface
structures proteins, e.g., bacteriophage tail ¢-
disposition of the two sites. Intramolecular recombination
between inverted or directly repeated sites will
bres, £agellar antigens or pilin, recombination provides
a source of genetic variability which helps the
invert or excise respectively the intervening DNA
host organism to adapt to environmental £uctua-

B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 161
tions. For these di¡erent systems, alternative gene
expression units located on various replicons or
within transposons [43]. Conjugative transposons
expression is achieved, either by £ipping the orientation
of a promoter between two structural genes or
by connecting variable C-terminal portions of a gene
have combined site-speci¢c recombination and conjugation
mechanisms to transfer a circular excised
to the constant N-terminal region located outside the
form of the element between a donor and a recipient
invertible segment [32]. An extreme example of the
cell [23]. A remarkable feature of integrons and conjugative
transposons is their low level of target site
latter is provided by the shu¥on system of R64 plasmid
in which a set of four invertible fragments controls
the selection of seven C-terminal segments for
selectivity when compared to other site-speci¢c recombination
systems, illustrating the potential for a
recombination mechanism to accommodate distinct
the pilV gene [33]. DNA splicing reactions by sitespeci¢c
recombination between directly repeated sites
functions.
has also been reported as a mechanism of gene activation
in developmentally regulated prokaryotic
processes, such as Bacillus subtilis sporulation [34]
The functional £exibility of site-speci¢c recombination
is also demonstrated by the fact that the various
biological functions listed above are ful¢lled by
and Anabaena sp. heterocyst di¡erentiation [35].
recombinases belonging to two major families of enzymes
using distinct biochemical mechanisms. The
Transposons of the Tn3 family encode a site-speci¢c
resolution system to reduce the cointegrate intermediates
generated by their replicative mode of
resolvase/invertase family, of which there are currently
approximately 40 di¡erent members, forms a
transposition [36]. Another widespread function of
rather homogenous group of related proteins in
site-speci¢c recombination is to resolve replicon
which a conserved serine residue plays a key catalytic
dimers arising from homologous recombination in
role [44,45]. Recombinases of the V integrase (V Int)
order to facilitate their segregation to daughter cells
family (V60 members) are much more divergent,
at the time of cell division. Whereas several di¡erent
replicons encode their own resolution systems (e.g.,
with only four completely invariant residues intimately
involved in catalysis : the RHRY tetrad [46^
Refs. [37^39]), plasmids of the ColE1 family utilise
48]. There is no functional segregation between the
two families and systems of both groups are used to
the multifunctional recombination system Xer encoded
by the Escherichia coli chromosome [40]. By
perform virtually all kinds of rearrangements [5]. As
acting at a speci¢c recombination site (dif) located in
for DDE transposases, these two families are not
the terminus region of replication, Xer recombination
also appears to play an active role in the partitioning
of the bacterial chromosome itself. Bacteria
exclusive and there are a few recombinases that appear
to belong to neither group. An interesting example
is the Piv invertase of Moraxella sp. which
de¢cient in Xer recombination form ¢laments with
together with the transposases of unusual IS elements
(the IS110/IS492 family) form what can be
aberrant nucleoids, presumably as a result the accumulation
of chromosome dimers [41]. This seems to
viewed as a third family of related recombinases
be an important physiological role of site-speci¢c
recombination that is highly conserved among bacteria.
[25]. Like conjugative transposons, several IS elements
of this family fail to generate target duplications
at the insertion site and/or produce an excised
circular form as a transposition intermediate.
Finally, the paradigm of site-speci¢c recombination
is the reaction by which bacteriophage V and
related phages choose whether they prefer to remain
3. Three mechanisms of DNA cut-and-paste
dormant as a prophage or to embark on the lytic
cycle of phage growth by integrating into and excising
from the host genome [4,42]. A similar excision/
integration reaction is used by di¡erent systems to
The establishment of a cell-free system for di¡erent
types of specialised recombination has allowed
translocate non-replicating genetic material, e.g.,
elucidation of the biochemical steps of the DNA
antibiotic resistance genes, between di¡erent genomic
strand exchange reactions. These in vitro studies
loci. Integrons are composite recombination systems
have identi¢ed three di¡erent mechanisms for cutting
that exchange mobile gene cassettes between speci¢c
and rejoining DNA molecules, corresponding to the

162
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
Fig. 3. Chemistry of transposition and site-speci¢c recombination reactions. Recombination DNA strand breakage and joining occur by
transesteri¢cation reactions in which the phosphate of the scissile phosphodiester bond is subject to nucleophile attack by a hydroxyl
group (arrows). Endonucleolytic cleavage at the transposon ends (A) and the strand-transfer reaction that join the ends to the target
DNA (B) are one-step transesteri¢cations in which the nucleophile is a water molecule and the 3P-OH end of the element, respectively.
Strand exchange catalysed by site speci¢c recombinases (C and D) occurs by two steps of transesteri¢cation (cleavage and rejoining) involving
a covalent protein-DNA intermediate. The nature of the catalytic residue and the line of entry of the nucleophile is di¡erent between
the two recombinase families. For cleavage catalysed by the invertase/resolvase family (C), the nucleophile hydroxyl is derived from
a serine and the leaving group is the 3P-OH of the deoxyribose. For the V integrase family (D), the catalytic residue is a tyrosine and the
leaving group is the 5P-OH. For both recombinase families, the rejoining step is the reverse of the cleavage step. Phosphate backbones are
drawn in thick and thin lines to distinguish the donor and target DNA (panel B) or the two recombination partner DNA strands (panels
C and D).
three major classes of proteins described above : the
DDE transposases, the resolvase/invertase, and the V
level of resolution by having obtained crystal structure
data for each of the three families of proteins.
