Genetic recombination and the cell cycle

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology0950-382XBlackwell Publishing Ltd, 2004? 200454511511160Review ArticleGenetic recombination and the cell cycleC. Lesterlin, F.-X. Barre and F. Cornet

Molecular Microbiology (2004) 54(5), 1151–1160

doi:10.1111/j.1365-2958.2004.04356.x

MicroReview
Genetic recombination and the cell cycle: what we have
learned from chromosome dimers
Christian Lesterlin,* François-Xavier Barre and
François Cornet
Laboratoire de Microbiologie et de Génétique Moléculaire,
118, route de Narbonne, F-31062 Toulouse Cedex,
France.
Summary
Genetic recombination is central to DNA metabolism.
It promotes sequence diversity and maintains
genome integrity in all organisms. However, it can
have perverse effects and profoundly influence the
cell cycle. In bacteria harbouring circular chromosomes, recombination frequently has an unwanted
outcome, the formation of chromosome dimers.

Dimers form by homologous recombination between
sister chromosomes and are eventually resolved by
the action of two site-specific recombinases, XerC
and XerD, at their target site, dif, located in the replication terminus of the chromosome. Studies of the
Xer system and of the modalities of dimer formation
and resolution have yielded important knowledge on
how both homologous and site-specific recombination are controlled and integrated in the cell cycle.
Here, we briefly review these advances and highlight
the important questions they raise.
Formation of dimers and control of crossing over
Several sets of experiments indicate that 10–15% of the
growing Escherichia coli cells require Xer recombination
to resolve chromosome dimers and allow monomer products to segregate correctly (Cornet et al., 1996; Steiner
and Kuempel, 1998a; Perals et al., 2000). This requirement for Xer recombination is not observed with recA
strains, no doubt because the vast majority of dimers form
by homologous recombination (Steiner and Kuempel,
1998a; Perals et al., 2000). The concentration of sister
chromatids, which varies with the growth rate, has a modAccepted 13 August, 2004. *For correspondence. E-mail Christian.
Lesterlin@ibcg.biotoul.fr; Tel. (+33) 561 33 59 85; Fax (+33) 561 33
58 86.

© 2004 Blackwell Publishing Ltd

est effect on dimerization. The apparent frequency of
dimerization may be increased after treatment with
DNA-damaging agents or in hyper-recombinant mutants
(Steiner and Kuempel, 1998a). Inactivation of RecA also
suppresses defective segregation that results from inactivation of the Xer system in Vibrio cholerae (Huber and
Waldor, 2002).
Homologous recombination may be required for the
completion of replication (Fig. 1A). For instance, the processing of replication forks that halt at a DNA lesion may
create DNA ends that need recombination to be resealed
so that replication can restart (Kuzminov, 1999). The
recombination process can either exchange the flanking
sequences (referred to as ‘Sister Chromatid Exchange’ or
‘Crossing Over’) to produce a dimer, or not, leaving monomeric chromosomes. Thus, the rate of dimer formation
depends both on the frequency of recombination between
sister chromosomes and on the frequency at which
recombination events lead to sister chromatid exchange.
Two major RecA-dependent recombination pathways exist

in E. coli, referred to as the RecBC- and the RecF-dependent pathways. One starts from the processing of doublestrand breaks by the RecBCD complex whereas the second depends on RecFOR and is thought to begin from
single-strand gaps (Cox et al., 2000). Both pathways produce a Holliday junction (HJ) that is normally resolved by
the RuvABC complex, although it may be processed by
other means in the absence of Ruv (Van Gool et al., 1999;
Cromie and leach, 2000; Michel et al., 2000; and references therein). The junction is displaced from the point of
strand invasion by the RuvAB strand migration activity and
is subsequently cleaved by RuvC. The orientation of
cleavage by RuvC determines whether sister chromatid
exchange occurs. In vitro, a strong bias in the sense of
resolution by RuvABC is dictated by the direction of migration of the complex (Van Gool et al., 1999), suggesting
that the outcome of recombination is determined by
events before resolution of the junction. Consistent with
this is the observation that whereas ruv and xer mutations
have modest effects on viability, they are nearly lethal
when combined (Michel et al., 2000). However, HJs are
not a compulsory intermediate of homologous recombination events. For instance, double-strand breaks resulting

1152 C. Lesterlin, F.-X. Barre and F. Cornet

Fig. 1. Chromosome dimers and the cell cycle. The cartoons represent a E. coli cell with chromosomes in red and blue. The green circle represents

oriC and the black and white squares represent the dif sites.
Left. Formation of a dimer during replication. The yellow flash represents recombination repair of a double-strand break leading to a crossing
over. This creates a dimer that persists until septation initiates (symbolized as the pale orange zone).
Middle. Dimer resolution. The two dif regions are thought to be loosely positioned in the septum vicinity, either on opposite sides (as represented
here) or on the same side of the septum. FtsK (yellow bouquet) loads onto DNA, translocates towards dif sites and activates XerCD/dif
recombination, allowing normal cell division.
Right. No resolution. In cells unable to resolve a dimer, septum constriction proceeds and breaks are induced in the entrapped DNA. This leads
to SOS induction, inhibition of further cell division and formation of twin filaments.

