We have purified and biochemically characterized
a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP
preferentially recombines ("swaps") sequences derived from Ig heavy
chain switch regions. We identified four of the proteins in the SWAP
complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose)
polymerase (PARP), and SWAP-70. The first three are proteins known to
be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Sµ and
S
sequences. SWAP-70 is a novel protein of 70 kDa. Its
cDNA was cloned and sequenced, and the protein was overexpressed in
Escherichia coli. SWAP-70 protein expression was found only
in B lymphocytes that had been induced to switch to various Ig isotypes
and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak
affinity for DNA, binds ATP, and forms specific, high affinity
complexes with B23, C23, and poly(ADP-ribose) polymerase. These
findings are consistent with SWAP being the long elusive "switch
recombinase" and with SWAP-70 being the specific recruiting element
that assembles the switch recombinase from universal components.
 |
INTRODUCTION |
The constant (C)1 region
of its heavy (H) chain determines the class of an Ig molecule. Mouse H
chains are designated µ,
,
3,
1,
2b,
2a,
, and
.
Upon stimulation by antigen, expression of the early IgM class usually
changes to that of another class (IgG3, IgG1, IgG2b, IgG2a, IgE, or
IgA). At the DNA level, the process is mediated by recombination that
generally involves DNA transfer between a switch (S) region within the
intron 5' to Cµ and another S region within the intron 5'
to the particular C-gene segment that is to be expressed
(1-5).
S regions, several kilobase pairs in length, contain short, G-rich
repetitive sequence elements, often arranged in tandem arrays. They may
adopt secondary structures, such as stem loops, and many break points
are located at such structures (6). In marked contrast to those
observed in V(D)J rearrangement (7-11), the switch recombination
break-and-rejoining points are imprecise. They lie scattered all over
the S regions or, less often, even outside the S regions. Thus,
class switch recombination is region-specific rather than
site-specific. Rearrangements combining three S regions are
occasionally detected (12), and, although switch recombination is
usually intrachromosomal (13), intermolecular rearrangements have
also been observed (14).
Following transcriptional activation (3, 15), switch rearrangements
generally result in deletion of the DNA sequences between the two
breakpoints. The mechanism for most class switching events can be
described by a loop-excision model (16). The exact structure of the
loop intermediate is unknown; it may resemble a crossed (
) or a stem
loop (
) conformation. In that model, after loop formation, four DNA
ends are generated by endonucleolytic cleavage of the two S regions to
be recombined; these ends are possibly protected or even fixed by
proteins. Either (i) the four ends can be rejoined in their original
configuration, (ii) the four ends can be inverted, or (iii) the
intervening sequence can be circularized and removed while the
remaining chromosomal DNA ends are joined together to yield the
switched gene. Both inversions (16) and excised circular DNAs, the
so-called switch circles of various sizes (17, 18), were observed
in vivo, but mechanistic details of the reaction remain
unknown.
To date, no isolation and biochemical characterization of an
S-region-specific recombination activity has been reported (19), although several S-region-binding proteins have been described, primarily in gel migration shift experiments (20-23). To our
knowledge, no recombination-specific function or recombination-related
enzyme activity has yet been described for these proteins. We have
screened extracts from switching B-cells for activities that
preferentially recombine S-regions. Our assay measures stable DNA
transfer, a central step of DNA recombination, from a plasmid
containing Sµ to a plasmid containing another S region. A
similar DNA transfer assay was instrumental in isolating a mammalian
protein complex that repairs DNA gaps and deletions through
recombination of homologous DNA substrates (24-26).
Here we report the isolation of a protein complex
we call it
SWAP
with a recombination activity that is specific for switching B-cells and prefers S-region substrates. We also report the identity of
four of the proteins in the SWAP complex: B23 (nucleophosmin), C23
(nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. B23
promotes single-strand DNA reannealing and the formation of joint
molecules in a D-loop assay between homologous sequences but also
between Sµ and S
sequences. SWAP-70 is a
novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70
protein expression was found only in B lymphocytes that had been
induced to switch and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and PARP.
 |
EXPERIMENTAL PROCEDURES |
DNA Transfer Assay (DTA)--
The DTA was done as described (24,
25). Input 3H radioactivity was between 150,000 and 350,000 cpm and the same for each experimental series. The Sµ
substrate consisted of an M13 double-stranded (ds) DNA carrying a
1.3-kilobase pair HindIII Sµ fragment (27),
and was labeled with digoxigenin (24). The S
plasmid
consisted of pSP72 containing a 3.7-kilobase EcoRI-HindIII S
2b fragment (28)
and was internally labeled with [3H]thymidine. For the
standard DNA transfer assay, 0.18 µg of the 3H-labeled
DNA substrate (e.g. pSP-S
) was incubated
together with 0.02 µg of the digoxigenin-labeled substrate
(e.g. M13-Sµ) and varying amounts of protein
in 50 µl containing 3 mM MgCl2, 30 mM EPPS, pH 7.4, 1 mM DTT, less than 50 mM ammonium sulfate, and 1 mM ATP. After 6 min,
the reaction was terminated by the addition of SDS to 0.05% and EDTA
to 50 mM. Further processing was as described (24) and
included a phenol-chloroform extraction step of the DNA prior to
binding the products to anti-digoxigenin beads. The activity is
expressed as a percentage of input 3H cpm, stably
transferred to the recipient and bound to the beads.
Purification of the SWAP--
The protein purification
procedures were carried out at 4 °C, and are summarized in Table I.
Nuclear extracts were prepared as described (25) from 1 × 108 to 8 × 108 lipopolysaccharide (LPS)
(50 µg/ml) blasts (0.7 mg of nuclear protein/108 cells).