Int families of site-speci¢c recombinases. A common
feature of the three mechanisms is that they proceed
3.1. The DDE transposases : a universal one-step
by transesteri¢cation reactions without requiring
reaction
transesteri¢cation
high-energy cofactors such as ATP (Fig. 3)
[3,31,49]. The energy of the broken phosphodiester
The biochemistry of reactions catalysed by the
bonds is conserved for the formation of new bonds.
DDE recombinases has been examined in detail for
several di¡erent systems including three bacterial
The DDE transposases use a one-step transesteri¢cation
mechanism, whereas the two distinct families of
transposable elements : the bacteriophage Mu, IS10
site-speci¢c recombinases use contrasting two-step
and Tn7 (for recent reviews, see [6,11,15,50]. In all
transesteri¢cation mechanisms involving di¡erent
amino acid residues in the formation of a covalent
cases, the transposase executes a set of critical reactions
that join the 3P ends of the element to the target
DNA-enzyme intermediate (Fig. 3). Understanding
of these mechanisms has now reached the atomic
DNA, whereas connection of the 5P ends and completion
of transposition requires processing events

Fig. 4. Biochemical steps underlying the non-replicative or replicative
transposition of three bacterial elements. The shaded rectangles
represent the DNA strands of the transposable elements
ends. For IS10 and Tn7, reactions occurring at a single end are
shown. Black and white rectangles are the £anking donor sequences
and the target DNA, respectively. The black and white
arrowheads show the 3P-end and 5P-end cleavage, respectively.
Curved arrows indicate the nucleophilic attack transferring the
3P-OH ends on staggered phosphates of the target DNA (black
dots). Crenellated lines represent the few target nucleotides that
are duplicated during the transposition process. For the three elements,
the biochemical steps are catalysed by the transposase in
a complex where the transposon ends are in synapsis. For IS10,
the target is captured after completion of the double-strand
breaks at the transposon ends, whereas for Mu and Tn7, the
presence of the target within the complex is required to activate
the cleavage reactions. The cross-hatching represents replication
events that complete transposition after complex dissociation.
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 163
strand is also cleaved to reveal the transposon 5P
ends and to excise the element from its initial genomic
locus. IS10 excision yields £ush transposons
ends [51], whereas the double-strand breaks at the
ends of Tn7 are staggered by three nucleotides,
with the 5P end cleavages occurring in the adjacent
donor backbone [52]. In sharp contrast, the second
strand cleavage does not occur at this stage of bacteriophage
Mu transposition which remains connected
at its 5P ends to the £anking DNA sequences.
In a second step, the 3P-OH ends of the excised
transposon (IS10 and Tn7) or the nicks in the donor
molecule (Mu) participate in a concerted strand
transfer reaction that join both ends of the element
to staggered phosphates of the two target DNA
strands (Fig. 4 ; see also Fig. 3B).
In the case of IS10 and Tn7, host processing of the
resulting strand transfer intermediate results in the
¢lling-in of the short single-strand gaps lying at
each transposon-target DNA junction. This repair,
which in the case of Tn7 is also presumed to remove
the overhanging three nucleotides at the 5P ends,
generates the short target duplications that £ank
the element in its new locus (Fig. 4). After eventual
cleavage of the donor backbone by an undetermined
mechanism, the strand transfer product of bacteriophage
Mu can be processed in a similar way during
the non-replicative integration of the phage. Alternatively,
in the absence of donor backbone dissociation,
complete replication of the element gives rise
to a cointegrate, which is the ¢nal product of replicative
transposition (Fig. 4).
Variations of the transposition pathway have been
reported for both prokaryotic and eukaryotic transposons.
In particular, the transposase of IS911, a
member of the IS3 family, has been found to carry
out a distinctive single-strand circularisation reaction
performed by host repair and replication functions
in which one transposon end is transferred to a target
site three nucleotides distant from the other end
(Fig. 4). An important variation between the three
[53,54]. In vivo, the resulting `¢gure-eight' molecule
bacterial systems is in the number and nature of cuts
is processed, giving rise to a circular excised form of
that sever the transposon from the £anking donor
the transposon thought to be a transposition intermediate
that can e¤ciently insert in a new locus.
Although mechanistically distinct, this `site-speci¢c'
DNA, resulting in di¡erent transposition end-products.
For the three transposons, transposition initiates
with a pair of speci¢c single strand cleavages
circularisation of IS911 is reminiscent of the excision
exposing the 3P-OH ends of the element (Fig. 4 ; see
reaction performed by site-speci¢c recombinases. Interestingly,
the formation of excised transposon
also Fig. 3A). For IS10 and Tn7 which use a nonreplicative
cut-and-paste mechanism, the other
circles has been observed for other members of the

164
IS3 family [54,55], and also for IS1 [56] and for diverse
eukaryotic transposons, suggesting that it may
represent a major mode of transposition [54].
As mentioned above, the initial cleavage that generates
the 3P-OH ends and the strand transfer reaction
catalysed by bacterial DDE transposases are
chemically equivalent to the reactions catalysed by
the retroviral integrase proteins (IN) for the integration
of the retrovirus cDNA into the genome of an
infected cell [6,7,57]. IN-mediated cleavages at the
ends of HIV cDNA and the subsequent strand transfer
step proceed with inversion of chirality at the
target DNA phosphates. The same result was obtained
for the strand transfer reaction catalysed by
the bacteriophage MuA transposase [58,59]. This
analysis indicates that the reactions catalysed by
the DDE recombinases occur by a one-step transesteri¢cation
mechanism in which the scissile phosphodiester
bond is directly attacked, either by a
water molecule (end cleavage), or by the 3P-OH
end of the element (strand transfer) without the formation
of a protein-DNA covalent intermediate
(Fig. 3). Both transesteri¢cation steps require diva-
cations (Mg
2‡
lent
or
2‡
) and it was postulated
Mn
that the role of the DDE residues was to form a
metal ion binding pocket in the active site [49].
This suggestion is strongly supported by the recent
¢nding that the catalytic domain of IN, MuA and
other enzymes involved in metal ion-dependent phosphoryl
transfers exhibit very similar structures.
