Tài liệu Báo cáo Y học: Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin Micha Schwikardi and Peter Droge ¨ potx

Use of site-specific recombination as a probe of nucleoprotein complex
formation in chromatin
Micha Schwikardi and Peter Dro¨ge
Institute of Genetics, University of Cologne, Germany
DNA transactions in eukaryotes require that proteins gain
access to target sequences packaged in chromatin. Further,
interactions between distinct nucleoprotein complexes are
often required to generate higher-order structures. Here, we
employed two prokaryotic site-specific recombination sys-
tems to investigate how chromatin packaging affects the
assembly of nucleoprotein structures of different complex-
ities at more than 30 genomic loci. The dynamic nature of
chromatin permitted protein–DNA and DNA–DNA inter-
actions for sites of at least 34 bp in length. However, the
assembly of higher-order nucleoprotein structures on targets
spanning 114 bp was impaired. This impediment was
maintained over at least 72 h and was not affected by the
transcriptional status of chromatin nor by inhibitors of histone
deacetylases and topoisomerases. Our findings suggest that
nucleosomal linker-sized DNA segments become accessible
within hours for protein binding due to the dynamic nature
of chromatin. Longer segments, however, appear refractory
for complete occupancy by sequence-specific DNA-binding
proteins. The results thus also provide an explanation why
simple recombination systems such as Cre and Flp are
proficient in eukaryotic chromatin.
Keywords: chromatin; DNA reactivity; nucleoprotein com-
plex; site-specific recombination; transcription.
Alterations in chromatin structure are involved in the regu-
lation of DNA transactions such as transcription and site-
specific recombination. Recently, chromatin remodeling and

histone acetylation/deacetylation were identified as import-
ant regulators of chromatin structure at specific loci
(reviewed in [1–4]). Fundamental questions in this context
concern the general reactivity of DNA sites packaged into
chromatin. For example, does the assembly of complex
nucleoprotein structures require active chromatin remodel-
ing throughout the genome, or is remodeling only required
at specific loci? Further, the transcription process itself
transiently alters the structure of chromatin (reviewed in [5]).
Little is known, however, whether these dynamic alterations
render sequences in vivo more accessible for DNA-binding
proteins and, thus, contribute to the formation of complex
nucleoprotein structures.
Site-specific recombination has been used as a powerful
method to investigate fundamental questions both in pro-
karyotic and eukaryotic cells [6–10]. In our present study,
we sought to address the questions outlined above by
employing two site-specific recombination systems that differ
markedly in their complexity. The less elaborate system is
represented by the Cre recombinase encoded by Escherichia
coli phage P1. This enzyme is a member of the integrase
family of conservative site-specific recombinases and
functions efficiently in eukaryotic cells (reviewed in [11]).
Two Cre monomers bind cooperatively to a 34-bp recom-
bination sequence termed loxP (Fig. 1A). Collision of two
loxP-bound dimers results in the formation of a recombino-
genic complex that catalyzes two reciprocal single-strand-
transfer exchange reactions. This leads to deletion of
intervening DNA if two loxP sites are positioned as direct
repeats.

The second system employed in this study is derived from
the E. coli gd transposon-encoded resolvase. The resolvase
system is more complex than the Cre system. In the first step
leading to recombination resolvase binds to a recombination
sequence called res. A single res is composed of three
binding sites (I–III) for resolvase dimers which together
occupy 114 bp (Fig. 1B). Three dimers bind cooperatively
to res with comparable affinities towards sites I and II in
order to generate a recombinogenic complex, termed resolvo-
some [12]. Two resolvosomes then synapse by random
collision [13]. Two res must be present as direct repeats on
the same negatively supercoiled DNA molecule. Only this
site orientation leads to the formation of a functional
synaptic complex, termed synaptosome, which entraps three
(–)supercoils [14]. Strand exchange is catalyzed by dimers
bound at paired sites I, while those bound at sites II and III
serve accessory roles in synaptosome formation and in the
activation of strand cleavage therein (Fig. 1B). This rather
complex architecture imposes directionality on recombina-
tion, i.e. strand exchange always results in deletion of DNA
between two res.
Recently, we have transferred the gd system to higher
eukaryotes [10]. Two resolvases containing activating
mutations (E124Q or E102Y/E124Q) and a SV40-derived
nuclear localization signal (NLS) at their C-termini are
recombination-proficient on episomal DNA. Full res are still
Correspondence to P. Dro
¨
ge, Institute of Genetics, University of
Cologne, Weyertal 121, D-50931 Cologne, Germany.