Fraction I (2 mg of protein) was loaded onto a Superdex 200 FPLC gel
filtration column (Amersham Pharmacia Biotech) and fractionated at a
flow rate of 1 ml/min in Buffer E (5 mM KCl, 5 mM MgCl2, 2 mM DTT, 0.2 mM EDTA, 15 mM Tris-HCl, pH 7.5 at 4 °C, and
1 mM PMSF, 10 mM
Na2S2O5, 1 µg/ml aprotinin, 0.5 µg/ml TLCK, 0.7 µg/ml pepstatin A) containing 80 mM
ammonium sulfate. 1.4-ml fractions were collected, active fractions
were pooled (5.6 ml, 0.6 mg of protein, Fraction II), diluted 1:4 with
Buffer E, and loaded at 1 ml/min onto a 1-ml Macro S cation exchange
FPLC column (Bio-Rad). After washing the column with 20 column volumes
of Buffer E-20 (E plus 20 mM ammonium sulfate), the
proteins were eluted at a 1 ml/min flow rate with a gradient from 20 to
600 mM ammonium sulfate in Buffer E in 1.2-ml fractions.
The switch-specific activity (2.4 ml, 0.011 mg of protein, Fraction
III) eluted in two fractions at around 280 mM ammonium
sulfate. For further purification, Fraction III was diluted 1:2 with
Buffer E and loaded at 0.3 ml/min onto a 1 ml blue Sepharose (HiTrap,
Amersham Pharmacia Biotech) FPLC column (pre-equilibrated in E-140).
Elution was with a linear gradient from 0 to 1000 mM
ammonium sulfate in Buffer E, and the activity eluted between 740 and
810 mM (0.6 ml, 6 ng of protein, Fraction IV). On ice or at
70 °C, the active fractions were stable for a short period only;
frozen in liquid nitrogen, samples remained active for at least several
weeks.
Cloning and Overexpression of the SWAP-70 cDNA--
The gene
encoding SWAP-70 was cloned using a polymerase chain reaction strategy
based on degenerate oligonucleotides derived from the N-terminal 26 amino acid residues and subsequent screening of a
-ZAP mouse spleen
cDNA library (Stratagene Inc.) with a nondegenerate
oligonucleotide. The cDNA contained in the plasmid vector
pBluescriptSK was excised from the phages, and the sequence of the
SWAP-70 cDNA was determined. The cDNA was subcloned into the pQE-30
vector (Qiagen, Inc.) for expression as a His-tagged protein in
E. coli. Purification of the protein was according to
standard methods, including lysozyme-lysis of the cells, chromatography of the clear cell extract on a nickel-Sepharose column and elution at
80 mM imidazole, followed by gel filtration on a Superdex75 FPLC column (Amersham Pharmacia Biotech).
DNA and ATP Binding--
Gel retardation were performed in
10-µl reaction mixtures containing 0.2-1 ng (3000-6000 cpm) of
32P-end-labeled DNA (230-bp EcoRI-KasI M13mp18
DNA fragment, or 49-nt ss oligonucleotide) in 20 mM HEPES
(pH 7.5), 1 mM DTT, 100 µg/ml bovine serum albumin, and
protein as indicated. After 20 min of incubation at room temperature,
DNA-protein complexes were resolved by electrophoresis at 4 °C in
nondenaturing polyacrylamide gels in 20 mM HEPES (pH 7.5),
0.1 mM EDTA. All gels were fixed (60 min in 10% acetic
acid, 10% ethanol), dried, and exposed for autoradiography.
For azido-ATP labeling (29), reactions were carried out in 200 µl of
20 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 0.1 mM DTT, 5 µM
8-N3-[
-32P]ATP (9-20 Ci/mmol;
ICN), and proteins (200 ng). Reaction mixtures were incubated on ice
for 60 min and then irradiated for 50 s at a distance of 10 cm
from a XL-1500 UV cross-linker (Spectronics Co.). After irradiation,
proteins were precipitated with trichloroacetic acid and analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography.
DNA Reannealing and D-loop Assays--
The DNA single-strand
reannealing assay (3' 32P-labeled, 422-nt DNA substrates)
was performed as described (26).
For the standard D-loop assay, 100 ng (24 fmol) of predominantly
supercoiled plasmid pUC-S
DNA or, for controls, DNA linearized outside the region of homology (Asp700 restriction enzyme
digest) and 0.8 ng (100 fmol) of 5'-32P-labeled, 49-nt ss
S
consensus sequence oligonucleotide (5'-GGGACCAGTCCTAGCAGCTGTGGGGGAGCTGGGGAAGGTGGGAGTGTGA;
G/C content, 65%) (21) were incubated together with protein for
30 min at 37 °C in the standard DNA transfer reaction buffer (20 mM EPPS, pH 7.4, 5 mM MgCl2, 1 mM DTT). Reactions were stopped by addition of SDS to 0.1%
and 4 µg of proteinase K and further incubation at 37 °C for 45 min. Routinely, products were analyzed in 0.7% agarose gels containing
3 mM magnesium acetate in the TAE buffer system. Gels were
run at 2 V/cm for 16-24 h, stained with ethidium bromide, photographed
to determine positions of the duplex DNA bands, dried, and exposed for
autoradiography for 2-24 h. In later experiments, the magnesium
acetate in the agarose gels was found not to be necessary, and the gels
could be run at 4 V/cm without reducing the yield of joint molecules.
For quantification of products, the ethidium bromide-stained bands were
excised, the agarose was melted, and the 32P radioactivity
was directly measured in a scintillation counter. The percentage of
input 32P-oligonucleotide paired with the duplex DNA in the
joint molecules was calculated. Many variations of the assay were
performed and are described under "Results." Some used an
oligonucleotide, CS-3, containing the first 25 nt of the 49-nt
S
sequence (G/C content, 68%) or the 22-nt T7
oligonucleotide (5'-GTAATACGACTCACTATAGGGC) (G/C content, 45%), which
is fully homologous to the plasmid pBluescriptSK. The pH variations
were based on compounds of the polysulfonate buffer family.
 |
RESULTS |
A DNA Transfer Activity with Preference for S Regions--
To
assay for a DNA transfer activity that prefers S-region substrates, we
labeled a plasmid (pSP72) containing S
2b sequences with
[3H]thymidine and ds M13 containing Sµ
sequences with a small number of digoxigenin ligands. Recombinant DNA
molecules were quantified by counting 3H in plasmid DNA
that had been immunoprecipitated by an anti-digoxigenin antibody (Fig.