from endonucleolytic cleavage may be repaired by a Ruvindependent pathway that involves the RecG helicase
(Meddows et al., 2004).
Both RecBC- and RecF-dependent recombination pathways may produce chromosome dimers during normal
growth (i.e. in the absence of DNA damaging agent or
recombination inducing mutations), at least when the
other pathway is inactivated (Steiner and Kuempel,
1998b; Perals et al., 2001). RecBC-dependent dimer formation can be explained by the application of the
observed bias of RuvABC cleavage to ‘canonical’ models

of RecBC-driven recombination (Cromie and Leach,
2000; Michel et al., 2000). However, the mechanism by

which RecF-dependent recombination produces dimers
during normal growth remains an open question. In addition, RecBC- and RecF-dependent recombination pathways may respond differently to induction of particular
DNA damage. This has been shown in the cases of UV
irradiation and of the processing of long palindromic
sequences inserted on the chromosome (Cromie and
Leach, 2000). In both cases, the induced dimers were
shown to be only produced by RecBC-dependent recom© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

Genetic recombination and the cell cycle 1153
bination. In contrast, RecBC-dependent repair of a double-strand break created by endonucleolytic cleavage or
ionizing radiations does not create dimers in wild-type
strains (Meddows et al., 2004). This bias towards the
absence of dimerization requires both RuvABC and
RecG. Clearly more investigations are required to understand the multiple ways dimers can form and the mechanisms that modulate their frequency.
Cell division-induced breakage of chromosome
dimers
Mutants unable to resolve chromosome dimers (because
of a defect in the Xer system) display a characteristic
phenotype, with increased generation time, frequent formation of filaments containing aberrantly segregated DNA
masses and partial induction of the SOS system (Blakely

et al., 1991; Kuempel et al., 1991). Inactivation of dimer
resolution also induces high frequencies of homologous
recombination between directly repeated sequences
inserted in the vicinity of the resolution site, dif, located in
the replication terminus. Pioneering work on this hyperrecombination phenomenon (denominated ‘terminal
recombination’ to its zone of occurrence) (Louarn et al.,
1991; 1994; Corre et al., 1997; 2000) and investigation of
the fate of dimer-containing cells (Hendricks et al., 2000;
Prikryl et al., 2001) led to the proposal that the following
sequence of events occurs in a cell unable to resolve its
chromosome dimer (Fig. 1C). (i) Replication and segregation proceed until the bulk of the sister chromosomes are
distributed to each daughter cell, but remain linked by
DNA passing through the division septum (Fig. 1). The
linking DNA is thought to be the dif region (see below).
(ii) Cell division proceeds and the septum constricts. This
process induces lesions that cause double-strand breaks
in the linking DNA. The mechanism that generates these
breaks (referred to as the ‘guillotine’ effect) remains
unclear. It certainly does not only involve physical shearing of the DNA by the closing septum or by physical
tension, as the force required to break a DNA duplex

appears out of the range of forces involved in chromosome segregation and cell division. An enzymatic activity
may be involved, either a nuclease induced by septum
closure or DNA lesions resulting from normal activities
such as an attempt to replicate the entrapped DNA. This
may occur, for instance, via a mechanism equivalent to
the processing of stalled replication forks by RuvABC
(Seigneur et al., 1998). However, no data support this
hypothesis at present. (iii) The RecBCD complex loads
onto the DNA ends and degrades DNA. Occasionally,
RecABC-dependent recombination may reseal one of the
sister chromosomes, giving rise to the terminal recombination phenomenon. In the absence of repair, degradation
proceeds and produces SOS-inducing signals, thus fur© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

ther inhibiting cell division and leading to formation of twin
filaments and cell death.
Xer: the house-keeping site-specific
recombination machine
Xer recombination is catalysed by two site-specific recombinases of the tyrosine recombinase family, XerC and
XerD (Blakely et al., 1993; Azaro and Landy, 2002).
Orthologues of XerC and XerD are found in most eubacteria that harbour circular chromosomes (Recchia and

Sherratt, 1999; Chalker et al., 2000) and have been
shown to be required for faithful segregation of the chromosome in Bacillus subtilis (Sciochetti et al., 1999) and V.
cholerae (Huber and Waldor, 2002). Xer mutants sometimes display intriguing and unexplained phenotypes, indicating that Xer recombinases may function in processes
other than chromosome dimer resolution. For instance,
XerD (but not XerC) appears essential for growth in Staphylococcus aureus (Chalker et al., 2000).
Xer recombination occurs between two ‘core’ recombination sites, which are 28- to 30-bp-long DNA sequences
containing one binding site for each recombinase separated by a 6- to 8-bp-long ‘central region’ (Fig. 2B).
Despite the peculiarity of using two different recombinases, whose raison d’être remains unclear but which has
proven to be very useful for mechanistic studies, the
mechanism of Xer recombination is thought to conform to
the tyrosine recombinase paradigm. Functional and structural data obtained with the Cre/loxP system have given
insight into this mechanism (Ghosh and Van Duyne, 2002;
and references therein) which is illustrated in Fig. 2C.
Recombination is catalysed inside a tetramer of recombinases bound to a pair of core sequences arranged in antiparallel configuration. Strand exchanges (located at the
edges of the central region) transit through covalent joints
between the cleaved 3¢ ends of the DNA and the conserved tyrosines of the recombinases. These covalent
joints are subsequently attacked by the free 5¢-OH ends
liberated by the cleavages. Exchange of a first pair of
strands is catalysed by a pair of recombinases (either
XerC or XerD, see below) and leads to an intermediate

containing an HJ. This complex then isomerizes to allow
exchange of the second pair of strands by the second pair
of recombinases.
Interestingly, the Xer system has been co-opted during
evolution by accessory genetic elements. XerCD catalyses resolution of multimers of certain plasmids that carry
Xer target sites (e.g. those of the ColE1 family and
pSC101) thereby contributing to their stability, and can
also mediate integration and excision of temperate phage
genomes into and out of their host chromosomes (e.g. the
filamentous phages CTX and VGJ of V. cholerae) (Huber
and Waldor, 2002; Campos et al. 2003a,b). How is Xer

1154 C. Lesterlin, F.-X. Barre and F. Cornet
Fig. 2. The Xer system.
A. The drawing shows the region from 1000 kb
to 2000 kb on the linear map of the E. coli
chromosome. dif (the black and white squares
represent the XerD and XerC binding sites
respectively) lies in the replication fork trap
delimited by two series of Ter sites (black flags).