The crystal structure of the DDE-containing catalytic
core of MuA shows that this region of the protein
contains two sub-domains [60]. The 70 C-terminal
residues form a L-barrel, on one face of which is
a large region of positive potential that could play a
role in the non speci¢c DNA binding activity associated
with the whole catalytic core. It was proposed
that this activity might be important for interactions
either with the DNA sequences surrounding the
cleavage points at the Mu ends or with the target
DNA, in order to position the substrate into the
enzyme active site [60]. Remarkably, in spite of a
very low level of sequence homology, the structure
of the N-terminal sub-domain containing the DDE
triad shows striking similarities, not only with the
catalytic domain of the IN proteins of HIV and
ASV retroviruses, but also with the structure of
more functionally distant enzymes such as the E.
HIV ribonucleases H (RNase H) and the
junction resolving enzyme RuvC
(reviewed in Refs. [61^63]). In the structure shared
by these di¡erent proteins (a central ¢ve-stranded L-
sheet surrounded by K-helices on either side) three or
four catalytically important acidic residues, i.e., the
DDE motif in the case of the MuA and IN proteins,
are clustered to form a possible two metal ion binding
pocket as actually observed in the structure of
HIV RNase H active site [64]. This structural similarity
between unrelated proteins strongly supports
the view that the DDE recombinases belong to a
larger group of enzymes, including polymerases and
ribozymes, that catalyse transesteri¢cation reactions
using a common `carboxylate-chelated two-metal
ion' catalytic mechanism [61,63]. In this mechanism,
initially proposed for the 3P-5P exonuclease activity
of DNA polymerase I, the two chelated metal ions
participate in the activation of the nucleophile hydroxyl
and the scissile phosphodiester bond by stabilising
the phosphate in a penta-coordinated form
[65].
However, DDE recombinases are not simple nucleases
or polynucleotidyl transferases. These enzymes
are distinguished by the fact that they consecutively
catalyse two (or three) transesteri¢cation
reactions at both transposons ends. Successful transposition
requires that these two sets of reactions be
temporally and spatially coordinated in order to prevent
incomplete recombination events which are
likely to be deleterious to the host and to the transposable
element itself. Recent work from di¡erent
laboratories has provided new interesting insight
into how this control is e¡ected in the case of Mu
(reviewed in Refs. [66,67]). A key step in Mu transposition
is the assembly of a synaptic complex into
which catalytically inert monomers of MuA become
activated by the formation of a tetrameric core
tightly bound to the two transposon ends. Assembly
of the tetramer thus implies a structural transition by
which the enzyme and the DNA cleavage sites become
engaged for catalysis ([6,15] ; see below). Complementation
experiments performed by mixing distinct
catalytic mutants of MuA have shown that the
tetramer active sites are built by interlocking separate
domains of distinct transposase subunits. In
each active site, one monomer provides the DDE
catalytic core (domain II), whereas a di¡erent mono-
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
coli and
E. coli Holliday

C-terminal part of the protein [68]. Although the
exact function of this second catalytic domain (domain
IIIA) is not known, it appears to play a role in
the activation and/or positioning of the DNA [69].
For the strand transfer reactions, the active site domain
IIIA is provided by MuA monomers occupying
the inner part of the Mu ends whereas the DEEcontaining
domain II is provided by transposase subunits
bound on the external sites [70,71]. By reciprocality
of domain sharing, this spatial arrangement
appears to be reversed for the ends cleavage reactions
[71]. Furthermore, in both reaction steps, the
DDE domain II operates in trans, i.e., the subunit
bound to one end catalyses the cleavage and joining
of the opposite end [70,72]. This intricate architecture
of the MuA enzymatic complex is viewed as a
mechanism which ensures that substrate synapsis
and catalysis are tightly coupled. The involvement
of a structural transition upon tetramerisation and
substrate binding is also consistent with the fact that
the DDE residues of the MuA catalytic core appear
to be in an inactive con¢guration [60]. Taking these
data into account, Yang et al. propose a pathway in
which the Mu ends are ¢rst nicked within the two
cleavage active sites and then swing onto two other
catalytic pockets to be transferred on the target
DNA [71].
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 165
As for Mu, a single transposase protein carries out
the three reaction steps underlying IS10 transposition
and the assembly of a precleaved synaptic complex
is required to ensure the coordination of the
processing events occurring at both ends [50,73,74].
IS10 transposition pathway is also highly ordered.
Cleavage of the transferred strand (3P end) always
precedes cleavage of the non-transferred strand (5P
end), and complete excision of the transposon is required
before a target DNA molecule is captured by
the complex for the strand transfer reactions [75,76].
In sharp contrast to Mu, a single transposase monomer
catalyses the three chemical steps of IS10 transposition
at each of the two transposon ends [77].
Thus, two monomers of transposase appear to carry
out the entire transposition reaction without sharing
domains. The mechanism which allows the repeated
use of a single DDE-containing catalytic pocket is
not known. Current models propose that consecutive
structural rearrangements must occur between each
reaction step to re-adjust the active site to novel
substrate con¢gurations [50,77].
A third and distinctive example of transposase architecture
is provided by Tn7 [11]. In contrast to Mu
and IS10, Tn7 transposition reactions are catalysed
by two DDE-containing enzymes with clearly separate
activities. TnsA mediates cleavage at the 5P ends
of the transposon whereas TnsB, which is also responsible
for the recognition and binding of Tn7
ends, promotes the 3P end cleavage and carries out
the strand transfer reaction [22]. Although the respective
functions of TnsA and TnsB can be selectively
blocked by mutation of their DDE residues,
the presence of both proteins is required for the formation
of an active complex. Tn7 transposase is thus
a heteromeric enzyme containing two di¡erent catalytic
subunits [22]. The possibility that the active sites
of the TnsA+B transposase core could be assembled
by contribution of distinct TnsA or TnsB protomers
has not been examined thus far. As with IS10, there
is currently no evidence for cis or trans activity by
TnsA and/or TnsB. Nevertheless, the biochemical
separation of the 5P and 3P ends processing reactions
has a remarkable implication. Inactivation of TnsA
converts the normal cut-and-paste transposition
pathway of Tn7 into a Mu-like replicative mechanism,
the 3P ends breakage and joining reactions catalysed
by TnsB within the transposase core being
functionally equivalent to those performed by the
MuA tetramer in Mu transposition [78].