1A); in this way, a direct and
quantitative measurement of the catalytic activity leading to DNA
transfer was obtained (19, 24). To an equimolar mixture of these two
constructs, we added nuclear extracts from untreated spleen cells or
from spleen cells cultivated for 2-3 days in the presence or absence of LPS (Fig. 1B). Because these cultures contain non-B
lymphocytes and other nonswitching cells, we separated the (switching)
blasts from the nondividing (nonswitching) cells according to
size by cell elutriation (30) and tested the nuclear extracts
prepared from both cell pools for DNA transfer activity on S substrates versus non-S substrates (Fig. 1B).

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Switch region DNA transfer assay.
A, schematic representation. The ds M13 DNA contains
Sµ and is digoxigenin-labeled; the pSP72 (or pUC19)
plasmid contains S and is 3H-labeled. The
two substrates were mixed together and incubated with a nuclear
extract, e.g. from switching B-cells. DNA transfer from the
S plasmid to the Sµ plasmid results in
plasmids containing both 3H and digoxigenin label. The
recombined plasmid is precipitated by a bead-bound antibody to
digoxigenin and its radioactive content is measured. B,
experimental design.
|
|
Despite the expected presence of general DNA transfer activities in
such an extract (24, 25), the crude nuclear extracts from LPS blasts
recombined S region substrates 2-3-fold better than non-S region
substrates (Fig. 2A);
i.e. a newly induced, specific activity is added on top of
the general DNA swapping. This activity, called SWAP, is not present in
extracts from nonswitching cells (Fig. 2A) or in extracts
from thymus cells (not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
SWAP in extracts and extract fractions from
switching and nonswitching spleen cells. A, nuclear extract
(Fraction I; 850 ng/reaction; striped) and SWAP Fraction II
(80 ng/reaction; white) tested with DNA substrates with or
without S-regions. B, SWAP Fraction IV (1 ng/reaction)
tested in two independent experiments (gray and
black) with DNA substrate combinations containing either
both S regions, one S region and an unrelated DNA (M13 RF, SV40, or
X174 RF), or no S regions (pSP + M13 RF).
|
|
We purified SWAP by eluting it from a sizing column at a position
corresponding to a molecular mass of globular proteins of 200-300 kDa
(Fraction II). This fraction showed a 4-fold preference for S region
substrates and was inactive when isolated from nonswitching cells (Fig.
2A); no other previously observed DNA transfer assay activity, e.g. from HeLa cells or calf thymus (24, 25),
eluted at that position. The SWAP was further purified by cation
exchange and blue Sepharose chromatography yielding Fraction IV with a preference for S region substrates of 10-fold (Figs. 2B and
3). With these purification steps, the
specific activity increased more than 1000-fold (Table
I). When an unrelated DNA,
e.g. from M13, SV40, or
X174, was paired with an S
region, SWAP activity was reduced, on the average, by 6-fold (Fig.
2B). Fully homologous DNA substrates (5.7 kilobase pairs)
recombined with less than half the efficiency of two S region
substrates (not shown).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Elution profiles, SWAP Fractions III and IV.
A, macro S anion exchange chromatography elution profile.
Open squares, DTA with 2 S region substrates; filled
circles, DTA with substrates lacking S regions. Dotted
line, salt gradient; %cpm, DTA activity. B,
blue Sepharose chromatography elution profile; labeled as in
A.
|
|
Because the DNA is treated with SDS/EDTA and phenol extraction after
the DNA transfer reaction, the linkage of the two substrates is
considered stable and independent of the continuous presence of protein
or Mg2+. More than 80% of the transfer products are
heat-stable (20 min at 85 °C); the rest probably represents
noncovalent reaction intermediates. Maximum product formation by
Fraction IV (1 ng) occurred after 6 min of incubation at 37 °C and 3 mM MgCl2. Omission of the four dNTPs did not
affect the reaction significantly, but lack of ATP rendered the
reaction 88% less efficient. Polymerase chain reaction analysis and
subcloning of the bead-bound recombination products confirmed the
presence of covalent S
-Sµ junctions among the DNA transfer products (not shown). Purified SWAP fractions had no
detectable DNA polymerase, DNA helicase, general endonuclease, or
general exonuclease activity (not shown).
Identification of Components of the SWAP Complex--
On
silver-stained SDS-polyacrylamide gels there were about 12 and 7 polypeptides left in Fractions III and IV, respectively (Fig.
4). When
heavily stained, species were seen in Fraction IV with molecular masses
of approximately 38, 50, 70, 75, 100, 115, and 160 kDa, of which the
70-kDa species is clearly the most prominent. We gel-eluted and
partially sequenced the 38-, 70-, and 115-kDa proteins. The 38-kDa
protein yielded tryptic peptides TVSLGAG and FINYVK, which are
identical to residues 46-52 and 266-271 of mouse B23 (nucleophosmin)
(31). The 115-kDa protein yielded tryptic peptides TLGDFLAEYAK and
TTNFAGILSQG, which are identical to residues 108-118 and 864-874,
respectively, of mouse PARP (32). These protein identifications were
confirmed by Western blotting experiments (not shown).

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 4.
SDS-polyacrylamide gel electrophoresis of
SWAP, Fractions I-IV. The fractions were loaded on a 7.5%
SDS-polyacrylamide gel, and the gel was stained after electrophoresis
with silver according to standard procedures. M,
marker.
|
|

View larger version (74K):
[in this window]
[in a new window]
|
Fig. 5.
Nucleotide and amino acid sequence of
SWAP-70. Boxed regions indicate nuclear localization
signals. The sequence has been submitted to GenBankTM
(accession number AF053974).
|
|
The N terminus of the 70-kDa protein was MRGLKDELLKAIWHAFTALDLDRS,
which was not present in the molecular biology data bases. The cDNA
of SWAP-70, the novel 70-kDa protein, was cloned and sequenced (Fig.