A zoom shows the ORFs surrounding dif.
B. Alignment of dif sites from different bacteria
(top) and core sequences of plasmid-borne Xer
sites (bottom). The central region and the XerC
and XerD binding sites are indicated.
C. Simplified model for XerCD-mediated strand
exchanges. (i) Synapse. The two core
sequences are in an anti-parallel configuration
bound with a recombinase tetramer. XerD is
shown in grey and XerC white. The small open
circles on the DNA represent the scissile phosphates that border the central region. The large
circles in front of the DNA represent the Nterminal DNA-binding domain. A flexible linker
connects this domain to the catalytic C-terminal
domain (the large ellipse with the catalytic
tyrosine residue shown) to form a C-shaped
clamp around the DNA. The main DNA deformation (the pronounced kink at the uncleaved
edge of the central region) is shown. In this
conformation, the two XerD monomers are in
an active conformation for cleavage. (ii) Transfer
of the first pair of strands by XerD-mediated

catalysis leads to the Holliday junction. This
four-way junction isomerizes (here symbolized
by changes in the angles between the four DNA
arms of the junction) to allow exchange of the
second pair of strands. (iii) Isomerization leads
to catalytic activation of the previously inactive
XerC pair of recombinase and inactivation of
the previously active XerD monomers and
allows exchange of the second pair of strands
by a mechanism similar to that involved in
exchange of the first pair of strands (iv).

recombination adapted to these different tasks? The outcome and the modalities of Xer recombination are influenced by the sequence of the recombination sites, by
intrinsic properties of the recombinases and also by modification of the nucleoprotein structure that are imposed
by additional factors. In plasmid multimer resolution,
accessory proteins and DNA sequences impose a ‘topological filter’ on Xer recombination, which ensures that it
occurs between sites that are directly repeated on the
same molecule (Alen et al., 1997; Colloms et al., 1997).
Xer recombination complexes are naturally inclined to initiate recombination by XerC-mediated catalysis (Arciszewska and Sherratt, 1995; Arciszewska et al., 1995; Hallet
et al., 1999; Barre et al., 2000). Recombination between
plasmid-borne sites naturally follows this route although
the order of strand exchange may be reversed if the orientation of the core relative to the accessory sequences
is inverted (Bregu et al., 2002). XerD-mediated catalysis
is even dispensable for recombination in certain cases

(Colloms et al., 1997). Chromosome dimer resolution follows a different route: it is initiated by XerD-mediated
catalysis and is controlled by features of chromosome
segregation and by a DNA translocase associated with
the division septum, FtsK (Aussel et al., 2002).
Coupling dimer resolution to cell division:
the FtsK protein
Although a pair of dif sites is available for recombination
after termination of replication, resolution of dimers is
postponed until about 20 min later, at the time of constriction of the division septum (Steiner and Kuempel, 1998b).
Moreover, inhibition of septation by drug treatment or by
shifting a thermosensitive ftsZ mutant to restrictive temperature inactivates dimer resolution (Steiner and Kuempel, 1998b). In the time period between termination and
septation [the G2 (D) period of the E. coli cell cycle], the
two dif regions remain close to each other in vicinity of the
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

Genetic recombination and the cell cycle 1155
forming septum (Niki et al., 2000; Li et al., 2002; Lau
et al., 2003). During this period, the sites are thought to
be bound by XerCD and are able to synapse and form HJ
intermediates (Barre et al., 2000). These HJ intermediates, formed by XerC-mediated catalysis, are not converted to products but can be reverted to substrates by
further XerC-mediated catalysis (Barre et al., 2000). Productive recombination depends on FtsK, a multifunctional
protein associated with the division septum (Recchia
et al., 1999; Steiner et al., 1999; Aussel et al., 2002).
FtsK is an essential protein required for cell division
(Begg et al., 1995). It is well conserved, even in bacteria
harbouring linear chromosomes, but its role in Xer recombination is less conserved than the protein itself. For
instance, the Haemophilus influenzae FtsK orthologue
conforms to the E. coli paradigm in that it is required for
Xer activation (Yates et al., 2003) but this is not the case
for either of the two FtsK homologues of B. subtilis (Sciochetti et al., 2001). The N-terminal domain of FtsK (ª200
amino acids, harbouring four transmembrane segments)
anchors the protein to the membrane and locates it at the
division septum. This domain is required for cell division
and is essential for viability (Draper et al., 1998; Wang
and Lutkenhaus, 1998; Yu et al., 1998). The C-terminal
domain, connected to the N-terminal domain by a ª600amino-acid linker of obscure function, is required for resolution of dimers (Recchia et al., 1999; Steiner et al.,
1999). This domain alone was shown to be sufficient for
activation of Xer recombination in vivo (Barre et al., 2000)
and a complete Xer recombination reaction between dif
sites could be reconstituted in vitro using a truncated form
of FtsK carrying an intact C-terminal domain (FtsK50C)
(Aussel et al., 2002).
FtsK does not activate Xer recombination by forcing a
topological filter on the complex as it does in resolution of
plasmid multimers (Aussel et al., 2002). FtsK-driven
recombination may occur between dif sites in direct or
inverted repetition, carried by the same molecule or not.
Nevertheless, FtsK conditions the recombination reaction
to yield topologically simple products, showing that it
imposes a defined conformation on the recombination
synapse. FtsK50C was shown to be an ATP-dependent
DNA translocase, which can create domains of superhelicity on circular and linear molecules. It was thus suggested that FtsK favours the encounter of recombination
sites by slithering when creating plectonemic loops on
DNA substrates (Ip et al., 2003). Direct evidence for loop
formation by FtsK50C was recently obtained from singlemolecule experiments (Saleh et al., 2004). This activity is
not sufficient to induce recombination. A local action
involving direct contact between the extreme C-terminal
domain of FtsK and the recombination complex is required
(Aussel et al., 2002; Yates et al., 2003). This activity also
requires ATP hydrolysis (Massey et al., 2004). It is thought
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