Fax: 1 49 221470 5170, Tel.: 1 49 221470 3407,
E-mail:
(Received 10 July 2001, revised 3 October 2001, accepted 5 October
2001)
Abbreviations: GFP, green fluorescence protein; Cre, cause of
recombination in phage P1; b-Gal, b-galactosidase; PGK, phospho
glycerate kinase; DMEM, Dulbecco’s modified Eagle’s medium; TRE,
tetracyclin-responsive-element; RLHRLZ, res-lox-hygromycin-
res-lox-lacZ.
Eur. J. Biochem. 268, 6256–6262 (2001) q FEBS 2001
required for efficient recombination indicating that, even
in the absence of substrate supercoiling, the reaction
proceeds through the normal synaptic complex [10]. This
has been demonstrated directly with topologically relaxed
substrates in a recent in vitro study employing correspond-
ing mutants of the related Tn3 resolvase [15]. We also
demonstrated that the gd resolvase double mutant E102Y/
E124Q (hereafter referred to as gd102NLS) and Cre recom-
bine episomal substrates in CHO cells with comparable
efficiencies [10].
A low level of recombination is observed with gd102NLS
on episomal substrates containing two isolated, directly
repeated copies of the 32-bp long site I of res [10]. Due to
the symmetry of the central two base pairs at site I, random
collision of two site I-bound gd102NLS dimers results, in
this case, in either deletion or inversion of the intervening
DNA segment (M. Schwikardi and P. Dro
¨
ge, unpublished
results; see Fig. 1C). Hence, depending on the presence or

on the accessibility of accessory sites in res, gd102NLS
employs two recombination pathways in eukaryotic cells
that can be distinguished by the resulting products.
In order to compare the activities of Cre and gd102NLS
on substrates packaged in chromatin, we have generated
reporter cell lines that carry target sites for both recom-
binases randomly integrated into the host genome. By
comparing the efficiencies of recombination on episomal
and on genomic targets, we have shown that the dynamic
nature of chromatin renders a site of at least 34 bp in general
reactive for recombination. However, the assembly of and/
or the interaction between more complex nucleoprotein
structures on 114-bp targets was significantly impaired in
chromatin. This impediment was not affected by the tran-
scriptional status of chromatin nor by inhibitors of histone
deacetylases and topoisomerases.
EXPERIMENTAL PROCEDURES
Vectors
Expression vectors for Cre, wild-type gd resolvase, and the
two resolvase mutants have been described previously [10].
pTRE-res-lox-hygromycin-res-lox-lacZ (-RLHRLZ) was
generated by PCR using the recombination cassette of
pCH-RLNRLZ as template [10]. The neomycin gene was
substituted by the hygromycin gene of pTK-Hyg (Clontech).
The entire cassette was introduced into the Bam HI site of
pTRE2 (Clontech). The derivate pTRE-SLHSLZ was gener-
ated by PCR using pTRE-RLHRLZ as template. The corre-
sponding recombined product vectors were generated through
transformation into E. coli strain DH5a or 294-Cre [16]. They
were subsequently characterized by restriction digestion and

DNA sequencing. Substrate vectors were isolated using
endotoxin free affinity chromatography (Qiagen, Germany).
Cell culture, cell lines, transfection, and recombination
assays
CHO-AA8 Tet-Off cells (Clontech) were grown in
Dulbecco’s modified Eagle’s medium (DMEM) containing
10% fetal bovine serum, 2 m
ML-glutamine, streptomycin
(0.1 mg
:
mL
21
), penicillin (100 U
:
mL
21
), and neomycin
(400 mg
:
mL
21
). Stable reporter cell lines were generated
with SapI-linearized pTRE-RLHRLZ. Three days post-
transfection, cells that had stably integrated the vector into
the genome were selected with 350 mg
:
mL
21
hygromycin.
Transfection was generally performed with 5 Â 10

