Previous Article | Next Article 
Molecular and Cellular Biology, April 1999, p. 3010-3017, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Roles of the "Dispensable" Portions of RAG-1 and RAG-2 in
V(D)J Recombination
S. B.
Steen,1,
J.-O.
Han,2
C.
Mundy,3,4
M. A.
Oettinger,3,4 and
D. B.
Roth1,2,5,*
Program in Cell and Molecular
Biology,1 Department of Microbiology and
Immunology,2 and Howard Hughes Medical
Institute,5 Baylor College of Medicine,
Houston, Texas 77030; Department of Molecular Biology,
Massachusetts General Hospital, Boston, Massachusetts
021143; and Department of Genetics,
Harvard Medical School, Boston, Massachusetts
021154
Received 14 August 1998/Returned for modification 29 September
1998/Accepted 22 January 1999
 |
ABSTRACT |
V(D)J recombination is initiated by introduction of site-specific
double-stranded DNA breaks by the RAG-1 and RAG-2 proteins. The broken
DNA ends are then joined by the cellular double-strand break repair
machinery. Previous work has shown that truncated (core) versions of
the RAG proteins can catalyze V(D)J recombination, although less
efficiently than their full-length counterparts. It is not known
whether truncating RAG-1 and/or RAG-2 affects the cleavage step or the
joining step of recombination. Here we examine the effects of truncated
RAG proteins on recombination intermediates and products. We found that
while truncated RAG proteins generate lower levels of recombination
products than their full-length counterparts, they consistently
generate 10-fold higher levels of one class of recombination
intermediates, termed signal ends. Our results suggest that this
increase in signal ends does not result from increased cleavage, since
levels of the corresponding intermediates, coding ends, are not
elevated. Thus, removal of the "dispensable" regions of the RAG
proteins impairs proper processing of recombination intermediates.
Furthermore, we found that removal of portions of the dispensable
regions of RAG-1 and RAG-2 affects the efficiency of product formation
without altering the levels of recombination intermediates. Thus, these evolutionarily conserved sequences play multiple, important roles in
V(D)J recombination.
| |
INTRODUCTION |
Immunoglobulin and T-cell receptor
gene segments are rearranged by V(D)J recombination to generate a
diverse repertoire of antigen binding domains. The recombinase
binds recombination signal sequences (hereafter termed signals) which
flank the gene segments and introduces a double-stranded
break (DSB) precisely between each signal and gene segment. This
cleavage event produces two types of DNA termini, signal ends that
terminate in signals and coding ends that contain the gene segment.
Signal ends join to form a signal joint, whereas coding ends join to
form a coding joint encoding the antigen binding domain (Fig.
1A) (24, 27, 28, 33, 38, 40).
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 1.
(A) Schematic diagram of V(D)J recombination
intermediates (coding ends and signal ends) and products (coding joints
and signal joints) generated from the plasmid substrate pJH290. Signals
are represented by triangles, and coding segments are represented by
rectangles. (B) Conservation of RAG-1 and RAG-2. Each protein is shown
as a rectangle, with individual amino acids represented as uniformly
sized dark or light bands. Dark bands represent amino acids that are
absolutely conserved in human, rabbit, mouse, chicken, xenopus, and
trout proteins (2, 5, 6, 8, 10, 22, 32). Full-length RAG-1
(FL1) contains 1,040 amino acids; full-length RAG-2 (FL2) contains 527 amino acids. Truncated RAG-1 (TR1) consists of amino acids 384 to 1040, and truncated RAG-2 (TR2) consists of amino acids 1 to 387.
|
|
The V(D)J recombinase minimally consists of the highly conserved,
lymphoid-cell-specific proteins RAG-1 and RAG-2 (22, 32). Transfection of the genes encoding RAG-1 and RAG-2 into cultured fibroblasts renders these cells competent to rearrange extrachromosomal recombination substrates, indicating that the RAG proteins are the only
lymphoid-cell-specific factors necessary for recombination (22). DSBs with the same characteristics as in vivo
intermediates (27, 28, 33, 35, 40) are generated in
cell-free reactions containing purified, truncated RAG-1 and RAG-2 and
the appropriate divalent metal ion (20). After cleavage, the
RAG proteins remain associated with the broken DNA ends. Stable
complexes have been isolated that contain the RAG proteins and a pair
of cleaved signal ends (1). More recently, complexes
containing the RAG proteins and all four DNA ends (two signal ends and
two coding ends) have been isolated (9). We and others have
suggested that disassembly or remodeling of these postcleavage
complexes may be necessary to allow the joining machinery to complete
formation of coding or signal joints (1, 39).