that FtsK resets the XerCD/dif synapse to a productive
complex within which XerD catalyses exchange of the first
pair of strands.
Chromosome polarization controls resolution
of dimers
A second crucial element in chromosome dimer resolution
(and, in fact, the first discovered) is the position of the dif
site on the chromosome. In E. coli, as in most bacteria,
dif is located opposite to oriC. This position is crucial for
dimer resolution (Leslie and Sherratt, 1995; Tecklenburg
et al., 1995; Cornet et al., 1996; Kuempel et al., 1996). To
be active, dif must be inserted within a narrow zone
around its natural position, the DAZ (dif activity zone). This
zone and the region where replication terminates,
although naturally coinciding, are functionally separate
(Cornet et al., 1996). The DAZ is the scene of specific
recombination between dif sites that occurs only in cells
that are able to form chromosome dimers (i.e. proficient
for homologous recombination) (Perals et al., 2000;
2001). This strongly suggests that the formation of an
active XerCD/dif–FtsK complex is restricted to cells with
a dimerized chromosome.
A search for the determinants of DAZ positioning
revealed an unexpected phenomenon, chromosome
polarization. The sequences surrounding dif appear to be
intrinsically polarized along the oriC-dif axis and their relative orientation is the main determinant of DAZ positioning. Notably, deletion of sequences surrounding dif is
harmless, whereas inversion of the same sequences
inhibits dimer resolution (Tecklenburg et al., 1995; Cornet
et al., 1996; Perals et al., 2000). The data suggest that the
polarization determinants are present throughout a large
terminal domain (more than 200 kb around dif) and are
highly repeated. The genome of bacteriophage l also
appears to be polarized by equivalent signals, as insertion
of l in one orientation near dif inhibits dimer resolution
whereas insertion in the other does not (Corre et al.,
2000).
These findings revealed a new aspect of chromosome
organization: the first functional implication of long-range
polarization of the chromosome. Identification of the polarization signals is complicated by the fact that inversion of
one or a small number of elements does not have a
detectable effect on dif activity (Perals et al., 2000). Chromosome sequences are oriented following the oriC/Ter
axis, defining the two replichores (Blattner et al., 1997).
Several types of short-sequence elements showing a
strongly biased orientation following the oriC-dif axis exist.
This results from the intrinsic biased orientation of chromosome sequences that define its replichore organization: strongly expressed genes, G/C skew, Chi sites and
numerous other oligomers (Salzberg et al., 1998; Lobry

1156 C. Lesterlin, F.-X. Barre and F. Cornet
and Louarn, 2003). Among these, a family of oligomers
containing the AGGG sequence appears the most likely
to underlie the polarization phenomenon observed. These
oligomers are over-represented in the chromosome and
exhibit a strong bias along the oriC-dif axis that increases
towards dif (Lobry and Louarn, 2003), and the orientation
of which switches at dif. The l genome is polarized by the
same motifs. However, the role of these elements in functional polarization remains to be proven experimentally.
Positioning the chromosome for dimer resolution
We do not know how polarization is recognized and put
to use in order to organize Xer recombination in vivo. A
good candidate for positioning of the dif sites before
recombination and polarization reading is the protein
FtsK. In this view, septum-associated FtsK would load
onto chromosomes and mobilize DNA according to its
intrinsic polarization. This process would stop when
encountering XerCD-bound dif sites, thereby ensuring a
proper sorting of chromosomal DNA in the sister cells and
synapse of the dif sites (Fig. 3). Then a physical contact
between XerCD/dif complexes and septum-borne FtsK
allows resolution of dimers to occur. As described in
Fig. 3, we postulate that, when a dimer is present, the
XerCD/dif complexes and FtsK colocalize at the division
septum at the time of septation. This restricts dif recombination to the septum region. Localization of FtsK is
ensured by its N-terminal domain and localization of the
dif sites by a chromosome polarization-dependent process. In a cell harbouring a chromosome dimer, two DNA

stretches persist between the two segregated chromosomes and are trapped by the forming septum. The
entrapped regions belong to the part of the chromosome
that is the last to be segregated. The fact that terminal
recombination concerns only the dif region and is maximal
at the dif position is the best evidence for this model and
corroborates the observed intracellular location of these
regions (Niki et al., 2000; Li et al., 2002; 2003; Lau et al.,
2003). Precise positioning of the dif sites is then achieved
by a polarization-dependent process, which allows formation of a productive synapse between dif sites.
FtsK may read chromosome polarization
The DNA translocase activity of FtsK makes it a good
candidate for positioning of the dif sites before recombination. In this view, septum-associated FtsK would load
onto chromosomes and mobilize DNA according to its
intrinsic polarization. This process would stop when
encountering XerCD-bound dif sites, thereby ensuring a
proper sorting of chromosomal DNA in the sister cells and
synapse of the dif sites (Fig. 3). Several lines of indirect
evidence support this hypothesis as they show that activation of recombination is not the only role of FtsK in dimer
resolution. (i) Although the Cre/loxP system recombines
independently of FtsK and may functionally replace
XerCD/dif, resolution of dimers by Cre/loxP depends on
FtsK (Capiaux et al., 2002). (ii) Expression of the C-terminal domain of FtsK (lacking the N-terminal domain) in a
strain lacking the C-terminal domain allows high frequency of recombination between dif sites (for instance,