6
cells.
Electroporation was in 800 mL RPMI medium without
phenolred and glutamate (Life Technologies) at 960 mF
and 280 V. Transfection efficiencies were determined by
FACS (FACS Calibur; Becton Dickinson) using the program
CELL QUEST and GFP as a marker. They were typically in
the range of 40–70%. Trichostatin A (ICN Biomedicals,
Germany), dissolved in ethanol, was added to culture
medium at 3 m
M final concentration. Butyrate (Sigma,
Germany), dissolved in sterilized water, was tested at 0,5
Fig. 1. Schematic representations of recombination pathways.
(A) Cre-loxP pathway. LoxP sites (arrows with open head) are present
as direct repeats on a circular substrate. Synapsis occurs by collision of
two loxP-bound dimers (filled circles). The two sites are aligned in an
antiparallel orientation. Strand exchange will then lead to deletion. (B)
Recombination on two full res by wild-type or mutant resolvase. The
res are depicted as direct repeats on a circular substrate. DNA super-
coiling, required for the reaction with wild-type resolvase, is omitted for
clarity. After all three cognate sites within res (I, II, and III) are bound
by resolvase dimers (filled circles) a synaptosome is generated. The
interaction between dimers bound at accessory sites II and III, and the
catalytically active ones at paired sites I that is required to trigger strand
exchange is indicated by arrows. Recombination will lead to deletion of
DNA between two res. (C) Recombination by mutant resolvases on
sites I of res. After resolvase dimers (filled circles) are bound to sites I,
random collision leads to two functional synaptic complexes. When
subsites I align in an antiparallel orientation (top), recombination leads
to inversion of DNA between sites I. If both sites align in a parallel

orientation, recombination leads to deletion (bottom). The fact that both
types of alignment leads to productive recombination complexes is due
to the symmetric nature of the site I of res.
q FEBS 2001 Site-specific recombination in chromatin (Eur. J. Biochem. 268) 6257
and 7 mM final concentration. Camptothecin (Sigma,
Germany) was dissolved in dimethylsulfoxide and applied
at 50 m
M, and EMD 50689, dissolved in dimethylsulfoxide,
was tested at 100 m
M. In order to inactivate the TRE-CMV
promoter, doxycyclin (20 ng
:
mL
21
) was added 2 weeks
prior to electroporation.
b-Galactosidase (b-gal) assays, Southern blotting and
PCR
b-Gal assays and Southern blotting were performed as
described previously [10,17]. The
32
P-labelled probe was
generated by PCR with oligonucleotides priming in the
N-terminal domain of the lacZ gene. Genomic PCR was
performed as described previously using 0.5 mg of purified
genomic DNA as template [10].
RESULTS
Cre- and gd102NLS-mediated recombination on episomal
substrates
We employed in this study a different cell line and different

recombination substrates than in our previous study. In order
to use episomal substrates as controls it was therefore
necessary to re-investigate how Cre and gd102NLS perform
under these conditions. Further, it was necessary to analyze
recombination on linearized episomal substrates as they
better resemble the topology of targets placed in chromatin
than circular substrates used before.
The recombination substrate, termed pTRE-RLHRLZ,
contains a tetracyclin-responsive-element (TRE)-CMV pro-
moter construct placed upstream of a recombination cassette
(Fig. 2A). Transcription from this promoter is regulated in
CHO-AA8 Tet-Off cells by doxycyclin. The promoter is
active in the absence of drug, while its presence leads to
rapid transcriptional inactivation [18]. The cassette is com-
posed of two directly repeated copies of res and of loxP
sites. They flank the coding region of the hygromycin gene
which serves as the resistance marker for the generation of
stable reporter cell lines (see below). We placed the coding
region of the lacZ gene downstream of the cassette.
Transcription is initiated 127-bp upstream of the first
nucleotide defining site III of the promoter proximal res,
Fig. 2. Recombination on episomal targets. (A) Diagram of substrate
vector pTRE-RLHRLZ. Relevant genetic elements are marked and
explained in the text. Start of transcription within the TRE-CMV
promoter is at 1374. (B) Normalized b-Gal activities as reporter for
recombination on episomal pTRE-RLHRLZ. The activity of the
reporter is expressed in (%) relative light units (RLU) and normalized to
the amount of protein in crude cell extracts. The activity resulting from
the recombined product (pTRE-RLZ) cotransfected with an expression
vector for a phage l integrase mutant was set as 100%. In each case,