Mutational analyses revealed that RAG-1 and RAG-2 proteins truncated by
30 and 25%, respectively, are still capable of recombining plasmid
substrates in fibroblasts, although generally with lower efficiency
than full-length RAG proteins (3, 12, 21, 26, 30, 31, 34).
These truncated proteins, which contain residues 384 to 1008 of 1,040 amino acids (RAG-1) and 1 to 387 of 527 amino acids (RAG-2) (Fig. 1B),
are more soluble than their full-length counterparts and are,
therefore, the forms of the RAG proteins used in cell-free
systems (4, 13, 20, 23, 37, 38).
Sequence analysis of the portions of RAG-1 and RAG-2 that have been
considered dispensable for recombination (amino acids 1 to 383 and 1009 to 1040 of RAG-1 and 388 to 527 of RAG-2) reveals many amino acid
residues that are conserved across diverse species (Fig. 1B),
suggesting that they may play an important role(s) in recombination.
This proposal is supported by recent work demonstrating that regions
within the N terminus of RAG-1 enhance signal joint formation (21,
26). However, these studies did not address which step of
recombination, cleavage or joining, is affected by truncation of RAG-1
and RAG-2. Specifically, the effects of the truncations on
recombination intermediates were not examined.
Here, we analyze the levels of both recombination intermediates (signal
ends and coding ends) and products (signal joints and coding joints).
In agreement with previous work (3, 12, 21, 26, 31, 34), we
found that the levels of V(D)J recombination products, coding and
signal joints, are reduced when truncated RAG proteins are used. We
expected that the levels of recombination intermediates would be
similarly reduced, since the characterized functions of the RAG
proteins affect the cleavage step. However, the truncated RAG proteins
consistently produced levels of signal ends that were 10-fold-higher
than those produced by the full-length RAG proteins. In contrast,
levels of coding ends were generally not affected. These observations
suggest that the truncated RAG proteins do not increase cleavage but,
rather, stabilize the signal end intermediates. Analysis of additional
RAG deletion mutants allowed us to separate the effects on
intermediates and products, showing that these effects map to distinct
regions of the RAG proteins. Thus, these studies have uncovered several
unexpected functions of the "dispensable" regions of the RAG
proteins, suggesting that they have roles both in the processing of
postcleavage complexes and in product formation.
| |
MATERIALS AND METHODS |
Plasmids.
Plasmid recombination substrates used in this
study are pJH290, which produces coding joints on the plasmid backbone,
and pJH289 (18), which produces signal joints on the plasmid
backbone. The expression vector encoding full-length RAG-1 is pJH548
(30). Full-length RAG-2 is encoded by pJH549
(30), which is not epitope tagged, and pMS201
(31), which is tagged with three copies of c-myc
epitope. The expression vector pMS127b (30) encodes
truncated core RAG-1, and pMS216 (31) encodes truncated
RAG-2. Vectors containing smaller deletions within RAG-1 are as
follows: pMS119C (30) encodes amino acids 1 to 1008; pMS126
(30) encodes 332 to 1008; pSK151 (12) encodes 331 to 1040. Vectors containing smaller deletions within RAG-2 are as
follows: pMS211 (31) encodes amino acids 1 to 517; pR2CC11
(3) encodes 1 to 481; pR2CC15 (3) encodes 1 to
425; pR2CC10 (3) encodes 1 to 418; pMS215 (31)
encodes del388 to 413.
Transient-transfection assay.
Transient transfections were
performed as previously described (35, 36). Briefly, 2 µg
of recombination substrate, 2.1 µg of full-length RAG-1 expression
vector (or the molar equivalent of smaller versions of RAG-1), and 2.5 µg of full-length RAG-2 expression vector (or the molar equivalent of
smaller versions of RAG-2) were transfected into Chinese hamster ovary
cells (RMP41, CHOK1, or xrs-6) by using either the calcium phosphate
method (CellPhect kit; Pharmacia) or FuGene6 (Boehringer Mannheim). DNA was harvested after 40 to 48 h. DNA was harvested according to the
method of Hirt as described previously (35, 36). All
substrates and RAG vectors were analyzed in a minimum of three
independent transfections.
PCR assays.
