Molecular Visualization of Immunoglobulin Switch Region RNA/DNA
Complex by Atomic Force Microscope*
Ryushin
Mizuta
§,
Kousuke
Iwai¶,
Masatsugu
Shigeno
,
Midori
Mizuta
,
Takeshi
Uemura
,
Tatsuo
Ushiki¶, and
Daisuke
Kitamura
**
From the
Research Institute for Biological Sciences
and the ** Genome and Drug Research Center, Tokyo University
of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan, the
¶ Department of Anatomy and Histology, Faculty of Medicine,
Niigata University, Asahimachi-dori, Niigata 951-8510, Japan, and
Seiko Instruments, Matsudo, Chiba 270-2222, Japan
Received for publication, September 10, 2002, and in revised form, December 2, 2002
 |
ABSTRACT |
Immunoglobulin heavy-chain (IgH) class switch
recombination (CSR) is initiated by DNA breakage in the switch (S)
region featuring tandem repetitive nucleotide sequences. Various
studies have demonstrated that S-region transcription and splicing
proceed to genomic recombination and are indispensable for CSR in
vivo, although the precise molecular mechanism is largely
unknown. Here, we show the novel physical property of the in
vitro transcribed S-region RNA by direct visualization using an
atomic force microscope (AFM). The S-region sense RNA, but not the
antisense RNA, forms a persistent hybrid with the template plasmid DNA
and changes the plasmid conformation from supercoil to open circle in
the presence of spermidine. In addition, the S-region transcripts
generate globular forms and are assembled on the template DNA into a
large aggregate that may stall replication and increase the
recombinogenicity of the S-region DNA.
 |
INTRODUCTION |
Two subsequent DNA recombination steps are involved in
immunoglobulin (Ig) production (1-3). First, V(D)J
recombination assembles the V, D, and J segments into a variable region
exon and contributes to the diversification of antigen-binding sites.
Second, class switch recombination
(CSR)1 converts the constant
(CH) region of the immunoglobulin heavy chain (IgH) from
Cµ to other classes and alters physiological functions that differ in
their properties of complement fixation, binding to class-specific Fc
receptors, and polymerization. Thus, class switching is a mechanism for
diversifying Ig effector functions with the same antigen specificity.
The mouse IgH locus of the IgM-expressing B lymphocyte contains a
rearranged V(D)J exon and 8 CH exons of the following
organization: 5'-V(D)J-Cµ-C
-C
3-C
1-C
2b-C
2a-C
-C
-3'. CSR is
initiated by DNA breakage in the S-region featuring tandem repetitive
nucleotide sequences that are located upstream of each CH
exon except for the C
exon. In the case of CSR from IgM to IgE, DNA
breakage initially occurs in both Sµ and S
and results in deletion
of the intervening region containing the Cµ to C
2a exons, and
religation of Sµ and S
causes juxtaposition of the V(D)J exon and
the C
exon. Despite the recent discovery of the
activation-induced deaminase (AID) as an indispensable factor for CSR
(2, 4), the molecular mechanism of CSR still remains elusive. In
particular, the initiation step of DNA cleavage is completely unknown.
Previous studies have revealed that the generation of germline
transcripts from the sites upstream of each S-region is a prerequisite for CSR, and the higher order DNA structure involving S-region RNAs
that are spliced out from the primary transcripts has been proposed as
a critical factor for CSR (1, 5-7). Moreover, biochemical studies,
including an agarose gel analysis, suggested that a persistent RNA/DNA
hybrid is formed between S-region transcripts and its template DNA
(8-11). Thus, the S-region RNA may be catalytically involved in DNA
breakage and CSR, and the characterization of the RNA/DNA hybrid may be
critical for the understanding of CSR. However, the precise structure
and the physical characteristic of both the RNA/DNA hybrid and the
S-region RNA are not yet fully established.