Fig. 3. Model for segregation of the Ter
domains and chromosome dimer resolution.
The cartoon represents the central part of a
dividing cell. The yellow bouquet represents
hexamers of FtsK bound at the constricting
septum. The chromosomes are shown as red
and yellow lanes, the dif sites as black and
white dumbbells and the recombinases as the
rose and green circles.
A. The two replicated dif regions are represented
in a cohesion state symbolized by the persistence of intercatenation links between them.
(AfiD) In the case of monomers, decatenation
allows final separation of the chromosomes with
no requirement for a contact between the recombination complexes and FtsK.
B. In the case of a dimer, decatenation leaves
the sister dif region trapped at the septum. FtsK
translocation towards the dif sites is oriented by
chromosome polarization (represented by the
arrowheads). This process is coupled with
removal of the catenation links by Topoisomerase IV that interacts with FtsK.
C. FtsK then contacts the recombination complexes and allows formation of a productive
synapse properly conformed for XerD catalysis.
D. Recombination resolves the dimer allowing
complete separation of the sister chromosomes.
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

Genetic recombination and the cell cycle 1157
between two sites inserted as direct repeats on the chromosome or on a plasmid) but does not support resolution
of dimers (Barre et al., 2000; Perals et al., 2001). (iii) In
strains harbouring displaced dif sites (but unable to
resolve dimers because the sites are outside of the DAZ),
overexpression of wild-type FtsK activates dif recombination but does not restore dimer resolution (Barre et al.,
2000). The last two findings also suggest that only septum-associated FtsK supports dimer resolution.
Most importantly, the outcome of terminal recombination in ftsK mutants supports a role for FtsK in reading
polarization. Frequencies of recombination between l
prophages in the terminal region depend on the orientation of the l prophage with respect to dif. A simple explanation is that the l genome carries polarization
determinants so that its presence may locally perturb
polarization and thus dimer resolution (Corre et al., 2000).
This orientation effect is abolished in ftsKC– cells (lacking
the C-terminal domain) (Corre and Louarn, 2002). Moreover, the distribution of DNA breaks resulting from unresolved dimers (inferred from terminal recombination
frequencies) is affected in ftsKC– cells. When compared
with xer strains, DNA breaks are less precisely located in
the immediate vicinity of dif and are spread more homogenously over a large terminal domain (Corre and Louarn,
2002). This can be taken to reflect two complementary
levels of sequence positioning. The first ensures approximate positioning of a large Ter domain around midcell.
Positioning of this domain is independent of FtsK and
chromosome polarization (see below). The second
involves reading of polarization elements by FtsK (and
possibly other factors) to sort DNA on either side of the
closing septum and thus to position the dif sites in the
case of dimeric chromosomes. How can DNA polarity
affect FtsK-dependent positioning of the dif sites? Monitoring DNA translocation by FtsK50C at the single molecule
level did not reveal any direct influence of the DNA
sequence, suggesting that the control effected by DNA
polarity on dif positioning is a complex phenomenon that
implies the activities of other proteins in vivo and/or of
other domains of the FtsK protein (Saleh et al., 2004).
Interestingly, the C-terminal domain of H. influenzae FtsK
can replace its E. coli counterpart for the in vivo processing of DNA polarity inside E. coli, which indicates conservation of the mechanism of polarity reading (Bigot et al.,
2004).
Folding and segregation of the Ter domain
The DAZ is contained within a larger structural entity
called the Ter macrodomain. A structural peculiarity of this
region was first suggested by the fact that it contains two
‘non-divisible zones’ that are regions refractory to inversion (Rebollo et al., 1988; Guijo et al., 2001). This Ter
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