Ligation-mediated PCR (LMPCR) was performed as
previously described (29, 35, 36). Briefly, ligations were
incubated overnight at 14°C with 2 U of T4 DNA ligase (Gibco
BRL), 200 pmol of annealed oligonucleotides DR19/DR20 (35),
and 1/50 of the DNA harvested from a single transfection. Ligated
samples were amplified for 24 cycles with primers specific to the
12-signal (DR20 and DR55) or 23-signal (DR20 and ML68) (36).
Primers ML68 and DR55 were used to amplify signal joints (24 cycles)
generated on plasmid pJH290. Blots were probed with the 32P
end-labeled oligonucleotide probe DR55, ML68, or the junction-specific probe DR69 (35).
Southern blot analysis.
Two- to three-fifths of each
transfection sample was subjected to PvuII digestion for
4 h. Samples were subjected to Southern blot analysis, and blots
were probed with a 32P random-primed 693-nucleotide (nt)
PvuII fragment of pJH290 (36). PhosphorImager
quantitation was performed with a Storm860 (Molecular Dynamics).
Western blot analysis.
Lysates from RMP41 cells transfected
with recombination substrate and RAG expression vectors were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed
with anti-human c-myc antibody (PharMingen). Detection
involved use of horseradish peroxidase-conjugated anti-mouse
immunoglobulin G (IgG) (PharMingen) as the secondary antibody and
enhanced chemiluminescence Western blotting detection reagents (Amersham).
| |
RESULTS |
Truncated RAG proteins generate high levels of signal ends.
Previously we developed an assay to measure levels of recombination
intermediates and products derived from extrachromosomal substrates
(35). A plasmid recombination substrate and expression vectors encoding RAG-1 and RAG-2 are transiently transfected into RMP41
fibroblast cells; after 40 to 48 h, products and intermediates are analyzed either by PCR amplification or directly by Southern blot analysis, as described previously (7, 35, 36).
Initial analysis by LMPCR revealed that truncated RAG proteins
generated approximately 10-fold-higher levels of signal ends
than
full-length RAG proteins (Fig.
2A,
compare lanes 3 and 6).
To confirm and extend these findings,
samples from transfections
of truncated or full-length RAG proteins
were analyzed directly
by Southern blotting. Cleavage at both signals
of the substrate
pJH290 generates a 329-nt excised fragment terminating
in signal
ends (diagrammed in Fig.
1A), whereas cleavage of the pJH289
substrate
generates a pair of coding ends on an excised fragment and a
pair
of signal ends on the plasmid backbone. As shown in Fig.
2B,
levels
of the excised signal end fragment derived from cleavage of
pJH290
were substantially increased in transfections containing
truncated
RAG proteins compared to full-length RAG proteins (329-nt
species
labeled SE; compare lanes 1 and 2). A similar effect was seen
with the pJH289 substrate, in which levels of the signal ends
on the
plasmid backbone were increased in the presence of truncated
RAG
proteins (232- and 208-nt species; compare lanes 6 and 7).
Quantitation
of data from several transfections revealed that
signal ends produced
by truncated RAG proteins were consistently
about 10-fold more abundant
than signal ends derived from full-length
RAG proteins (Table
1). Similar results were obtained in
another
wild-type CHO cell line, CHOK1, and in the mutant CHO cell
line,
xrs-6, which fails to carry out signal joint formation due to
lack of functional Ku86 (
7) (data not shown).
View larger version (50K):
[in this window]
[in a new window]
|
FIG. 2.
Signal end levels are 10-fold higher in transfections
with truncated RAG proteins compared with full-length proteins. (A)
LMPCR analysis of a signal end derived from pJH290. DNA from a
transfection containing truncated RAG proteins (TR1+TR2) was diluted,
as indicated above each lane, prior to ligation. DNA from a
transfection containing full-length RAG proteins (FL1+FL2) was
undiluted (1×). The blot was hybridized with radiolabeled
oligonucleotide DR69 (35). The expected position of the
LMPCR product (141 nt) is marked. The signal analyzed in this blot
contains a 12-nt spacer; LMPCR analysis of a signal with a 23-nt
spacer gave comparable results (data not shown). (B) Southern blot
analysis of the two recombination substrates pJH290 and pJH289
(diagrams shown above blot). Samples were cut with restriction enzyme
PvuII. Molecular species generated by cleavage and/or
restriction enzyme digestion are identified to the side of each blot
(restriction site represented by P). Lanes 5 and 10 contain
transfections of recombination substrate in the absence of RAG
constructs. Each lane contains three-fifths of the total DNA harvested
from a transfection sample; all samples in panel B were transfected in
parallel. The blot was hybridized with a radiolabeled PvuII
fragment of pJH290 containing the unrearranged signal pair
(36). Open triangle, signal with 12-nt spacer (12-signal);
closed triangle, signal with 23-nt spacer (23-signal); SE, fragments
containing a signal end(s); CE, fragments containing a coding end(s).