To directly determine the potential role and the physical
characteristic of both the S-region transcript and the RNA/DNA hybrid at the single molecular level, we examined the S-region RNA transcribed in vitro using an atomic force microscope (AFM) (12). This
approach revealed the specific characteristic of the nascent S-region
transcript and the higher order structure of the RNA/DNA hybrid at the
single molecular level.
 |
EXPERIMENTAL PROCEDURES |
Plasmid--
The pGD231 plasmid, a generous gift from M. Lieber
(9), contains an immunoglobulin S
3-region DNA (2.2 kb) in a pBSKS
vector (Stratagene).
In Vitro Transcription--
Standard reactions were carried out
using a RiboProbe in vitro transcription system (Promega).
Briefly, the pGD231 plasmid DNA (1 µg) was transcribed with 10 units
of T3 or T7 RNA polymerase in 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, 10 mM dithiothreitol, 2 mM spermidine, 0.05%
Tween 20, 20 units of recombinant ribonuclease inhibitor, and 0.5 mM each of rATP, rGTP, rCTP, and rUTP in a volume of 20 µl for 1 h at 37 °C. Reactions were stopped by heat inactivation for 20 min at 65 °C. Some samples were incubated with 1 µg of RNase A (Wako, Osaka, Japan) for an additional 30 min at
37 °C. Where indicated, 2 units of RNase H (Toyobo, Osaka, Japan)
were added. Samples for AFM analysis were purified with MicroSpin S-400
HR columns (Amersham Biosciences). Unless otherwise specified, all
samples were subjected to phenol/chloroform extraction and ethanol
precipitation and suspended in TE buffer (10 mM Tris-HCl, pH 7.9, and 1 mM EDTA).
AFM Imaging--
Samples were diluted in 10 mM
Tris-HCl (pH 7.9) and 1 mM NaCl to 1 ng/µl DNA
concentration. 5 µl of each solution was deposited onto the center of
a freshly cleaved mica disk. Just before deposition, the sample
solution was supplied with MgCl2 to a final concentration of 2.5 mM. After 5 min, the mica was blown dry with
compressed air. To remove salts on the mica surface, 10 µl of
distilled water was dropped on the mica followed immediately by a quick
blow for drying. This rinsing step was repeated twice. AFM studies were performed using the SPM-400 scanning probe microscope controlled by an
SPI 3700 probe station (Seiko Instruments). This microscope was
equipped with a piezo translator with maximum x-y
scan range of 20 µm width and a z range of 1.2 µm. The
cantilevers used were rectangular, with a force constant of 35-40
N m
1 and a resonance frequency of 320-400
kHz (SI-DF40, Seiko Instruments). Unless otherwise specified, all
images were obtained as height mode images in a dynamic force mode in
the air at room temperature. The resonance frequency shift was measured
using the slope detection method at a slightly higher frequency of the
cantilever than its resonance frequency.
 |
RESULTS |
Altered DNA Mobility upon in Vitro Transcription of Repetitive
Sequence--
To confirm previous results (8-11), we examined the
conformational change of the plasmid (pGD231, Fig.
1A) containing an
immunoglobulin S
3-region DNA after inducing S
3-region
transcription on either strand of the DNA. The guanine-rich (G-rich)
sense RNA and the cytosine-rich (C-rich) antisense RNA were transcribed
by T7 and T3 RNA polymerases, respectively. Thereafter, free RNA was
digested with RNase A. Consistent with the previous report (9), almost all of the plasmids transcribed with T7 RNA polymerase migrated slowly
in an electrophoretic gel as smears (Fig. 1B, lane
3), but those transcribed with T3 RNA polymerase migrated normally as supercoiled plasmids (Fig. 1B, lane 1).
Treatment with RNase H, which specifically degrades the RNA in the
RNA/DNA hybrid, restored those plasmids transcribed with T7 RNA
polymerase to their original mobility (Fig. 1B, lane
4). These results suggest that the sense RNA, but not the
antisense RNA transcribed from the S
3 region, forms the RNA/DNA
hybrid and is responsible for the mobility shift. However, from results
of this analysis, it was not sufficiently clear whether the mobility
shift is caused by the conformational change of the plasmids or merely
by the added molecular weight of the associated transcripts.