data were collected from six separate transfection assays, each
employing two wells containing about 2 Â 10
5
cells. (C) Normalized
b-Gal activities as reporter for recombination on pTRE-SLHSLZ. This
substrate contains two isolated sites I of res replacing the full res in
pTRE-RLHRLZ. The recombined product, termed pTRE-SLZ,
cotransfected with pPGKIntss was used again as control (100%). The
graph shows the mean values of two assays with standard deviations
indicated by vertical lines.
6258 M. Schwikardi and P. Dro
¨
ge (Eur. J. Biochem. 268) q FEBS 2001
and proceeds through the entire cassette and the downstream
lacZ gene (Fig. 2A). b-Gal assays performed with crude
extracts prepared from CHO-AA8 Tet-Off cells transfected
with pTRE-RLHRLZ confirmed that the lacZ gene is not
expressed (data not shown). Recombination by either Cre
or resolvase leads to deletion of the resistance gene and
to expression of b-Gal. Recombination by either Cre or
resolvase thus generates identical product vectors, termed
pTRE-RLZ (Fig. 2A).
Linearized substrate and product vectors were cointro-
duced with an expression vector for either Cre (pPGKCrebpa)
or gd102NLS (pPGKgd102NLS). Substrate and product
vectors cotransfected with an expression vector for the
phage lambda integrase mutant Int-h (pPGKInthss) served
as controls. It is important to emphasize here that recom-
binases are expressed from the same eukaryotic promoter
(PGK) in the same vector background, and that Cre and

gd102NLS contain identical NLS [10].
Normalized b-Gal activities were determined in cell
extracts prepared 72 h after transfection. The results show
that Cre and gd102NLS efficiently recombine linearized
pTRE-RLHRLZ (Fig. 2B). In contrast to our previous study,
however, we found that recombination by gd102NLS is
reduced in this cell line to a level of 60% of that observed
with Cre. Identical results were obtained when (–)super-
coiled instead of linearized substrates were cotransfected
with recombinase expression vectors (data not shown).
We also analyzed recombination on a linearized deriva-
tive of pTRE-RLHRLZ, termed pTRE-SLHSLZ. This sub-
strate contains two isolated sites I of res as direct repeats,
instead of two full res. gd102NLS is also proficient to
recombine sites I in the absence of accessory sites. The
efficiency of this reaction is significantly reduced, however,
reaching 10% of that observed with Cre (Fig. 2C).
Recombination on genomic substrates
Hygromycin-resistant cell lines were generated with linear-
ized pTRE-RLHRLZ. Southern blot analysis, PCR, and
DNA sequencing revealed that they contain between one
and about 20 copies of the substrate vector at different
genomic locations. Hence, the vector integrated probably
randomly into the host genome (data not shown). We first
analyzed recombination in cell line TRE2/3 which contains
a single copy of the vector. The analysis was performed in
the absence of doxycyclin, i.e. the TRE-CMV promoter is
active and transcription proceeds through the entire
recombination cassette.
The expression vectors for Cre, wild-type resolvase