domain was defined following the observation that
sequences belonging to a large part of the chromosome
around the terminus display similar intracellular location,
suggesting that these sequences behave as a structural
unit during the cell cycle (Niki et al., 2000). In slowly
growing cells, sequences of the Ter macrodomain move
to the new cell centre after division and remain there until
the next division. The same study revealed an equivalent
1 Mb Ori macrodomain that encompasses oriC. Macrodomain organization of the chromosome is also supported
by recent genetic data on the capacity for remote
sequences to collide (M. Valens et al., submitted). By this
criterion, macrodomains, within which communication
between sequences is frequent, appear insulated from the
rest of the chromosome. The Ter macrodomain defined by
both approaches extends from about 25¢ (1150 kb) to 45¢
(2050 kb) on the chromosome. It is noteworthy that
sequences belonging to the Ter macrodomain display several peculiarities (Pedersen et al., 2000): they are particularly poor in repeated elements (REPs, BIMEs) and show
a general trend for intrinsic curvature and low flexibility.
No biological significance has been yet attributed to any
of these features.
Two recent series of experiments based on live fluorescence labelling techniques provide a closer view of Ter
macrodomain segregation (Li et al., 2002; 2003; Lau
et al., 2003). Whereas sequences outside Ter appear as
two separate foci in the cell soon after their replication,
Ter sequences most often remain together as a single
fluorescent focus during the time between termination of
replication and septum constriction (the D period, about
20¢). The replicated Ter region frequently remains asymmetrically colocalized in the cell (as represented in Fig. 3),
on one side of the division septum, until just before septum closure. Time-lapse microscopy showed that, at least
in certain conditions, one of the Ter regions may be
pumped through the closing septum (Lau et al., 2003). It
is not known at present whether FtsK is involved in this
process. Interestingly, a region of stronger cohesion
appears embedded in the Ter macrodomain (about 250 kb
containing the DAZ) (Li et al., 2003). Our recent data
reveal that FtsK acts preferentially in this cohesive region,
rather than elsewhere on the chromosome (unpublished).
An other interesting aspects is that FtsK interacts with
Topoisomerase IV, the major chromosome decatenase
that acts at a late stage of the cell cycle, apparently to
remove residual catenation links between sister chromosome after termination of replication (Espeli et al.,
2003a,b). We thus propose that the inferred cohesive
domain is a structural entity that encodes both cohesion
and polarization signals and in which catenation links
persist after termination of replication (Fig. 3). Resolution
of catenation may release the two Ter regions and allow
them to migrate. Cohesion is released during septation

1158 C. Lesterlin, F.-X. Barre and F. Cornet
and allows separation of sister chromosomes involving
oriented translocation of FtsK towards dif that facilitates
removal of the last catenation links and eventually induces
XerCD/dif recombination in the case of a dimer.
Acknowledgements
We want to thank Bénédicte Michel, Jean-Yves Bouet,
Leonara Poljak and David Lane for critical reading of this
manuscript. C.L. is funded by a PhD fellowship from the
French ‘Ministère de la Recherche’. F.X.B. received an ATIPE
from the CNRS.

References
Alen, C., Sherratt, D.J., and Colloms, S.D. (1997) Direct
interaction of aminopeptidase A with recombination site
DNA in Xer site-specific recombination. EMBO J 16: 5188–
5197.
Arciszewska, L.K., and Sherratt, D.J. (1995) Xer site-specific
recombination in vitro. EMBO J 14: 2112–2120.
Arciszewska, L., Grainge, I., and Sherratt, D. (1995) Effects
of Holliday junction position on Xer-mediated recombination in vitro. EMBO J 14: 2651–2660.
Aussel, L., Barre, F.X., Aroyo, M., Stasiak, A., Stasiak, A.Z.,
and Sherratt, D. (2002) FtsK is a DNA motor protein that
activates chromosome dimer resolution by switching the
catalytic state of the XerC and XerD recombinases. Cell
108: 195–205.
Azaro, M.A., and Landy, A. (2002) l integrase and the l Int
family. In Mobile DNA II. Craig, N.L., Craigie, R., Gellert,
M., and Lambowitz, A. (eds). Washington, DC: American
Society for Microbiology Press, pp. 118–148.
Barre, F.X., Aroyo, M., Colloms, S.D., Helfrich, A., Cornet,
F., and Sherratt, D.J. (2000) FtsK functions in the processing of a Holliday junction intermediate during bacterial
chromosome segregation. Genes Dev 14: 2976–2988.
Begg, K.J., Dewar, S.J., and Donachie, W.D. (1995) A new
Escherichia coli cell division gene, ftsK. J Bacteriol 177:
6211–6222.
Bigot, S., Corre, J., Louarn, J.-M., Cornet, F., and Barre,
F.-X. (2004) FtsK activities in Xer recombination, DNA
mobilization and cell division involve overlapping and
separate domains of the protein. Mol Microbiol
doi:10.1111/j.1365-2958.2004.04335.x.
Blakely, G., Colloms, S., May, G., Burke, M., and Sherratt,
D. (1991) Escherichia coli XerC recombinase is required
for chromosomal segregation at cell division. New Biol 3:
789–798.
Blakely, G., May, G., McCulloch, R., Arciszewska, L.K.,
Burke, M., Lovett, S.T., and Sherratt, D.J. (1993) Two
related recombinases are required for site-specific recombination at dif and cer in E. coli K12. Cell 75: 351–361.
Blattner, F.R., Plunkett, G., 3rd, Bloch, C.A., Perna, N.T.,
Burland, V., Riley, M., et al. (1997) The complete genome
sequence of Escherichia coli K-12. Science 277: 1453–
1474.
Bregu, M., Sherratt, D.J., and Colloms, S.D. (2002) Accessory factors determine the order of strand exchange in Xer
recombination at psi. EMBO J 21: 3888–3897.