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Fig. 1.
Conformational change of plasmid DNA upon
in vitro transcription of the mouse IgH repetitive
switch sequence. A, diagram of the plasmid DNA (pGD231)
containing S 3 sequence (2.2 kb). The long
arrow and the bent arrows indicate the
direction of physiological transcription in the IgH gene and the
direction of transcription by T3 or T7 RNA polymerase, respectively.
The S 3 sequence is indicated by a box. B,
altered mobility of pGD231 on an agarose gel after in vitro
transcription. Supercoiled pGD231 plasmids were transcribed by T3
(lanes 1 and 2) or T7 RNA polymerase (lanes
3 and 4), treated with RNase A (+,
lanes 1-4) and/or RNase H (+, lanes 2 and 4), and run on an agarose gel. Untranscribed pGD231
plasmids and a 1-kb ladder (M) were run on lanes
5 and 6, respectively. Positions of supercoiled
(SC) and open circle (OC) forms of the plasmids
are indicated. C, AFM views of the conformational change of
pGD231 after transcription by the T3 or T7 RNA polymerase. The samples
examined above (B, lanes 1 and 3) were
purified and visualized under AFM. Scale
bars, 200 nm.
|
|
Conformational Change of Plasmid DNA upon in Vitro
Transcription--
To visualize a possible conformational change of
the pGD231 plasmid at single molecular resolution, the same samples
used in the agarose gel analysis were subjected to AFM analysis after purification by phenol/chloroform extraction and ethanol precipitation. As clearly seen in Fig. 1C, plasmids treated with T3 RNA
polymerase formed the supercoiled structure (left
panel), but those treated with T7 RNA polymerase formed the
relaxed open circle structure to various extents (right
panel). These data clearly indicate that the sense RNA
transcribed from the S-region forms a persistent hybrid with its
substrate DNA and relaxes its conformation, which may represent a
phenomenon known as chromatin opening. Chromatin opening in the
S-region, despite the lack of direct evidence, is proposed as the
initial step for the access of some endonucleases to this region in CSR
(1, 3, 5).
Spermidine Stabilizes the RNA/DNA Hybrid--
In the course of the
purification of the RNA/DNA hybrid, we found that a cation and some
salt is indispensable for establishing and maintaining this
hybrid. In our in vitro transcription, the standard
buffer contains spermidine, a trivalent cation at normal cellular pH.
Spermidine is one of the polyamines (putrescine, spermidine, and
spermine) and is known to be critical for cell cycle progression and
induction of nucleic acid and protein syntheses (13). Polyamines are
also known to preferentially bind to the GC-rich region of RNA and to
change its structure (13). We found that spermidine at a physiological
concentration in a reaction significantly increases the extent of
RNA/DNA hybrid formation (Fig. 2,
top panel) without affecting total RNA production
(bottom panel). Furthermore, the purified RNA/DNA
complex was found to be unstable in water, and the addition of some
salt increased its stability (data not shown). These results suggest
that the formation and maintenance of the RNA/DNA hybrid is regulated
by spermidine and some salt.

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Fig. 2.
Spermidine is required for RNA/DNA hybrid
formation. Spermidine (SP) concentration was varied
(0-2 mM) in the in vitro transcription reaction
(lanes 1-4). Aliquots of samples before (bottom
panel) and after RNase A treatment (top
panel) were run on agarose gels and stained with ethidium
bromide. A 1-kb ladder was used as a molecular weight marker
(M).