(gdNLS), and the resolvase single (gd124NLS) and double
mutant (gd102NLS) were introduced separately into TRE2/3
cells by electroporation. In addition, an expression vector
for GFP was used to determine transfection efficiencies.
Genomic DNA isolated 72 h after electroporation was
digested with Bam HI and analyzed for recombination by
Southern blotting using a probe specific for the N-terminal
region of the lacZ gene (compare Fig. 2A). The results show
that Cre efficiently recombines the genomic substrate
(Fig. 3). Considering the transfection efficiency indepen-
dently determined in each experiment, a quantitative
analysis by phosphorimager from four different experiments
revealed that recombination occurred, on average, in about
66% of cells transfected with pPGKCrebpa. However,
neither gdNLS nor gd124NLS generated a detectable amount
of products. Only gd102NLS produced a faint signal. In this
case, quantitation revealed that recombination occurred in
about 2% of cells transfected with pPGKgd102NLS (see
also Table 1). Hence, compared to the reactions on episomal
targets, the efficiency of recombination by gd102NLS is
severely reduced on genomic res.
We then tested whether treatment of TRE2/3 cells with
doxycyclin affects recombination. The drug was added to
the medium 2 weeks prior to transfection. This treatment
leads to the rapid inactivation of the TRE-CMV promoter
and should provide time for a potential re-setting of the
chromatin structure. Control experiments using cell line
TRE2/3R containing one copy of recombined pTRE-RLZ,
which was subcloned from Cre-treated TRE2/3 cells, con-
firmed that b-Gal activity was reduced 200- to 300-fold

compared to a control lacking the drug. Further, the residual
activity detectable in doxycyclin-treated TRE2/3R cells was
only threefold to fivefold higher than that in parental TRE2/3
cells, indicating that the TRE-CMV promoter was efficiently
inactivated by the drug (data not shown). When we tested
recombination in doxycyclin-treated TRE2/3 cells, however,
quantitation of Southern blots revealed that Cre and
gd102NLS remained unaffected by the transcriptional status
of the recombination cassette (Table 1).
The entire set of experiments exemplified above with
TRE2/3 cells expressing either Cre or gd102NLS was
performed with five different cell lines, thus investigating
recombination on more than 30 genomic copies of pTRE-
RLHRLZ. While Cre reproducibly recombined between 40
Fig. 3. Cre, but not resolvase, efficiently recombines genomic
targets. Genomic DNA was prepared from TRE2/3 cells 72 h after
electroporation with recombinase expression vectors. DNA was
digested with Bam HI, separated on a 0.8% (w/v) agarose gel, trans-
ferred to nitrocellulose membrane, and hybridized to a probe derived
from the N-terminal region of lacZ. Bam HI-digested pTRE-RLHRLZ
and pTRE-RLZ were used as unrecombined and recombined controls,
respectively.
q FEBS 2001 Site-specific recombination in chromatin (Eur. J. Biochem. 268) 6259
and 80% of targets, gd102NLS exhibited only a residual
activity (Table 1). We conclude that Cre, irrespective of the
transcriptional status of loxP sites in chromatin, efficiently
recombines pTRE-RLHRLZ at the majority of genomic
loci. However, gd102NLS appears to be severely impaired
when targets are packaged into chromatin. Further, this
impediment is maintained irrespective of the transcriptional

status of target sequences.
gd102NLS Recombines at genomic
res
through random
collision of I sites
The residual recombination activity observed in the Southern
analyses with gd102NLS (Fig. 3; Table 1) could be due to
recombination at genomic sites I without synaptosome
formation. This would be similar to the reaction on episomal
site I substrates. To investigate this possibility, we used
b-Gal activity and PCR as reporters for recombination. The
results exemplified with cell line TRE2/3 show that a
residual b-Gal activity is indeed detectable when gd102NLS
is expressed (Fig. 4A). This activity (about 5% compared to
Cre) is in the range of that observed with episomal site I
substrates (about 10%; Fig. 2C). Further, genomic PCR
employing primer pair P1/P2 (see Fig. 2A) confirmed that
Cre and gd102NLS catalyzed deletion in TRE2/3 cells
(Fig. 4B). If the activity observed with gd102NLS results
from a simple synapse generated by random collision
between dimers bound solely at sites I, inversion of the DNA
segment located between res should also be detectable. PCR
analysis with primer pair P3/P4 and DNA sequencing of
products revealed that gd102NLS also catalyzed inversion
on genomic res (Fig. 4C). Faint PCR signals indicative of
inversion and deletion were also observed in some
experiments with gd124NLS.
Recombination is not affected by inhibitors of histone
deacetylases or topoisomerases
Histone modifications such as acetylation and phosphoryl-