This blot is representative of more than four experiments that all gave
similar results. Although the 12-signal end fragment in lane 6 appears
to be present at slightly higher levels than the 23-signal end
fragment, three independent experiments (data not shown) show no
difference between the levels of the 12- and 23-signal end fragments.
In this and some subsequent gel photographs, all samples were run on
the same gel, but some lanes were removed for clarity.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Quantitation of levels of signal end intermediates
in several transfections containing truncated and/or
full-length RAG proteins
|
|
To determine whether truncated RAG-1 and RAG-2 are jointly responsible
for the difference in signal end levels, we transfected
combinations of
truncated and full-length RAG proteins (TR1 plus
FL2 or FL1 plus TR2).
Both combinations gave moderately (two-
to threefold) increased levels
of signal ends on the excised fragment
(Fig.
2B, lanes 3 and 4) and on
the plasmid backbone (lanes 8
and 9), as summarized in Table
1. These
results indicate that
both RAG-1 and RAG-2 contain elements that affect
signal end
levels.
One potential explanation for increased levels of signal end
intermediates is that truncated RAG proteins may simply perform
DNA
cleavage more efficiently than their full-length counterparts.
The
transient transfection system allows detection of coding ends,
which
have also been observed previously in lymphoid cells expressing
high
levels of full-length RAG-1 and RAG-2 (
24). Unlike signal
ends, levels of coding ends on neither the plasmid backbone (Fig.
2B,
199- and 165-nt species labeled CE in lanes 1 and 2) nor the
excised
fragment (253-nt species in lanes 6 and 7) were affected
by the use of
truncated RAG proteins. This experiment was repeated
more than 10 times, with similar results. These data suggest that
the truncated RAG
proteins do not generate increased levels of
signal ends simply by
performing cleavage more efficiently. An
alternative possibility is
that the high levels of signal ends
reflect altered processing, such as
increased stability of these
recombination intermediates or increased
protection from degradation,
as discussed
below.
Truncated RAG proteins generate low levels of signal and coding
joints.
Since truncated RAG proteins unexpectedly
generated increased levels of signal ends, we measured the
abundance of coding and signal joints by Southern blotting. Coding
joints were reduced in transfections of truncated RAG proteins (Fig.
3A; ~364-nt species labeled CJ; compare
lanes 1 and 2). Quantitation of five independent experiments revealed
an average sevenfold decrease in levels of coding joints generated by
truncated RAG proteins in comparison to full-length RAG proteins.
Signal joints were also reduced three- to fivefold in the presence of
truncated RAG proteins (460-nt species labeled SJ; compare lanes 4 and
5). Thus, although we observed increased signal ends in transfections
containing truncated RAG proteins, levels of recombination products
were actually decreased.
View larger version (62K):
[in this window]
[in a new window]
|
FIG. 3.
Coding joint and signal joint levels are reduced in
transfections containing truncated RAG proteins. (A) Southern blot
analysis of the two recombination substrates (pJH290 and pJH289).
Molecular species are identified on the sides of the blot. Unlabeled
species present between the coding joint and unrearranged fragments
correspond to singly cleaved species (36). Techniques were
performed as described for the experiment shown in Fig. 2B. (B) PCR
analysis of signal joints generated by truncated and full-length RAG
proteins. Lane 2, a 10-fold dilution of the transfection in lane 1. PCR
samples were subjected to gel electrophoresis and Southern
blot analysis as previously described (36). The blot was
hybridized with the radiolabeled oligonucleotide DR55. These same
transfection samples were analyzed in panel C. (C) Southern blot
analysis of coding joints generated by truncated and full-length RAG
proteins. Techniques were performed as in the experiment shown in Fig.
2B. SE, signal end fragment; CJ, coding joint fragment; SJ, signal
joint fragment; P, PvuII restriction site.
|
|
We next asked whether truncated RAG-1, RAG-2, or both are responsible
for the effects on coding and signal joints. The PCR
analysis shown in
Fig.