These three di¡erent examples of `division of labour'
within a transposition complex illustrate the
remarkable £exibility by which a common chemical
mechanism can be adapted in di¡erent ways to accomplish
complex and highly controlled recombination
reactions.
Unlike transposases, site-speci¢c recombinases, at
least in principle, execute all recombination DNA
breakage and joining reactions without involving
host repair or replication functions [3^5,31]. These
reactions are biochemically related to those catalysed
by the topoisomerase enzymes that regulate the intracellular
level of DNA supercoiling [79]. Indeed,
site-speci¢c recombinases often exhibit type I topo-
mer donates another catalytic region located in the
3.2. Site-speci¢c recombinases : two-step
transesteri¢cations by distinct mechanisms

166
proteins. In the E. coli inversion system Fim, the two
Fig. 5. Concerted DNA breakage and rejoining reactions catalysed
by resolvase/invertase family enzymes. The subunit rotation
model is shown. The ovals represent recombinase subunits with
the conserved catalytic serine `S'. Thick and thin lines are the
top and bottom strands of the recombination sites, respectively.
The short vertical bars are the 2 bp of the overlap region between
the two cleavage points. Black arrows represent the nucleophilic
attacks of phosphates (black dots) by hydroxyl groups
(arrowheads). The four DNA strands are cleaved (a), exchanged
by 180³ rotation of the half-site bound subunits (b) and religated
in the recombinant con¢guration (c).
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
isomerase activity by which they can relax supercoiled
DNA substrates. However, in a recombination
reaction between two sites, DNA strands are not
only broken and rejoined, they are also exchanged.
Therefore, as discussed above for the DDE recombinases,
site-speci¢c recombination requires the assembly
of a synaptic complex containing multiple recombinase
subunits and the relevant DNA recombining
partners.
For most of the systems of the two major families
(i.e., the resolvase/invertase and the V Int families),
recombination takes place within a short (V30 bp)
DNA segment called the `core' or `crossover' site
onto which two recombinase subunits bind, usually
by recognising speci¢c sequences with dyad symmetry
[3,80,81]. In the synaptic complex, the two core
sites are brought in close proximity. It is generally
admitted that the four recombinase subunits bound
on the two duplexes participate in the recombination
reaction (Figs. 5 and 6). A few systems, all belonging
to the V Int family, involve two distinct recombinase
recombinases, FimB and FimE, act independently on
the same recombination sites to control the `on' or
`o¡' position of this particular genetic switch [82]. By
contrast, the XerC and XerD recombinases cooperate
in all Xer-mediated DNA rearrangements and
the two proteins bind with distinct speci¢cities to
separate regions of the di¡erent recombination sites
of this system [83,84].
The recombination locus of all systems, even those
working with a single recombinase, contain a certain
degree of asymmetry so that `top' and `bottom'
strands can be distinguished. In the simplest cases,
this asymmetry is entirely encoded within the core
site whereas in other systems, external elements
may contribute to the recombination site polarity
by imposing a speci¢c geometry on the synaptic
complex ([80,81] ; see also below). The catalytic
mechanism used by the two families of site-speci¢c
recombinases is di¡erent as is the structural organisation
of the enzymes.
and QN transposons [3,32,36,80,81,85]. Although the
3.2.1. The resolvase/invertase family : concerted
breakage and rejoining of four DNA strands
The best characterised recombinases of this family
chemistry of strand exchange used by these recombinases
appears to be highly conserved, important variation
is found in the assembly of the recombination
are the invertases Gin from bacteriophage Mu and
synapse determining the selectivity of the reaction,
Hin from Salmonella sp. and the resolvases of Tn3
i.e., inversion or resolution (see below).

B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 167
arm region that connects the two domains. The small
In a recombination reaction catalysed by resolvases
or invertases, double strand breaks staggered
C-terminal DNA binding domain is involved in the
by 2 bp occur at the middle of the two paired core
recognition of the outer part of the core site consensus
sequence by making speci¢c contacts in both the
sites, giving rise to recessed 5P ends and 3P-OH overhangs
(Fig. 5 ; see also Fig. 3C). One recombinase
major and minor grooves. Its structure, containing a
subunit is linked to each of the 5P ends through the
helix-turn-helix DNA binding motif, is similar to
conserved serine residue of the family [86,87]. This
that previously found for the DNA binding domain
serine presumably provides the primary nucleophile
of Hin invertase [96]. The arm region that joins the
hydroxyl group in the cleavage reaction [45]. The
two globular domains also contributes to DNA
ligation step that follows strand exchange can be
binding through interactions in the minor groove.
viewed as the converse of the cleavage : the protein-
The large N-terminal catalytic domain contains the
DNA phosphoseryl bond of one strand is attacked
active site serine and other catalytic residues, as well
as a set of residues forming a hydrophobic core at
the dimer interface. This domain also appears to be
by the 3P-OH end of the partner to release the enzyme
and reseal the DNA backbone in the recombinant
con¢guration (Fig. 5 ; see also Fig. 3C).