5). SWAP-70 does not belong to a known protein family, and its
586-amino acid sequence contains nuclear localization signals at
positions 222-225, 281-284, and 361-376. There is a possible
coiled-coil region between amino acids 320 and 450, as well as a
potential O-glycosylation site at amino acids 314-315, a
continuous hydrophilic region near its C terminus, and several potential phosphorylation sites for several protein kinases. A fourth
SWAP component is described in the next section.
High Affinity Binding of SWAP Components to the SWAP-70
Protein--
The 200-300-kDa gel filtration elution position of SWAP
suggests a protein complex, and we looked for high affinity
interactions between B23, PARP, and SWAP-70. His-tagged SWAP-70 was
overexpressed from E. coli and purified to near homogeneity
as described under "Experimental Procedures" (Fig.
6A). Purified SWAP-70 protein (Fraction II) was bound to Sepharose beads as an affinity tag for
proteins contained in a nuclear extract from LPS-induced, switching
B-cells. About 1 mg of extract was loaded at 25 mM ammonium sulfate. The bound material was stepwise eluted with 80, 120, 300, 600, and 1200 mM ammonium sulfate, and the fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting. The 300 and 600 mM fractions contained about 10 polypeptides, as judged from silver-stained gels (not shown). On
immunoblots, PARP peaked in the 300 mM fraction (Fig.
6B), and B23 peaked in the 300 and 600 mM
fractions (Fig. 6C), corresponding to 600 and 1200 mM ionic strength, respectively, of a monovalent salt; this result suggests high affinity binding. Preincubation of the SWAP-70 affinity column with polyclonal anti-SWAP-70 antibodies completely abolished its ability to retain any of the other components of the
complex (not shown).

View larger version (48K):
[in this window]
[in a new window]
|
Fig. 6.
SWAP-70 affinity chromatography.
A, purification of SWAP-70 from E. coli. Proteins
were stained with silver. Un, uninduced; In,
induced; I and II, fractions I and II.
M, marker. B, immunoblot of affinity
column-eluted fractions, probed with anti-PARP antibodies.
C, as in B, but probed with anti-B23 antibodies.
D, as in B, but probed with anti-C23 antibodies.
I, nuclear extract, load. FT, flow-through,
80-1200 mM ammonium sulfate.
|
|
Using the affinity column to look for additional proteins forming a
complex with SWAP-70, we identified the 100-kDa polypeptide observed in
Fractions III and IV of the SWAP complex (Fig. 4). Because it
associates with B23 (33) and has a similar molecular mass, C23
(nucleolin) was a good candidate for this protein. Using anti-C23
antibodies on Western blots, we detected two cross-reacting polypeptides of 95-100 kDa in the 300 mM ammonium sulfate
fraction, likely representing two phosphorylation forms of this heavily modified protein. A third band of lower molecular mass may be a
degradation product (34) (Fig. 6D). Thus, a fourth component of the SWAP complex is C23, which also binds to SWAP-70 with high affinity. C23 has been described as a subunit of LR1, a B-cell-specific heterodimer that specifically binds switch region DNA (21, 35).
Expression Pattern of SWAP-70--
Equal amounts of nuclear
extracts of various tissues from 4-week-old C57BL/6 mice were analyzed
by Western blotting with affinity purified polyclonal anti-SWAP-70
antibodies. Although there may be low level expression in other
tissues, SWAP-70 is strongly expressed only in B-cells that have been
induced to switch (Fig. 7A).
Low expression in spleen and in lymph nodes presumably results from the
few activated B-cells present in any 4-week-old mice. However, only
spleen cells stimulated with LPS and concanavalin A (ConA) express
SWAP-70 at high levels. Importantly, SWAP-70 is not expressed in
thymocytes, nor in activated, proliferating T-cells. ConA stimulates
T-cells, and these in turn stimulate B spleen cells, which then express
SWAP-70 at high levels (Fig. 7, A and B); about
half of the cells in ConA-stimulated spleen cell cultures are activated
B-cells (not shown). In mice lacking B-cells (36), ConA did not induce
the expression of SWAP-70 (Fig. 7B). However, LPS-stimulated
cells from mice lacking T-cells (34) contain high levels of SWAP-70
(Fig. 7B). In a culture of proliferating pre-B-cells (37),
SWAP-70 is highly expressed only when the cells are stimulated with
anti-CD40 antibodies and IL-4 to induce switching primarily to the IgE
isotype (38) (Fig. 7C). Similarily, high expression of
SWAP-70 was seen 3-5 days after induction of switching to IgA in the
cell line I.29-D22 by treatment with LPS and transforming growth
factor-
(39) (not shown). SWAP-70 is also expressed at high levels
expressed in the pre-B-cell line 18-81, which undergoes class
switching (40, 41), but only at low levels in the pre-B-cell line
70Z/3, which does not switch (Fig. 7D). In these cell lines,
an approximately 45-kDa polypeptide cross-reacts with the antibodies
and might be a proteolytic degradation product of SWAP-70.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 7.
Expression pattern of SWAP-70 on Western
blots of nuclear extracts (10 µg of protein each) using
affinity-purified rabbit antibody against SWAP-70. A,
various tissues; B, Con A- and LPS-stimulated spleen cells
from CD3 / (i.e. T-cell-negative) mice;
membrane (mem) Cµ /
(i.e. B-cell-negative) mice; and wild-type (wt)
mice; extracts prepared at day 3 after induction. C,
pre-B-cells induced to switch to IgE by anti-CD40/IL-4 treatment (38);
d, day after induction; the cells proliferated
undifferentiated in the presence of IL-7. D, mouse B-cell
lines 18-81 (switching, 5 µg of protein) and 70Z/3 (nonswitching, 20 µg of protein).
|
|
Molecular Properties of SWAP-70--
To directly study DNA binding
properties of SWAP-70, gel shift experiments were performed. As shown
in Fig. 8A, SWAP-70 bound to
DNA. This binding does not require Mg2+ and is even
somewhat reduced by its presence. Increasing amounts of SWAP-70 (20 and
40 ng) bound more DNA, but only about 5% of the input DNA was bound,
even under optimal conditions. The oligonucleotide used for the
experiment shown in Fig. 8A contains the 49-nt
S
consensus signal (28). The same binding efficiency,
however, was observed with other, non-S oligonucleotide substrates.