Campos, J., Martinez, E., Marrero, K., Silva, Y., Rodriguez,
B.L., Suzarte, E., et al. (2003a) Novel type of specialized
transduction for CTX phi or its satellite phage RS1 mediated by filamentous phage VGJ phi in Vibrio cholerae. J
Bacteriol 185: 7231–7240.
Campos, J., Martinez, E., Suzarte, E., Rodriguez, B.L., Marrero, K., Silva, Y., et al. (2003b) VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same
chromosomal site as CTX phi. J Bacteriol 185: 5685–5696.
Capiaux, H., Lesterlin, C., Perals, K., Louarn, J.M., and Cornet, F. (2002) A dual role for the FtsK protein in Escherichia
coli chromosome segregation. EMBO Rep 3: 532–536.
Chalker, A., Lupas, A., Ingraham, K., So, C., Lunsford, R., Li,
T., et al. (2000) Genetic characterization of gram-positive
homologs of the XerCD site-specific recombinases. J Mol
Microbiol Biotechnol 2: 225–233.
Colloms, S.D., Bath, J., and Sherratt, D.J. (1997) Topological
selectivity in Xer site-specific recombination. Cell 88: 855–
864.
Cornet, F., Louarn, J., Patte, J., and Louarn, J.M. (1996)
Restriction of the activity of the recombination site dif to a
small zone of the Escherichia coli chromosome. Genes
Dev 10: 1152–1161.
Corre, J., and Louarn, J.M. (2002) Evidence from terminal
recombination gradients that FtsK uses replichore polarity
to control chromosome terminus positioning at division in
Escherichia coli. J Bacteriol 184: 3801–3807.
Corre, J., Cornet, F., Patte, J., and Louarn, J.M. (1997)
Unraveling a region-specific hyper-recombination phenomenon: genetic control and modalities of terminal recombination in Escherichia coli. Genetics 147: 979–989.
Corre, J., Patte, J., and Louarn, J.M. (2000) Prophage
lambda induces terminal recombination in Escherichia coli
by inhibiting chromosome dimer resolution. An orientationdependent cis-effect lending support to bipolarization of
the terminus. Genetics 154: 39–48.
Cox, M.M., Goodman, M.F., Kreuzer, K.N., Sherratt, D.J.,
Sandler, S.J., and Marians, K.J. (2000) The importance of
repairing stalled replication forks. Nature 404: 37–41.
Cromie, G.A., and Leach, D.R. (2000) Control of crossing
over. Mol Cell 6: 815–826.
Draper, G.C., McLennan, N., Begg, K., Masters, M., and
Donachie, W.D. (1998) Only the N-terminal domain of FtsK
functions in cell division. J Bacteriol 180: 4621–4627.
Espeli, O., Lee, C., and Marians, K.J. (2003a) A physical and
functional interaction between Escherichia coli FtsK and
topoisomerase IV. J Biol Chem 278: 44639–44644.
Espeli, O., Levine, C., Hassing, H., and Marians, K.J. (2003b)
Temporal regulation of topoisomerase IV activity in E. coli.
Mol Cell 11: 189–201.
Ghosh, K., and Van Duyne, G. (2002) Cre-loxP biochemistry.
Methods 28: 374–383.
Guijo, M.I., Patte, J., del Mar Campos, M., Louarn, J.-M., and
Rebollo, J.E. (2001) Localized remodelling of the Escherichia coli chromosome. The patchwork of segments refractory and tolerant to inversion near the replication terminus.
Genetics 4: 1413–1423.
Hallet, B., Arciszewska, L.K., and Sherratt, D.J. (1999) Reciprocal control of catalysis by the tyrosine recombinases
XerC and XerD: an enzymatic switch in site-specific recombination. Mol Cell 4: 949–959.
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

Genetic recombination and the cell cycle 1159
Hendricks, E.C., Szerlong, H., Hill, T., and Kuempel, P.
(2000) Cell division, guillotining of dimer chromosomes
and SOS induction in resolution mutants (dif, xerC
and xerD) of Escherichia coli. Mol Microbiol 36: 973–
981.
Huber, K.E., and Waldor, M.K. (2002) Filamentous phage
integration requires the host recombinases XerC and
XerD. Nature 417: 656–659.
Ip, S.C., Bregu, M., Barre, F.X., and Sherratt, D.J. (2003)
Decatenation of DNA circles by FtsK-dependent Xer sitespecific recombination. EMBO J 22: 6399–6407.
Kuempel, P.L., Henson, J.M., Dircks, L., Tecklenburg, M., and
Lim, D.F. (1991) dif, a recA-independent recombination
site in the terminus region of the chromosome of Escherichia coli. New Biol 3: 799–811.
Kuempel, P., Hogaard, A., Nielsen, M., Nagappan, O., and
Tecklenburg, M. (1996) Use of a transposon (Tndif) to
obtain suppressing and nonsuppressing insertions of the
dif resolvase site of Escherichia coli. Genes Dev 10: 1162–
1171.
Kuzminov, A. (1999) Recombinational repair of DNA damage
in Escherichia coli and bacteriophage lambda. Microbiol
Mol Biol Rev 63: 751–813, table of contents.
Lau, I.F., Filipe, S.R., Soballe, B., Okstad, O.A., Barre, F.X.,
and Sherratt, D.J. (2003) Spatial and temporal organization
of replicating Escherichia coli chromosomes. Mol Microbiol
49: 731–743.
Leslie, N.R., and Sherratt, D.J. (1995) Site-specific recombination in the replication terminus region of Escherichia coli: functional replacement of dif. EMBO J 14:
1561–1570.
Li, Y., Sergueev, K., and Austin, S. (2002) The segregation
of the Escherichia coli origin and terminus of replication.
Mol Microbiol 46: 985–995.
Li, Y., Youngren, B., Sergueev, K., and Austin, S. (2003)
Segregation of the Escherichia coli chromosome terminus.
Mol Microbiol 50: 825–834.
Lobry, J.R., and Louarn, J.M. (2003) Polarisation of prokaryotic chromosomes. Curr Opin Microbiol 6: 101–108.
Louarn, J.M., Louarn, J., Francois, V., and Patte, J. (1991)
Analysis and possible role of hyperrecombination in the
termination region of the Escherichia coli chromosome. J
Bacteriol 173: 5097–5104.
Louarn, J., Cornet, F., Francois, V., Patte, J., and
Louarn, J.M. (1994) Hyperrecombination in the terminus region of the Escherichia coli chromosome: possible relation to nucleoid organization. J Bacteriol 176:
7524–7531.
Massey, T.H., Aussel, L., Barre, F.-X., and Sherratt, D.J.
(2004) Asymmetric activation of Xer site-specific recombination by FtsK. EMBO Rep 4: 399–404.
Meddows, T.R., Savory, A.P., and Lloyd, R.G. (2004) RecG
helicase promotes DNA double-strand break repair. Mol
Microbiol 52: 119–132.
Michel, B., Recchia, G.D., Penel-Colin, M., Ehrlich, S.D., and
Sherratt, D.J. (2000) Resolution of Holliday junctions by
RuvABC prevents dimer formation in rep mutants and UV
irradiated cells. Mol Microbiol 37: 181–191.
Niki, H., Yamaichi, Y., and Hiraga, S. (2000) Dynamic organization of chromosomal DNA in Escherichia coli. Genes
Dev 14: 212–223.
© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160