Although cleavages of the four DNA strands or
important for higher order interactions between resolvase
dimers in the recombination complex [97^99].
the religation steps can be experimentally uncoupled
The DNA in the co-crystal is bent by 60³ away from
the enzyme catalytic domain.
by using recombination site variants or mutated recombinases,
both types of reaction are normally
The QN resolvase-DNA complex appears to be in
highly coordinated [88,89]. The cleaved complex in
an inactive con¢guration, the catalytic serines of the
which four enzyme-linked recombination half-sites
two resolvase subunits being too far away from the
scissile phosphates to cleave the DNA [95]. However,
are held together by recombinase subunit interactions
seems to be an obligate intermediate [88].
the active serine of either monomer is closest to the
DNA cleavage position proximal to the half-site
Thus, recombination by the resolvase/invertase family
occurs by a mechanism in which four DNA
bound by the recombinase subunit. This correlates
with the fact that the active serine and several other
strands are broken and rejoined in a concerted manner.
catalytic residues of QN resolvase act in cis on the
nearest scissile phosphodiester bond [88]. The crystal
The standard substrates for invertases and resolvases
are supercoiled molecules (see below) and the
structure also suggests that, as in MuA, catalytic
topological change induced by recombination has
residues may be shared between the two monomers
been found to be equivalent to a right-handed 180³
to form the active site. While there is currently no
rotation of one pair of cleaved half-sites relative to
experimental evidence to support the possibility for a
the other prior the rejoining step (Fig. 5) [90^92]. In
composite catalytic site, mutations in one resolvase
subunit seem to activate the adjacent subunit in
this mechanism, the 2 bp `overlap' regions that separate
the top and bottom strand cleavage positions
need to be identical in the two core sites to stably
trans, presumably by altering inter-subunit interactions
at the dimer interface ([88] ; M.R. Boocock
reconnect the recombinant DNA duplexes [93,94]. It
has been shown recently that in reactions involving
and N.D.F. Grindley, unpublished). Likewise, mutations
in the equivalent dimerisation domain of the
non-homologous overlap regions, strand exchange
DNA invertases Gin, Hin and Cin, make the recombinases
more reactive and independent of structural
mediated by Tn3 resolvase proceeds through apparent
360³ (2U180³) rotation of the half-sites without
rejoining the mis-paired strands in the recombinant
elements controlling the formation of the recombination
synaptic complex ([92,100^103] ; see also below).
(180³ rotation) con¢guration [89].
These observations indicate that activation of the
The crystal structure of the QN resolvase dimer
active site serine recombinases appears to require
complexed to the core recombination site has been
conformational changes during the assembly of an
determined recently [95]. The DNA-bound resolvase
enzymatically competent recombination structure as
monomer contains two globular domains lying on
discussed above for the DDE transposases. Indeed,
opposite faces of the DNA helix and an extended
some level of structural £exibility as revealed by the

168
Fig. 6. Sequential strand exchange by the V Int family site-speci¢c recombinases. The DNA strand swapping/isomerisation model is presented.
The letter `Y' refers to the conserved catalytic tyrosine. Other symbols are as in Fig. 5. The top strands (thick lines) are cleaved
¢rst (a), swapped between the two partners (b), and then religated (c). The branch point of the generated Holliday junction intermediate
is positioned at the middle of the (6-bp) overlap region and the top strands are crossed. Isomerisation of the Holliday junction to a recombinant
con¢guration in which the bottom strands are crossed requires the reorganisation of the DNA helices and the four half-sitesbound
recombinase subunits within the complex (d). The resulting Holliday junction isoform is resolved by repeating steps a to c in order
to exchange the bottom strands (e).
QN resolvase-DNA co-complex could be compatible
with limited distortions [95].
The conformational changes that take place during
strand exchange appear less straightforward.
Since resolvase cleaves the DNA in cis and seems
not to dissociate from its binding site during recombination
[104], two models have been proposed to
account for the apparent 180³ half-site rotation in
the reaction. In the `subunit rotation' model, strand
exchange is coupled to a rotational rearrangement of
the DNA-linked recombinase subunits within the tetramer
[91]. A major di¤culty with this mechanism is
that the recombinase dimer interface holding the
cleaved ends in the complex must be disrupted transiently
during the dissociation/reassociation process
(Fig. 5). An alternative `static subunit' model postulates
that the recombinase tetramer does not dissociate
and that recombination occurs by localised
conformational and topological rearrangement of
the DNA molecules within the complex [95,99].
This second model is di¤cult to reconcile with the
recent observation that Tn3 resolvase brings about
multiple rounds of rotations without rejoining the
single 180³ rotation intermediate. It is argued that
in a static subunit mechanism, such an iteration of
the reaction would entangle the DNA within the
catalytic complex to an unacceptable level [89,105].
Unlike the recombinases of the resolvase/invertase
family, site-speci¢c recombinase related to V Int such
as the Cre recombinase of phage P1, E. coli XerC
and XerD and the Flp protein from the yeast 2W
plasmid, exchange the two pairs of DNA strands
separately and sequentially (Fig. 6) [3,4,31,106].
To initiate the ¢rst strand exchange, the tyrosine
residue of the conserved catalytic motif RHRY
attacks a speci¢c scissile phosphate in one strand
(de¢ned here after as the top strand) of each recombination
core sites, thereby forming a 3P phosphotyrosyl-linked
recombinase-DNA complex and
generating a free 5P-OH end (Fig. 6). The polarity
of this cleavage reaction is thus reversed when compared
to that of the resolvase/invertase-mediated
cleavages (compare also Figs. 3C and 3D). In a sec-
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
3.2.2. The V Int family : sequential pairs of DNA
exchange
strand

B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 169
ond step, the recombinase-DNA phosphotyrosyl
bond is attacked by the 5P-OH end from the partner
bottom strand exchange [111,112]. Also, in the recombinase-Holliday
junction complex, the centre of
duplex to generate a four-way branched structure, or
the overlap region is partially unstacked [113].