|
|
RNA/DNA Hybrid Formation Proven by RNase H
Binding--
Biochemical studies have suggested that the S-region
RNA/DNA hybrid forms a structure called R-loop, a protrusion of a
non-template DNA strand as a result of RNA/DNA hybridization (10, 11). The single-stranded region of the R-loop is supposed to serve as a
substrate for some endonucleases that may cause DNA cleavage and
subsequent recombination. To inspect the RNA/DNA hybrid without the
helical stress from the double-stranded circular DNA, we digested the
plasmid with SacI or KpnI, and then subjected the
linearized plasmid to in vitro transcription and AFM
analysis (Fig. 3A). However,
we did not detect any visible alteration in structure, including an
R-loop, by the transcription on either strand (Fig. 3B,
top panels). Possibly, the R-loop collapsed and
clamped onto the nascent RNA, forming an intramolecular triplex as
suggested by Reaban et al. (10), and/or the R-loop is too
small to be visualized using AFM. To detect RNA/DNA hybrid formation in
a linear plasmid, we incubated the purified sample with RNase H. As
mentioned above, RNase H specifically degrades the RNA in the RNA/DNA
hybrid, and this catalytic activity requires Mg2+.
Therefore, RNase H is supposed to maintain its association with the
nascent RNA of the RNA/DNA hybrid in the absence of Mg2+.
After incubation with RNase H without MgCl2, we detected
some aggregation on each T7 (but not T3) RNA polymerase-treated DNA (Fig. 3B, bottom panels). Also, the
apparent length of the linear DNA in the T7 RNA polymerase-treated
sample became shorter, and the aggregation was often associated with
DNA loop or branch formation. These data suggest that the sense
S-region RNA actually forms a persistent hybrid with the template DNA
and that the binding of RNase H to each hybrid facilitates the
secondary structure formation.

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Fig. 3.
Evidence of RNA/DNA hybrid formation after
in vitro transcription. A, diagram of the
linear pGD231 plasmid after digestion by SacI or
KpnI restriction enzymes. The long
arrow and bent arrows indicate the
direction of physiological transcription and the direction of
transcription by T3 or T7 RNA polymerase, respectively. B,
RNA/DNA hybrid formation probed by RNase H binding. Transcribed and
RNase A-treated linear plasmids were incubated with (+,
bottom panels) or without ( , top
panels) RNase H and then purified with spin columns and
examined under AFM. Scale bars, 200 nm.
|
|
Direct Visualization of S-region RNAs--
In the above
experiments, we used RNase A-treated samples to eliminate the
nonhybridized free RNA. We next sought to determine the physical
properties of the free S-region RNA transcribed in the sense or
antisense direction. Thus, the in vitro transcribed samples
were subjected to AFM analysis without RNase A treatment (Fig.
4). We visualized RNA molecules and
identified a clear difference between the sense and antisense RNAs. An
elongated form of the antisense RNA, possibly forming an intramolecular
secondary structure, was released from the substrate DNA (Fig. 4,
left T3 panels). In contrast, the
sense RNA formed a large aggregate (Fig. 4, right T7 panels). The RNA/DNA hybrid examined in Fig. 3
was supposed to serve as a core of this aggregate. The height profiles
of the RNA, DNA, and RNA/DNA molecules are shown in the bottom
panel of Fig. 4. The largest height of the right
side T7 panel (~3.3 nm) was
significantly greater than that of the left side
T3 panel (~1.3 nm) or the height of the
double-stranded DNA (~0.4 nm). The large aggregate of the RNA/DNA
complex consisted of small globular molecules, which was evident from
the phase mode image (Fig. 5,
right panel). Interestingly, several DNA
fragments were linked via this aggregate (Fig. 5), raising a
possibility that the RNA aggregate functions as an anchor for synapsing
two S-region DNAs. From these results, we propose that the S-region
RNA, transcribed in the sense direction, forms a persistent hybrid with
the template DNA, as well as a globular form, and forms a large complex
with other RNA molecules to juxtapose the other S-region DNAs.

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Fig. 4.
Elongated form of the antisense RNA and the
aggregated form of the sense RNA. Linear pGD231 plasmids were
transcribed in vitro, purified without RNase A treatment,
and visualized under AFM. Plasmids transcribed by T3 RNA polymerase
(antisense orientation) or T7 RNA polymerase (sense orientation) are
shown in the left and right panels,
respectively, and enlarged images are shown in the middle
panels. The height profiles, indicated as lines
and arrows in the middle panels, are
shown in the bottom panel. The dotted
line at 0.4 nm indicates the height of a double-stranded
DNA. Scale bars, 200 nm.