SWAP-70 binds ds and ss DNA with about the same efficiency (not shown). Addition of ATP after preincubation of the protein with DNA abolished the signal, as did incubation with the nonhydrolyzable analog ATP-
-S. Binding of SWAP-70 to 32P-azido-ATP was not
inhibited by DNA (Fig. 8B). Thus, either ATP has a higher
affinity than DNA to the same binding site of SWAP-70, or ATP induces a
conformational change such that DNA cannot bind anymore. At any rate,
ATP is a potential modulator of DNA binding of SWAP-70.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 8.
DNA and ATP binding by SWAP-70. A,
0, 20, or 40 ng of SWAP-70 was incubated for 20 min with a
32P-labeled 49-nt oligonucleotide either without
MgCl2 (lanes 1-3 and 10-12) or with
MgCl2 (lanes 4 and 5, 1 mM; lanes 6 and 7, 5 mM;
lanes 8 and 9, 10 mM); with 1 mM ATP (lane 10) or 1 mM ATP- -S
(lane 12); or with 1 mM ATP added for the last 5 min of incubation (lane 11). One major binding product
(top arrow) and one minor binding product
(bottom arrow) are labeled. B,
32P-azido-ATP was incubated with either E. coli
RecA (2; 38 kDa, 0.3 µg), or SWAP-70 (1; 70 kDa, 1 µg, 2 µg) proteins. DNA was added to (+) or omitted from
( ) the reaction.
|
|
Promotion of DNA Reannealing by B23--
It is unknown whether the
short stretches of homology found between different S regions play a
role in switch recombination, but DNA transfer of switch regions
probably requires juxtaposition and possibly pairing of the substrate
DNAs (19). Pairing of two complementary DNA single strands is promoted
by most known recombination proteins (42), and we tested individual
components of the SWAP complex for this activity. A 422-bp, linear,
3'-labeled ds DNA fragment was heat-denatured and reannealing to the
duplex form visualized by gel electrophoresis (26). Although SWAP-70 was not active in this pairing assay (not shown), purified B23 was at
least as efficient as the E. coli RecA protein (Fig.
9). A titration of B23 into the reaction
showed that 15 ng of B23 (390 fmol) yielded as much ds DNA as 24 ng of
RecA (600 fmol). The B23 mediated reaction is ATP-independent. There
is, however, a strict dependence on Mg2+ (Fig. 9).

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 9.
DNA reannealing activity of B23 protein.
A heat-denatured 422-bp 32P-end-labeled DNA fragment was
incubated with various amounts of B23 protein, with E. coli
RecA, or without protein ( ), and with or without ATP or
MgCl2, as indicated (described in Ref. 26). ss,
single-stranded substrate; ds, double-stranded reannealing
product.
|
|
Formation of D Loops and Joint Molecules by B23--
For a more
complex three-strand pairing reaction (probably closer to the switch
recombination reaction) we investigated formation of joint molecules
generated by invasion of ss oligonucleotide into a ds DNA target, the
so-called D-loop formation (42-50). For most reactions, a
5'-32P-labeled ss oligonucleotide (49 nt; S
consensus sequence (28)) was incubated with a ds supercoiled pUC
plasmid carrying a 3.7-kilobase pair fragment of the S
region (27) and various amounts of B23 protein. In some assays, a known
unspecific pairing product (the so-called "Birnboim band") appears,
resulting from annealing of the oligonucleotide to partially and
irreversibly denatured plasmid DNA, which is generated in alkaline
lysis plasmid preparation methods (51) (band 2 in Fig.
10, A and B, and
see Fig. 13; band 3 in Figs. 11 and 13 indicates the free
oligonucleotide). This band migrates at the position of the supercoiled
plasmid and does not represent a true D-loop formation product, which migrates slower. The side product, however, is not present if the
supercoiled plasmid was purified under nondenaturing conditions (Figs.
10 and 11, C-E).

View larger version (83K):
[in this window]
[in a new window]
|
Fig. 10.
Formation of joint molecules: analysis of
reaction characteristics. Formation of joint molecules (band
1) with the 49-nt S oligonucleotide and the
superhelical pUC-S duplex target. A,
titration of B23 into the reaction; amounts (in ng) of B23 are
indicated. B, varying reaction conditions. The pH variations
range from 4.3 to 9.0 (indicated at the top). C,
titration of MnCl2 or MgCl2 in the presence or
absence of 1 mM ATP into the reaction, which lacked any
metal cofactor.
|
|

View larger version (86K):
[in this window]
[in a new window]
|
Fig. 11.
Formation of joint molecules in various
reactions. 1, joint molecules; 2, unspecific
annealing product (Birnboim band). Equal amounts of ss (80 fmol; 49-nt
S or noted otherwise) and ds (22 fmol;
pUC-S or noted otherwise) substrates were incubated for
30 min at 37 °C with protein. A, formation of joint
molecules by B23, SWAP-70 (S70), and B23 plus SWAP-70.
B, SWAP, Fraction III (left panel) or B23
(right panel) incubated with linear or supercoiled
pUC-S target substrates. C, B23 incubated
with either the ss (left) or the ds (right)
49-nt/bp S oligonucleotide; D, B23 or SWAP-70
(S70) incubated with a 25-nt S oligonucleotide substrate
and pUC-S target duplex DNA; E, B23 or SWAP,
Fraction III, incubated with a 22-nt oligonucleotide (T7, 100%
homology) and supercoiled pBluescript target DNA.
|
|
B23 promotes the formation of D-loops under a variety of conditions.
Titration of B23 into the reaction is shown in Fig. 10A. With 48 fmol of ss and 22 fmol of ds substrate DNAs, product formation required at least about 5 ng (130 fmol) of B23 protein, and saturation was reached at about 100 ng (2.6 pmol) of B23. The optimum pH of the
reaction buffer was about 7.0-7.4, with very little product visible at
pH 8.2 (Fig. 10B). A metal cofactor is required.