`Holliday junction' intermediate, in which only two
Although variations of both models can be envisioned,
the strand swapping mechanism seems more
DNA strands have recombined. To resolve this intermediate
and complete the recombination reaction,
suitable for other recombination systems of the V Int
the two other (bottom) strands are exchanged by
repeating the cleavage/religation process 6^8 bp
family, such as conjugative transposons and integrons,
which are less strict in their requirement for
downstream of the ¢rst strand cleavage position
sequence homology between partner recombination
(Fig. 6).
sites [23,43]. These systems may be less sensitive to
DNA mispairing by catalysing both strand exchanges
without annealing recombinant strands. Alternatively,
the Holliday junction iso-form generated
As in the resolvase/invertase-mediated recombination,
sequence homology in the 6^8 bp overlap region
that separates the top and bottom strand cleavage
positions in the two partner recombination core
sites is also essential for most (but not all) of the
by a single strand exchange may be a stable recombination
product that is not processed back by the
systems belonging to the V Int family. Although
recombinases but could be well resolved by some
other host function (as discussed in Ref. [113]).
the homology appears to play a role after the synapsis
of the two core sites, the exact mechanism by
Another matter of divergence within the V Int
which it in£uences the reaction remains unclear. The
family concerns the catalytic role of the di¡erent
recombinase subunits within the synaptic complex.
classical model supposes that sequence identity between
recombination sites is required for a reversible
A wealth of data indicate that the yeast recombinase
process called `branch migration' that moves the
Flp uses a trans cleavage mechanism in which the
branch point of the Holliday junction from its site
catalytic RHR triad of one monomer activates the
of formation at one end of the overlap region to the
scissile phosphodiester adjacent to its binding site,
site of resolution at the opposite end. This is
while the tyrosine nucleophile is contributed by a
achieved by stepwise melting and reannealing of
di¡erent Flp monomer bound on a di¡erent halfsite.
As for MuA, this active site sharing observed
for Flp was proposed as a mechanism for coupling
catalysis and recombination site pairing [31,114].
the complementary strands of the parental and partner
DNA duplexes, [5,107]. This view is now challenged
by an alternative `strand swapping/isomerisation'
mechanism (Fig. 6) [108]. The model proposes
However, this view was weakened by experiments
that, after cleavage, two or three nucleotides from
showing that the two collaborating monomers are
the parental overlap sequences are melted and then
bound on the same core site and that cleavage may
actually precede synapsis [115,116]. An alternative
swapped between the partner duplexes. In this mechanism,
sequence homology is required in the reannealing
reaction that orients the 5P-OH end of the
model based on an asymmetrical (head to tail) assembly
of Flp monomers has been proposed, again
suggesting that active site-dependent molecular
invading strand for ligation. Movement of the Holliday
junction is limited to the 1^3 central bp of the
bridges may be required [117]. In contrast, all current
data indicate that XerC and XerD recombinases
overlap region. This movement would simply be unstacking-restacking
events whereby the Holliday
act on the closest cleavage site by providing all catalytic
residues in cis [118]. For V Int, experiments
supporting both cis and trans mechanisms have
junction DNA helices are reorganised from a `parental'
(top strands crossed) con¢guration to a `recombinant'
(bottom strands crossed) con¢guration
been reported [119,120]. These apparently con£icting
results have raised a number questions that have
in which the bottom strand cleavage sites are adequately
positioned for strand exchange (Fig. 6)
been debated in the recent literature [48,121^123].
[108^110]. This view is supported by recent work
Although the details of the molecular interactions
between recombinases subunits are not known, some
showing that one Holliday junction isomer preferentially
undergoes top strand exchange, whereas resolution
of the other iso-form predominantly occurs by
new insight has been provided by the recent acquisition
of structural data for the C-terminal catalytic

170
attP is wrapped in an `intasome' complex in which
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
moieties of V Int and the Haemophilus phage 1 integrase
(HP1 Int) and for the complete 298 aa XerD
These systems contain additional accessory DNA sequences,
to which further recombinase subunits and/
or other proteins bind thereby forming part of the
recombinase [124^126]. The C-terminal catalytic domain
of these three recombinases exhibits a similar
functional nucleoprotein complex. In many cases,
fold where the conserved residues RHR are exposed
DNA supercoiling is also required for the assembly
in a basic groove likely to contact the DNA for the
and/or activity of the recombination complex. The
activation of the scissile phosphodiester bond. However,
the structure of the region containing the active
tyrosine (the extreme C-terminal part of the domain)
is very di¡erent in the three structures. This region
precise architecture of the complex provides a mechanism
for controlling the outcome of a recombination
reaction, while a requirement for cellular proteins
may provide a means of relating the frequency
of a recombination reaction to host metabolism.
also seems to be involved in recombinase inter-subunit
interactions. In V Int, the tyrosine is positioned
Thus, the requirement for higher order interactions
on a disordered £exible loop in a con¢guration
represents an additional level of regulation ensuring
which, by allowing conformational changes, could
that recombination only occurs at the correct time,
be consistent with either a cis or trans cleavage
between the correct sites [5,9].
[124]. In contrast, the structures of both HP1 Int
and XerD support a cis mechanism, the tyrosine
For example, bacteriophage V integration and excision
need to be irreversible and separate steps of
being docked at a ¢xed position within the catalytic
phage development to permit a committed decision
pocket. In the HP1 Int catalytic domain, which crystallised
as a dimer, the tyrosine is in a position ready
between lysogeny and lytic growth [42]. Both recombination
reactions are mediated by the formation of
for in line nucleophilic attack of the scissile phosphate
[125], whereas in the model proposed for
nucleoprotein structures that arrange the recombination
partners in a complementary con¢guration for
XerD-DNA complex, some local alteration of the
synapsis [4,42,127]. In addition to the core site DNA
structure would be required to bring the residue in
binding activity, V Int has a second DNA binding
an active con¢guration [126]. As for QN resolvase, it is
domain that recognises accessory `arm sites' in the
thought that this conformational activation is induced
by protein interactions between XerD and
£anking regions of the recombination loci. This enables
a single recombinase protomer to form a molecular
bridge between adjacent sites on the same
the partner recombinase XerC (our unpublished results).