Pedersen, A., Jensen, L., Brunak, S., Staerfeldt, H., and
Ussery, D. (2000) A DNA structural atlas for Escherichia
coli. J Mol Biol 299: 907–930.
Perals, K., Cornet, F., Merlet, Y., Delon, I., and Louarn, J.M.
(2000) Functional polarization of the Escherichia coli chromosome terminus: the dif site acts in chromosome dimer
resolution only when located between long stretches of
opposite polarity. Mol Microbiol 36: 33–43.
Perals, K., Capiaux, H., Vincourt, J.B., Louarn, J.M., Sherratt,
D.J., and Cornet, F. (2001) Interplay between recombination, cell division and chromosome structure during chromosome dimer resolution in Escherichia coli. Mol Microbiol
39: 904–913.
Prikryl, J., Hendricks, E.C., and Kuempel, P.L. (2001) DNA
degradation in the terminus region of resolvase mutants of
Escherichia coli, and suppression of this degradation and
the Dif phenotype by recD. Biochimie 83: 171–176.
Rebollo, J., Francois, V., and Louarn, J. (1988) Detection and
possible role of two large nondivisible zones on the Escherichia coli chromosome. Proc Natl Acad Sci USA 85: 9391–
9395.
Recchia, G.D., and Sherratt, D.J. (1999) Conservation of Xer
site-specific recombination genes in bacteria. Mol Microbiol 34: 1146–1148.
Recchia, G.D., Aroyo, M., Wolf, D., Blakely, G., and Sherratt, D.J. (1999) FtsK-dependent and -independent
pathways of Xer site-specific recombination. EMBO J 18:
5724–5734.
Saleh, O.A., Perals, C., Barre, F.X., and Allemand, J.F.
(2004) Fast, DNA-sequence independent translocation by
FtsK in a single-molecule experiment. EMBO J 23: 2430–
2439.
Salzberg, S.L., Salzberg, A.J., Kerlavage, A.R., and Tomb,
J.F. (1998) Skewed oligomers and origins of replication.
Gene 217: 57–67.
Sciochetti, S.A., Piggot, P.J., Sherratt, D.J., and Blakely, G.
(1999) The ripX locus of Bacillus subtilis encodes a sitespecific recombinase involved in proper chromosome partitioning. J Bacteriol 181: 6053–6062.
Sciochetti, S.A., Piggot, P.J., and Blakely, G.W. (2001) Identification and characterization of the dif site from Bacillus
subtilis. J Bacteriol 183: 1058–1068.
Seigneur, M., Bidnenko, V., Ehrlich, S.D., and Michel, B.
(1998) RuvAB acts at arrested replication forks. Cell 95:
419–430.
Steiner, W.W., and Kuempel, P.L. (1998a) Sister chromatid
exchange frequencies in Escherichia coli analyzed by
recombination at the dif resolvase site. J Bacteriol 180:
6269–6275.
Steiner, W.W., and Kuempel, P.L. (1998b) Cell division is
required for resolution of dimer chromosomes at the dif
locus of Escherichia coli. Mol Microbiol 27: 257–268.
Steiner, W., Liu, G., Donachie, W.D., and Kuempel, P. (1999)
The cytoplasmic domain of FtsK protein is required for
resolution of chromosome dimers. Mol Microbiol 31: 579–
583.
Tecklenburg, M., Naumer, A., Nagappan, O., and Kuempel,
P. (1995) The dif resolvase locus of the Escherichia coli
chromosome can be replaced by a 33-bp sequence, but
function depends on location. Proc Natl Acad Sci USA 92:
1352–1356.

1160 C. Lesterlin, F.-X. Barre and F. Cornet
Van Gool, A.J., Hajibagheri, N.M., Stasiak, A., and West, S.C.
(1999) Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of Holliday junction resolution.
Genes Dev 13: 1861–1870.
Wang, L., and Lutkenhaus, J. (1998) FtsK is an essential cell
division protein that is localized to the septum and induced
as part of the SOS response. Mol Microbiol 29: 731–740.

Yates, J., Aroyo, M., Sherratt, D.J., and Barre, F.X. (2003)
Species specificity in the activation of Xer recombination
at dif by FtsK. Mol Microbiol 49: 241–249.
Yu, X.C., Tran, A.H., Sun, Q., and Margolin, W. (1998) Localization of cell division protein FtsK to the Escherichia coli
septum and identification of a potential N-terminal targeting
domain. J Bacteriol 180: 1296–1304.

© 2004 Blackwell Publishing Ltd, Molecular Microbiology, 54, 1151–1160