ation play important roles in the regulation of chromatin
structure. In particular acetylation of the N-terminal tails
of histones are thought to render chromatin more accessible
for DNA-binding proteins. In fact, DNA transactions such
as V(D)J recombination and transcription are enhanced
when histone deacetylases are inhibited [19,20]. Further,
eukaryotic topoisomerases appear to be involved in
chromatin organization, perhaps through direct interaction
with histone deacetylases [21]. We decided therefore to test
whether inhibitors of histone deacetylases and eukaryotic
topoisomerases might render genomic res more accessible
for gd102NLS, which could then lead to a significant
increase in recombination activity.
Two reporter cell lines were incubated with histone
deacetylase inhibitors butyrate or trichostatin A (reviewed in
[22]) at 24 h after transfection of recombinase expression
vectors. Cells were treated for 24 h, after which drugs were
removed and cells were incubated for additional 24 h.
Untreated cells served as controls. Further, controls with
b-Gal-expressing TRE2/3R cells treated in the same way
with trichostatin A showed that b-Gal activity increased up
to fourfold. This might indicate that the TRE-CMV pro-
moter becomes more accessible for the transcriptional
machinery. However, Southern analysis and b-Gal assays
revealed that the efficiencies of recombination by Cre and
gd102NLS remain unaffected by these inhibitors. Following
the same protocol, treatment of TRE2/3 cells with topo-
isomerase type I inhibitor camptothecin and with the
flavonoid EMD50689, the latter inhibits both type I and type
II topoisomerases [23], also showed no effect on recom-

bination efficiencies (data not shown).
DISCUSSION
We have investigated the reactivity of chromosomal DNA
for two prokaryotic site-specific recombinases. Episomal
substrates were used first as controls because previous
studies indicated that transfected, nonreplicating plasmid
DNA is either not packaged into chromatin [10] or exhibits
an atypical chromatin structure even at 4 days after trans-
fection [24]. Our comparison between episomal and
genomic substrates revealed that chromatin packaging of
loxP sites did not significantly affect the activity of Cre at
more than 30 different genomic loci. Likewise, the rather
inefficient gd102NLS recombination pathway employing a
synapse consisting solely of two site I-bound dimers
appeared to be functional to about the same extent on
episomal and on genomic substrates. The latter result is
particularly important. It implies that the steady-state intra-
cellular concentration of gd102NLS at genomic and at
episomal targets must be in the same range.
The centers of the loxP site and of the site I of res located
next to each other in the recombination cassette are separ-
ated by 45 bp. Hence, the two sites together occupy 78 bp.
The fact that both sites remained reactive when packaged in
chromatin implies that the nucleosomal structure of
chromatin must be rather dynamic, at least over the time
frame of our experiments. Assuming that the nucleosomal
positioning is not determined by the sequence of our
Table 1. Recombination on genomic targets. The values represent mean values of (%) recombination determined from two to four experiments.
An asterisk indicates data from one experiment. ND, not determined.
Cell line