Another major conformational change would
DNA molecules or between the two recombining
be required to move the globular N-terminal domain
of XerD (absent from the V and HP1 Int structures)
duplexes in the synaptic complex. For both integration
and excision, Int subunits bound to the arms of
that blocks the access to the active site. The fact that
one partner are delivered in trans to the core site of
the other partner.
the peptide connecting the two XerD domains is disordered
in the crystal is again indicative of structural
However, V Int-mediated integration and excision
£exibility [126].
employ distinct subsets of proteins acting on di¡erent
DNA binding sites and, therefore, the two reactions
are not the simple reverse of each other. For
integration, the 240-bp phage recombination locus
4. Higher order nucleoprotein complexes: an
additional level of control
the relevant Int subunits can capture the naked 25-
The strategies used by transposable elements and
bp core region of the bacterial attachment site, attB
site-speci¢c recombination systems to assemble their
recombination machinery share many similarities. In
[127,128]. Formation of the intasome involves precisely
positioned DNA bends induced by the host
particular, for the majority of elements, the minimal
number of recombinase molecules bound to their
protein IHF and a set of Int-mediated bridges between
the core site and speci¢c arm binding sites in
binding sites on DNA dictated by the chemistry of
the £anking DNA segments of attP. Excision utilises
two other proteins in addition to Int and IHF,
the reaction is usually insu¤cient to mediate a `normal'
recombination reaction at a `normal' frequency.
namely the phage-encoded Xis and the host factor

B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 171
7. Models for the assembly of a synaptic complex for di¡erent specialised recombination systems showing topological selectivity. a:
Fig.
in resolvase/res system. b: Invertasome of DNA inversion systems. c: Synaptic complex assembled by Xer recombination
Synaptosome
at the ColE1 plasmid resolution site cer. d: Possible structure of the LER synaptic complex intermediate during bacteriophage Mu
system
formation. Grey arrows represent the directly repeated (DR) or inverted repeated (IR) recombination core sites, or the
transpososome
as well as a di¡erent set of arm site/core sites
FIS,
to assemble a synaptic complex between
interactions,
and attR.
this complex, the four Int subunits are now contributed
In
by both partners (three from attL and one
distinct and mutually exclusive `lock and key'
use
which allow the phage to become com-
interactions
to either lysogeny or lytic growth [5,42]. Furthermoremitted
the higher order interactions responsible
the assembly of the integrative and excisive synaptic
for
structures also in£uence the resolution of the
Mu left (L) and right (R) ends. Open ribbons are the res site accessory subsites II and III, the cer site accessory sequences
bacteriophage
or the recombinational and transpositional enhancers (E). In the Mu LER complex, the thick and thin lines represent the phage ge-
(AS),
and the donor DNA molecule, respectively. The wavy line is the target DNA. Only the MuA subunits that will form the active tetramenome
after conversion of the LER complex are represented. Additional molecules of MuA and of the proteins HU and IHF which participate
in the formation of the LER complex are not shown.
the ends of the integrated prophage attL
from attR) [129]. Thus, V integration and excision

172
maintained (reviewed in Refs. [6,15,66,67]). Like V
be
MuA has two distinct DNA binding domains
Int,
B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178
to determine the right-handed rotational direction of
Holliday junction intermediates, thereby reinforcing
the directionality of both recombination reactions
strand exchange [90^92,132]. As mentioned above,
[130].
single amino acid changes in resolvase and invertase
enzymes allow the requirement for supercoiling and
During V integration and excision, DNA supercoiling
acts as an additional architectural element
facilitating directional wrapping of the DNA around
higher order nucleoprotein interactions to be overcome,
underlining the regulatory function of these
interactions ([92,100,101,103] ; M.R. Boockock and
the complex, but recombination then occurs by random
collision between sites in various con¢gurations.
In contrast, it is crucial for the in vivo function of
N.D.F. Grindley, personal communication). By promoting
deletions and inversions as well as fusion
site-speci¢c resolution and DNA inversion systems
products, the behaviour of these relaxed recombinase
to recombine selectively sites which are in direct or
variants is similar to that of recombinases such as
Cre and Flp which mediate recombination with little
inverted repeat on the same DNA molecule, respectively.
For these systems, such selectivity is achieved
selectivity for a particular site organisation and with
through a recombinational synapse of a precise local
no requirement for a speci¢c DNA topology [4,106].
geometry [80].
The Xer recombination system provides an intriguing
example of functional £exibility. Recombination
at the E. coli chromosome site dif which can
The 120-bp resolvase recombination site res contains
three subsites, each of which binds two resolvase
subunits. In the recombination complex, strand
occur intermolecularly and intramolecularly appears
to be achieved solely by the action of the XerC and
exchange occurs at subsite I, while the eight resolvase
molecules bound to the other two subsites
XerD recombinases on a 28-bp core recombination
of the two synapsed res sites form a protein sca¡old
site. In contrast, Xer recombination at sites present
around which three plectonemic DNA supercoils are
in natural multicopy plasmids (e.g., cer in ColE1
interwrapped (Fig. 7a) [36]. Such a 33 synapse, in
plasmid) is preferentially intramolecular and has
which three negative supercoils are trapped, can only
the requirement for
V200 bp of accessory DNA
form readily on directly repeated sites on the same
sequence and for additional host proteins (ArgR,
DNA molecule, thereby limiting recombination to
the repressor of the arginine regulon ; and PepA,
intramolecular resolution. To form a 33 synapse
the aminopeptidase A) [40]. These are involved in
between inverted repeated sites, or between sites on
forming a synapse of de¢ned geometry in which, as
separate circular molecules, unfavourable compensating
positive supercoils would have to be introduced
elsewhere. This system therefore constitutes a
in the resolvase synaptosome, three negative supercoils
are trapped (Fig. 7c) [133]. It is likely that functionally
equivalent topological ¢lters have evolved
so-called `topological ¢lter' [80]. A similar topological
¢lter is used in the inversion reaction catalysed by
independently in a number of di¡erent recombination
systems by multiplying the recombinase binding
the DNA invertases like Gin and Hin. In this case
however, two negative supercoils are trapped in the
sites within the recombination locus (as in the resolvase
system) and/or by recruiting di¡erent host DNA
three-branched `invertasome' complex in which the
binding proteins (as in Xer recombination or in Gin
two inverted repeated recombination sites bound by
and Hin inversion systems).
the recombinase are aligned on a third and distant
element, the recombinational enhancer, which is
bound by the host factor FIS (Fig. 7b) [32,80,85].