MgCl2 can be replaced by MnCl2 (Fig.
10B), but CaCl2 does not support the reaction,
and addition of Zn2+ strongly inhibits it (not shown).
Preincubation of B23 with the ss oligonucleotide was unnecessary.
Initially, an inhibitory effect of ATP added to a reaction was observed
(Fig. 10C, right two lanes). Titration of the metal cofactor
in the presence or absence of 1 mM ATP, however, showed that the inhibitory effect is most likely due to the well-known metal
ion chelating effect of ATP. Increasing the concentration of
MnCl2 reversed the inhibitory effect of ATP (Fig.
10C). Roughly the same amounts of joint molecules were
formed in the presence of 5 mM MgCl2 and 1-2
mM MnCl2. ATP inhibition was also reversed when
PARP enzyme was added to the reaction, independent of whether NAD was
added or not (not shown). The nature of this reversion remains to be
determined.
In Fig. 11A, joint molecules were formed in a standard
reaction with 50 ng (1.3 pmol) of B23 in a buffer containing 5 mM MgCl2. SWAP-70 is not active in the assay,
and titration of increasing amounts of SWAP-70 into the B23 mediated
D-loop reaction had no effect (Fig. 11A). The complete SWAP
fraction, which contains B23, was also active in the assay (Fig.
11B). For both the complete SWAP complex and purified B23
protein, no joint molecules were formed with the linear duplex DNA
substrates, but they were easily detectable with the supercoiled
substrate (Fig. 11B). Linear duplex DNA is also not an
efficient substrate for D-loop formation mediated by E. coli
RecA (42). If the ss oligonucleotide was replaced by a ds
oligonucleotide of the same sequence, the reaction efficiency was
markedly reduced, and the remaining minor signal was probably caused by
unannealed oligonucleotides (Fig. 11C). B23 also mediated D-loop formation with a shorter 25-nt oligonucleotide of the
S
sequence (68% G/C; Fig. 11D) as well as
with a 22-nt oligonucleotide (T7, 45% G/C) fully homologous to a
different duplex DNA substrate (pBluescriptSK) (Fig. 11E).
SWAP-70 was inactive in this reaction.
Joint Molecule Formation: Heat Stability, Reaction Kinetics, and
Protein Titrations--
Once they are formed by B23, the joint
molecule products do not require the presence of protein. The
quantitative results in Fig. 12 were
obtained by excising gel bands of joint molecules and direct
scintillation counting. Measurement of the free 32P
oligonucleotides excised from the same lane of the gel, permitted calculation of percentage of ss substrate converted into product. Variations among experiments were less than ±15%.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 12.
Quantitation of joint molecules. Joint
molecules (JM) were formed with the 49-nt S
oligonucleotide and pUC-S duplex DNA substrates.
Radioactive joint molecule and free oligonucleotide bands were excised
from the agarose gel, and the gel was melted and directly counted in
the scintillation counter. A, titration of B23 and % of
oligonucleotide converted into joint molecules (30-min incubation, 22 fmol of ds, 48 fmol of ss substrates). B, kinetics of joint
molecule formation using 1.3 pmol of B23, 100 fmol of ss substrate, and
44 fmol of ds substrate. Input ss substrate radioactivity was 85,000 cpm. Dotted line, idealized linear curve. C, heat
stability of the joint molecules. Input radioactivity was 88,000 cpm.
|
|
Under reaction conditions with 22 fmol of duplex and 48 fmol of ss
substrates, about 3.2% of the S
oligonucleotide (1.5 fmol) was converted by 100 ng (2.64 pmol) of B23 protein into joint
molecules (Fig. 12A). The reaction appears to be linear over more than 1 h, and some product formation could be detected after 1 min incubation (Fig. 12B). In this set-up, with 50 ng of
B23, 100 fmol of oligonucleotide, and 44 fmol of duplex DNA, after 20 min 3.0% and at the 90-min time point, 5.7% of the ss oligonucleotide was converted into joint molecules. Similar to the negative controls shown in Figs. 10 and 11, little oligonucleotide was incorporated into
the duplex target upon omission of protein, even in the 90-min reaction
(1.1%).
The joint molecules were tested for heat stability (Fig.
12C). After treatment with proteinase K and SDS, the
reaction mixture was heated for 20 min to various temperatures.
Although none could be detected at 95 °C, the amount of joint
molecules was not reduced at 55 °C. Considering the 92% homology
between the S
consensus sequence and the
pUC-S
duplex, the joint molecules possess the stability
expected for true base pairing (Fig. 12C). Using the formula
Tm = 81.5 + 16.6 (log[Me2+]) + 0.41 × P
600/N, where
P = fraction G/C; N = number of nt, with [Me2+] = 2 mM MnCl2, and
deducting 2 °C for each percent of nonhomology, we calculate the
approximate Tm to be 58 °C. At 58 °C, the
number of joint molecules is reduced by
(Fig.
12C). The slight increase in joint molecules after heating to 45-55 °C is probably due to protein-independent annealing of the oligonucleotide to duplex DNA, which began to denature at these
temperatures. The joint molecules do not require the continuous presence of Mg2+ or Mn2+ ions, because addition
of 50 mM EDTA during the proteinase K/SDS treatment did not
reduce the joint molecule signal (not shown).
Varying the amount of the ds DNA substrate also had an effect on the
reaction. Most efficient product formation was found using 1.1 µg
(245 fmol) of duplex DNA in a reaction containing 50 ng (1.3 pmol) of
B23 protein and 100 fmol of ss oligonucleotide (5.1% ss substrate
converted into joint molecules after 20 min of incubation). Based on
the binding site size of B23 on ss DNA of 11 nt (52), the 49-nt ss
substrate can theoretically be bound by 4.5 molecules of B23; the
binding site size on ds DNA is not known, but if it is similar, the
6800-bp duplex substrate may bind at least 618 molecules of B23. The
affinity of B23 for ds DNA, however, is much lower than that for ss DNA
(52), so that the ss substrate would be covered first. About 450 (4.5 × 100 fmol) monomeric B23 molecules may be bound by the ss
substrate, leaving roughly 850 (of 1.3 pmol) molecules of B23 available
for the duplex molecule. This results in three or four B23 molecules per duplex DNA molecule. Thus, the ds DNA does not need to be coated
with the protein. An alternative calculation of the reaction efficiency
based on the amount of duplex substrate, which is limiting, converted
into joint molecules yielded a 4-fold higher efficiency (up to
20%).