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Fig. 5.
Synapsed DNA fragments via a large aggregate
composed of small globular sense RNA molecules. Left
panel, height mode image. Right panel,
phase mode image. Scale bars, 200 nm.
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|
 |
DISCUSSION |
Here, we show the novel physical property of the in
vitro transcribed S-region RNA by direct visualization using an
AFM. The S-region sense RNA, but not the antisense RNA, forms a
persistent hybrid with the template plasmid DNA and changes the plasmid
conformation from supercoil to open circle in the presence of
spermidine. In addition, the S-region transcripts generate globular
forms and are assembled on the template DNA into a large aggregate.
The globular form and the large RNA/DNA complex formed by the sense
S-region RNA are intriguing, because nucleic acids are negatively
charged, and electric repulsion should have an adverse effect on the
aggregation. Several possibilities might explain the aggregation. One
possibility is that some cations can neutralize the negative charge. As
mentioned earlier, spermidine, a trivalent cation in solution, is
indispensable for the S-region RNA/DNA hybrid formation (Fig. 2), and
the addition of salt also increases the stability of the purified
RNA/DNA hybrid (data not shown). Thus, neutralization of a charge by a
cation may be indispensable for the generation of the globular RNA or
RNA/DNA complex. Another possibility is that the unique structure of
the S-region might facilitate the aggregate formation. Two possible
structures might be critical for this phenomenon. One is a
four-stranded structure (G4-DNA) in which G-rich DNA strands are linked
by a Hoogsteen-bonded guanine quartet (14, 15). G-rich RNA strands may
be linked in the same manner and form G4-RNA and/or G4-RNA/DNA. The
second is the stem-loop structure generated from palindromic sequences of the S-region, which has recently been proposed as a possibility because of its role in CSR (2, 16). Further analysis of the sequence requirement for the complex formation will verify these possibilities.
What is the physiological role of this RNA/DNA complex in CSR? We
propose the following possibilities. 1) The RNA/DNA complex maintains
the S-region of the IgH gene open, as shown in Fig. 1C,
which facilitates the access of some endonucleases and CSR machinery.
2) This complex serves as an anchor for synapsing S-region RNA/DNA
complexes, as shown in Fig. 4, which is a critical step in CSR. 3) This
complex stalls the movement of the replication fork, which may also
create a target for some endonucleases. It was reported previously that
some repetitive sequences become highly recombinogenic by stalling the
replication fork with their transcription and that a stable higher
order structure of DNA and RNA, despite the lack of direct evidence, is
the cause of the stalling (17). Our data directly showed that the
RNA/DNA complex at repetitive sequences causes such a higher order
structure, which may well explain the replication-coupled recombination
of repetitive sequences. This is particularly relevant in CSR, because several reports have suggested the correlation between CSR and replication (5, 18) and the increased level of spermidine, a critical
factor for the RNA/DNA hybrid, in cells entering replication (19).
Here, at the single-molecular level, we showed the unique physical
characteristics of S-region transcripts that may be crucial for CSR. A
similar intron-delivered repetitive RNA may be important in various
aspects of cellular functions.
 |
ACKNOWLEDGEMENTS |
We thank M. R. Lieber for the pGD231
plasmid and T. Kawai and H. Tanaka (the Institute of Scientific and
Industrial Research (ISIR)-Sanken, Osaka University) and M. J. Shulman
for discussions.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 81-4-7123-9883;
Fax: 81-4-7124-1561; E-mail: mizuta@rs.noda.tus.ac.jp.
Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M209262200
 |
ABBREVIATIONS |
The abbreviations used are:
CSR, class switch
recombination;
CH, constant region of IgH;
S-region, switch
region;
AFM, atomic force microscope.
 |
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