Cre gd102NLS
(–)Doxy (1)Doxy (–)Doxy (1)Doxy
TRE2/3 (single copy) 66.8 74.2 2.0 2.5
TRE3/2 (single copy) 42.3 39.1 2.2 1.3
TRE3/5 (single copy) 75.0 ND ND ND
TRE2/2 (. 20 copies) 50.2 50.2 2.5* ND
TREII/3 (. 10 copies) 84.0 76.2 2.2 2.3
6260 M. Schwikardi and P. Dro
¨
ge (Eur. J. Biochem. 268) q FEBS 2001
substrate, the loxP site and the site I of res may be located
either in the nucleosomal core or in the linker DNA; the
length of the latter can vary significantly in vivo. In addition,
productive encounters readily occurred between sites I of
res and between loxP sites separated by 1.2 kb, thus further
strengthening the view of a rather flexible chromatin struc-
ture [2,9]. Importantly, our results have shown that these
basic properties of chromatin are not significantly altered by
the transcriptional status of DNA.
In contrast to loxP sites and sites I of res, the reactivity of
episomal and genomic full length res differed markedly in
our analysis. Genomic res were about 30-fold less reactive
for recombination than their epsiomal counterparts. We
consider two explanations for this result. Firstly, the ordered
nucleosomal organization of chromatin prevents the co-
operative binding of gd102NLS to all three sub-binding
sites. Even the repeated passage of the transcriptional
machinery does not affect this accessibility limit. Further-
more, as a significant amount of gd102NLS is present in
CHO cells throughout the time course of our experiments

[10], neither changes in chromatin structure occurring
during the cell cycle nor the passage of a replication fork
render two 114-bp spanning res simultaneously accessible
for the recombinase.
Secondly, if we assume that two resolvosomes form
simultaneously on genomic substrates, their interaction to
generate a functional synaptosome may be prevented by
unknown structural features of chromatin. It is possible, for
example, that the toroidal wrapping of DNA in eukaryotic
chromatin somehow precludes the plectonemic intertwining
of res sites, which is required to assemble a functional
synaptosome [14]. Nevertheless, sites I of res aligned in
two different orientations, leading to deletion or inversion
of the intervening DNA (Fig. 4). This implies that the 1.2-kb
long DNA segment connecting two res sites must be rather
flexible despite its nucleosomal organization.
Our analysis employing inhibitors of histone deacetylase
and topoisomerases revealed that the reactivity of res
remained unaffected, even though the transcriptional activity
of the TRE-CMV promoter was enhanced several-fold by
both types of inhibitors. Apparently an opening of the
chromatin structure through hyperacetylation of histones
and possible changes in DNA and/or chromatin topology
resulting from topoisomerase inhibition were not sufficient
to render genomic full length res accessible for resolvase
binding. It will nevertheless be interesting to test in the
future a wider range of substances for their potential to
activate resolvase-mediated recombination on genomic res.
Our results thus suggest that there is a general requirement
for substantial active chromatin remodeling in order to

assemble multicomponent nucleoprotein complexes. This
requirement significantly increases the stringency with
which DNA transactions are regulated in higher eukaryotes.
ACKNOWLEDGEMENTS
We thank members of our and of K. Rajewsky’s laboratory for critical
comments on the manuscript. The flavonoid EMD50689 was a kind gift
of Dr J. Ko
¨
hrle, Wu
¨
rzburg, Germany. Special thanks go to K. Rajewsky
for support with cell culture facilities. This work was financed through
SFB 274 and Deutsche Forschungsgemeinschaft grant Dr187/8–2
(PD).
Fig. 4. Resolvase recombines at genomic res via random collision
of site I-bound dimers. (A) Normalized b-Gal activities as reporter
for recombination in pTRE2/3 cells. The graph shows the mean values
from five transfection experiments. The 100% reference was obtained
with crude extracts prepared from pTRE2/3R cells transfected with
pPGKCrebpa. Note that the RLU are plotted in a logarithmic scale.
(B) PCR to analyze deletion in TRE2/3 cells. Genomic DNA was
prepared 72 h after transfection. The PCR product indicative of deletion
is marked (del.). The product resulting from unrecombined genomic
pTRE-RLHRLZ is also indicated (unrec.). (C) PCR to analyze inversion
in TRE2/3 cells. Only one PCR product (inv.) is generated. Products
were analyzed on 0.8% agarose gels and visualized by UV after
ethidium bromide staining.
q FEBS 2001 Site-specific recombination in chromatin (Eur. J. Biochem. 268) 6261
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