Joint Molecules Formed with Different S Regions--
The homology
between the 49-nt S
consensus sequence oligonucleotide
and the pUC-S
target is 92%, and four mismatches were
generated upon pairing. Because there exist patches of homologies between different S regions and switch recombination occurs between two
different S regions, we tested joint molecule formation using the
S
oligonucleotide and a pUC-Sµ or
M13-Sµ ds target DNA (Fig.
13A). The maximal homology
here was 75% over the entire 49-bp sequence or 90% in a 11-bp
stretch. Although not as efficient as the
S
-S
pairing (Fig. 13A, right two
lanes), B23 was able to produce joint molecules with the two
different S regions. No joint molecules were obtained with the pUC
plasmid DNA or the M13 ds DNA as target; in these cases, the homologies
were lower (46% for pUC19, 51% for M13; maximum of 70% in stretches
of 15-20 bp). Comparable results were obtained using substrates with
an S
, an Sµ, or no S region cloned
into a different plasmid vector (Fig. 13B). Thus, small
patches of homologies in the S regions may support pairing of different
S regions, but possibly other sequence or structural elements or a
minimal length of multiple homology patches (like that present in S
regions) is necessary to form stable products.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 13.
Formation of joint molecules with different
S region DNA substrates. The S oligonucleotide (160 fmol) was incubated in A with (+) or without ( ) 50 ng (1.3 pmol) of B23 protein and the plasmid DNAs with or without S regions as
indicated; in B without ( ) or with 50 or 100 ng of B23
protein and another set of plasmid DNAs (50 fmol each) containing
either S , Sµ, or no S region sequence. The
left portion (pSP/pSP-Sµ) of
B was exposed twice as long (34 h). 1, joint
molecule product; 2, unspecific band; 3, free
oligonucleotide substrate.
|
|
 |
DISCUSSION |
The SWAP Complex--
We have used a modified version of a DNA
transfer assay (19, 24-26) to identify activities that preferentially
recombine Ig H-chain S-region sequences. Such an activity, the SWAP
complex, was isolated from nuclear extracts of switching B-cells.
So far, four components of SWAP have been identified. Three of these, B23, C23, and PARP, are previously sequenced proteins that are present
in many sorts of cells. SWAP-70 is a novel protein that, like the SWAP
complex, is only present in the nucleus of switching B-cells.
Although SWAP works best with two S regions, it does work at a lower
level in reactions in which only one of the substrates contains an S
region. In this case, SWAP might use the S region as a preferred
substrate and recombine it with whatever partner is available, a
process similar to the myc translocations in plasmacytomas and
Burkitt's lymphoma (53), which are thought to be mediated by the
switch recombinase, as are other illegitimate translocations into S
regions (54). The preference of purified SWAP for S regions in the
cell-free system is about 10-fold. Although this might be sufficient
for the switch recombinase, additional specificity for a particular S
region is probably contributed by transcriptional activation of the
locus and/or the germ line switch transcripts (3-5, 15). It is also a
matter of debate whether or not limited homologies contribute to the
in vivo switch rearrangement. But there is no a
priori reason why a switch activity has to be inactive on
homologous substrates. The short patches of homology that exist between
different switch regions may well support the switch recombination reaction in vivo, and, as we show, they seem to be used
by B23 in the formation of joint molecules.
SWAP-70 binds only weakly to DNA, but it interacts strongly enough with
the other SWAP components to be used in affinity chromatography. The
possibility that only one or a few components of the SWAP complex bind
directly to SWAP-70, whereas others may bind indirectly via
interactions with these components, cannot be ruled out. In any case,
all known components in the fraction elute from a SWAP-70 affinity
column only at high salt concentrations; that is, the binding
affinities are high.
The binding of ATP and the inhibition of DNA binding by ATP or a
nonhydrolyzable analog suggest a regulative role of ATP binding on
SWAP-70 activity. The DNA transfer reaction requires ATP, but the role
of ATP in this reaction, catalyzed by the full SWAP fraction only,
remains to be determined. Based on the ATP effect, direct interactions
between SWAP-70 and DNA do not seem to be essential for the transfer
reaction. The amino acid sequence of SWAP-70 (Fig. 5) does not contain
a canonical Walker A NTP binding motif (55), but amino acid residues
183-190 resemble that motif to some extent.
Protein B23, also called nucleophosmin, numatrin, or NO38, has become
known as a predominantly nucleolar phosphoprotein bound to the nuclear
matrix (60). Although the precise function(s) of B23 remain unknown, it
was hypothesized to act as a protein shuttle involved in ribosome
assembly with migration between the nucleus and the cytoplasm (60).
However, B23 seems to have other functions as well. The protein binds
nucleic acids cooperatively with a preference for ss over ds DNA, but
without preference for either RNA or ss DNA (52, 61). Similar to other
ss binding proteins, B23 exhibits a helix-destabilizing activity,
disrupting the base stacking, observed on synthetic ss nucleic acids
such as poly(rA) and poly(rC) (52). A recent study showed increased phosphorylation and poly(ADP)ribosylation of B23 after x-ray treatment of mammalian cells (58). These observations and the higher affinity of
B23 to ss DNA foster speculation about a possible involvement in DNA
repair and recombination.
Since it promotes DNA single-strand reannealing, B23 contributes
previously unknown recombination activities to the SWAP complex. Promotion of reannealing of ss DNA is a feature common to most established recombination proteins (42). However, other proteins that
are very unlikely to be involved in DNA recombination, e.g. fatty acid synthase, also support the reaction. Furthermore, certain nonprotein agents, such as polyethylene glycol, also stimulate DNA
reannealing (42). Thus, although the presence of a reannealing activity
in a protein does not necessarily mean it is a recombination factor,
reannealing activity might well be expected of a recombination protein.