In transposition, the higher order interactions controlling
the assembly of the active complex, or `transpososome',
not only dictate the con¢guration with
The FIS-bound enhancer is required for both for
which the transposon ends can interact, but they
the synaptic complex assembly and the activation of
also in£uence target DNA site selection. Multiple
the recombinase subunits. The latter may be
achieved by inducing a conformational change at
checkpoints in the assembly and progression of bacteriophage
Mu transpososome ensure that each cycle
the dimer interface [92,103,131]. Likewise, in both
resolvase and invertase-mediated recombination,
of replicative transposition will be successfully executed
and that the integrity of the phage genome will
DNA supercoiling energy is used after initial synapsis
to provide the driving force for the reaction and

B. Hallet, D.J. Sherratt / FEMS Microbiology Reviews 21 (1997) 157^178 173
to the transposase TnsA+B core, three other proteins
enabling a single transposase subunit to interact with
speci¢c sequence elements at the ends of Mu, as well
(TnsC, D and E) participate in Tn7 transposition
as with a transposition enhancer located within the
[11]. The use of either TnsD or TnsE in the complex
Mu genome, about 1kb from the left end. At an
directs the transposon to di¡erent types of target
early stage of the transposition reaction, a complex
sites. TnsD is responsible for the recognition of the
circuit of interactions between MuA subunits bound
speci¢c chromosome integration site attTn7, whereas
to the left end (L), the enhancer (E), and the right
end (R) promotes the formation of a transient threesite
synaptic complex called `LER' (Fig. 7d) [134].
TnsE allows Tn7 to transpose with little target speci¢city
into di¡erent sites. TnsC mediates communication
between the transposon ends and the target
Assembly of this complex requires DNA supercoiling
as well as precisely-positioned DNA bends induced
by the host proteins HU and IHF. Upon assembly,
DNA complex, and has an immunity function analogous
to that of MuB. In the TnsD-dependent pathway,
no cleavage is observed in the absence of the
target DNA, suggesting that all participants of the
the catalytically inert components of the LER complex
undergo a conformational transition converting
reaction, i.e., TnsA, B, C and D, the transposon ends
and the target site attTn7, must be assembled in the
the inactive pre-transpososome into the stable synaptic
complex in which the two Mu ends and the active
transpososome to initiate recombination [22,139].
sites of the MuA tetramer are engaged for catalysis.
As for other recombination systems, the geometry
By contrast, in IS10 transposition, the doublestrand
breaks severing the element from the donor
of the LER complex may act as a topological ¢lter
restricting synapsis to two adjacent inverted ends
locus must occur before the transpososome can interact
with the target DNA [76]. IS10 transposase is
[135]. DNA supercoiling may also provide the free
also able to use transposon ends located on distinct
molecules or in directly repeated con¢guration rather
energy required for the DNA and/or protein conformational
changes likely to occur during conversion
than the canonical inverted orientation, to carry-out
transposition [140]. Finally, when compared to Mu
of the LER complex [136]. In transpososome assembly,
the enhancer acts as a platform from which
and Tn7, IS10 is also more promiscuous with respect
MuA monomers are delivered to form the active
to target site selection. Intra-transposon insertion
events are observed and may be favoured through
tetramer at the Mu ends [68,137,138]. These MuApromoted
bridges between the enhancer and the two
a regulation pathway involving the host factor IHF
ends are reminiscent of those formed by V Int for the
synapse of two recombination sites.
A similar role as a sca¡old has been proposed for
the Mu transposition cofactor protein, MuB [137].
([141] ; R. Chalmers and N. Kleckner, personal communication).
This relative £exibility of IS10 transposition
accounts for how IS10-promoted adjacent deletions
and replicon fusions occur, as well as how
MuB is an allosteric activator of MuA that is also
new transposable element based on IS10 can form
involved in target capture and in the mechanism of
[50].
target immunity whereby self-integration by Mu is
prevented. Such immunity is conferred by a set of
interactions between MuA and MuB that precludes
5. Conclusion
Recent advances in our understanding of transposition
and site-speci¢c recombination have provided
the formation of a MuB-DNA complex in the vicinity
of a transposase-bound end. Although association
of a MuB-bound target DNA to the transpososome
is not absolutely required to initiate
new pictures of specialised recombination machines.
transposition, its presence strongly stimulates both
Some of these snapshots must now be viewed with
the end cleavage and strand transfer reactions
[6,15]. This ensures that Mu transposition will only
stereoglasses ! The emerging theme is that recombinases
are built by combining simple and conserved
chemical mechanisms of DNA cut and paste with
occur in an appropriate target, thus avoiding selfdestruction.
speci¢c DNA binding activities and the ability to
The coordination of the transposition reactions
form a complex in which the DNA substrates are
seems even tighter in the case of Tn7. In addition
brought together in order to trigger the recombina-

174
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Although the £exibility of the conservative recombination
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Acknowledgments
We are grateful to Sean Colloms, Francois Cornet
and Finbarr Hayes for critical reading of the manuscript
and for helpful comments. Work in D.J.S.'s
laboratory was supported by grants from the Wellcome
Trust and the Medical Research Council. B.H.
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the European Molecular Biology Organization and
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