This expectation is fulfilled by B23, which promotes the reaction in an
ATP-independent but Mg2+-dependent manner with
an efficiency even higher than that of E. coli RecA
protein.
The formation of joint molecules constitutes a more complex
three-strand reaction, because it entails the invasion of a
single-strand DNA into a DNA duplex. This reaction reflects an early
and specific step in DNA recombination, and only a few known
recombination proteins promote this reaction (43-49). Our data show
that protein B23 mediates D-loop formation in a protein
concentration-dependent manner. B23 can use ss
"invader" substrates varying in length from 22 to 49 nt, in G/C
content from 45 to 65%, and in homology with the ds target DNA from
about 75 to 100%. Various linear duplex DNA substrates did not support
the reaction and serve as negative controls for unspecific,
e.g. helicase or nuclease-driven pairing. The reaction
requires Mg2+ or Mn2+ but not ATP. In theory,
formation of joint molecules with superhelical acceptor DNA does not
necessarily need an external source of energy, and strand exchange in
the absence of ATP or in the presence of nonhydrolyzable ATP analogs
has been demonstrated for E. coli RecA protein (42). The
reaction may be driven by the gain in entropy as a consequence of loss
of superhelicity (42, 50). Considering the degree of homology between
the standard DNA substrates, the joint molecules are remarkably stable;
heating to 55 °C, close to the Tm, leaves the
joint molecules intact (Fig. 12C). This argues for full base
pairing between the ss and ds substrates. Conversion of about 6% of
the oligonucleotide substrate into joint molecules after a 90-min
incubation of a standard reaction appears to be less efficient than
what has been reported for D-loop formation by E. coli RecA
protein or the meiotic human homolog HsDmc1 (49, 59). The kinetics are
also different; RecA-mediated formation of joint molecules decreased
significantly after the initial 2-min incubation (59), whereas no such
decrease was seen in the B23 mediated reaction.
Is SWAP the Switch Recombinase?--
The expression pattern of
SWAP and of the SWAP-70 protein is consistent with it being involved in
the process of class switching. The nuclear localization signals in
SWAP-70 suggest its presence in the nuclei, and in immunoblotting
experiments, it has not been seen at any comparable level in
cytoplasmic extracts. It is strongly expressed only in switching
B-cells. Importantly, the expression pattern of SWAP-70 could not be
explained by activation of cell proliferation, which accompanies
switching in spleen cell cultures. Indeed, the 70Z/3 and 18-81 cell
lines proliferate at the same rate, yet SWAP-70 is expressed in the
switching line 18-81 only; several of the tissues (e.g.
thymus) tested in Western blotting experiments contain proliferating
cells but are SWAP-70-negative, as are ConA-stimulated, proliferating
spleen cells from mice lacking B-cells. Moreover, pre-B-cells induced
to switch by anti-CD40 and IL-4 treatment actively proliferate before
induction and cease to do so afterward. However, SWAP-70 is highly
expressed several days after switch induction, at a time when switching
is most prominent (35). There is no detectable expression of SWAP-70 during V(D)J recombination, which occurs after removal of IL-7 from
these pre-B-cell cultures (35). It should be noted that SWAP-70 has
been observed to be expressed in B-cells switching to several different
Ig isotypes: IgG2b (after LPS treatment), IgE (after treatment with
anti-CD40 and IL-4), and IgA (after treatment with transforming growth
factor-
and LPS).
Although their precise functions remain to be determined, all four
components of the SWAP complex thus far identified seem to fit into
roles one may expect from a switch recombinase complex. (i) The role of
SWAP-70 is likely to reside in protein-protein interactions. Thus,
SWAP-70 might act as a specific class switch recombinase assembly
factor. (ii) C23 is identical to a component of the heterodimeric LR1,
which has been reported earlier in independent studies to be
B-cell-specific and to bind specifically to switch region DNA (21, 35).
Thus, C23 might act as a switch region targeting factor in the SWAP
complex. (iii) B23 might provide the activities necessary to perform
the first steps in a recombination reaction: pairing of the DNA
substrates and strand invasion with formation of stable joint
molecules. (iv) PARP may regulate components of the complex (39). Based
on switch induction end point studies, PARP-negative mice seem to have
no serious deficiency in class switching
(56).2 Although PARP is
probably not essential for class switching, it is difficult to assess
the impact of selection for isotypes other than IgM. A decreased
efficiency in switched isotype production may be counterbalanced by
greater expansion of cells that are switched. PARP may have a
regulatory role in switching, because treatment of switching I.29
B-cells with PARP inhibitors results in an increase in switch frequency
(39). The possible existence of a second enzyme similar to PARP has not
entirely been ruled out and would obscure the phenotype of the knockout
mouse. PARP has also been shown to bind not only to DNA ends but also
to bind DNA secondary structures such as stem loops (57). Such
structures have been suggested to be substrates for the switch
recombinase (6). It is notable that B23 protein has been found to be
modified by PARP (58).
In terms of class switch recombination, the utilization by B23 of two
different S region DNA sequences, Sµ and
S
, as ds and ss DNA substrates is most remarkable. It
was observed with S regions cloned in several vector DNAs. No other
mammalian protein has been described to mediate such a reaction, which
may resemble formation of an early recombination intermediate in the class switch reaction (7, 10).
The lines of evidence discussed in this section strongly suggest that
the SWAP complex is involved in at least one of the genetic events that
take place at the Ig loci during the later stages of the
differentiation of B-cells, most likely the Ig H-chain class switch. A
cogent conclusion will have to await the outcome of genetic knockout
experiments.
We thank C. Steinberg, F. McBlane, J.-M.
Buerstedde, and L. Du Pasquier for discussion of the manuscript; F. Alt, G. Siebenkotten, and A. Radbruch for S region probes; P. K. Chan for antibodies against B23; P. Bouvet for anti-C23 antibodies; M. Olson for recombinant B23; F. Althaus for PARP; and D. Avila for
peptide sequencing.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF053974.