3B indicates that transfections of both
combinations of truncated
plus full-length RAG proteins moderately
reduced levels of signal
joints (lanes 4 and 5). Therefore, both
truncated RAG-1 and RAG-2 are
responsible for the reduced signal
joint levels. These data are
summarized in Table
2.
Southern blot analysis of coding joints is shown in Fig.
3C.
Transfection of truncated RAG-1 plus full-length RAG-2 (lane
4)
generated levels of coding joints as high as those seen with
full-length RAG proteins (lane 2). However, transfection of full-length
RAG-1 and truncated RAG-2 (Fig.
3C, lane 5) gave low levels of
coding
joints, as seen with truncated RAG proteins (lane 1). These
results
establish that truncated RAG-2, but not RAG-1, is responsible
for
decreased coding joint formation and provide the first evidence
specifically implicating RAG-2 in coding joint
formation.
Different regions of the RAG proteins are responsible for increased
signal ends and decreased joints.
Since our data show that removal
of the dispensable regions of the RAG proteins affects levels of both
ends and joints, one might expect to find a correlation between the
two; that is, decreased joining might result in increased ends. Such a
correlation is not always observed in vivo. Signal ends are not
increased in mice (39) or in cultured cells (7)
bearing mutations in the DNA repair machinery that virtually eliminate
signal joint formation. Nevertheless, we considered the possibility
that decreased formation of signal joints seen with truncated RAG
proteins might be directly related to the increased levels of signal
ends. To explore the relationships between the effects of the
truncations on signal ends, signal joints, and coding joints, we used a
series of RAG constructs containing smaller deletions within the
"dispensable" portions of these proteins. In this manner, we mapped
the regions responsible for each effect. In these experiments, the
various truncations are tested paired with the standard truncated form of RAG-1 or RAG-2. The same results were obtained when the deletion mutants were paired with full-length RAG proteins (data not shown).
To determine whether the same regions of RAG-2 are responsible for
decreased signal and coding joints and increased signal
ends, we used
the deletion mutants diagrammed in Fig.
4A. An expression
vector encoding amino
acids 1 to 517 of RAG-2 is sufficient to
increase signal ends to levels
seen with truncated RAG proteins
(Fig.
4B, compare lanes 1 and 3).
Therefore, the region responsible
for the increased signal ends
generated by truncated RAG-2 is
localized to the 10 C-terminal amino
acids. However, unlike truncated
RAG-2, this construct (amino acids 1 to 517) does not cause decreased
coding joint formation (compare lanes
1 and 3) or signal joint
formation (data not shown), indicating that
the reduced levels
of joints are not necessarily associated with
increased levels
of signal ends.
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 4.
Deletion analysis of the regions of RAG-2 responsible
for reduced coding joint levels and increased signal end levels. (A)
Schematic diagram of the RAG-2 constructs analyzed. Proteins are
represented by lines, with spaces indicating regions deleted from the
construct. Numbers above each line identify the terminal amino acids.
The gray box between amino acids 414 and 481 illustrates the area that
affects coding joint and signal joint formation, and the dark box
between amino acid 517 to 527 illustrates the area that affects signal
end levels, as demonstrated in panel B. Drawings are not to scale. (B)
Representative Southern blotting data that map the regions responsible
for the decreased levels of coding joint products and increased levels
of signal end intermediates produced by truncated RAG proteins.
|
|
Additional deletion mutants were used to further map the regions of
RAG-2 responsible for decreased coding and signal joints.
Coding joints
generated by a RAG-2 construct retaining amino acids
1 to 481 are not
decreased to the low levels seen with truncated
RAG-2 (Fig.
4B, compare
lanes 1 and 4). However, removal of an
additional 56 amino acids (1 to
425) reduces coding joint levels
to those seen with truncated RAG-2
(compare lanes 1 and 5). Similar
results are generated by a construct
retaining amino acids 1 to
418 (data not shown). Since a mutant with an
internal deletion
(del388-413) did not decrease coding joint levels to
the extent
seen with truncated RAG-2 (compare lanes 1 and 6), we
conclude
that a region within amino acids 414 to 481 is responsible for
the lower levels of coding joints observed with truncated RAG-2.
This
region, which contains many conserved residues, is also responsible
for
the decreased signal joint levels seen with truncated RAG-2
(data not
shown). Additional evidence supports our conclusion
that this region of
RAG-2 is important for joint formation, as
alteration of amino acids
436 to 441 causes a greater-than-15-fold
reduction in both signal and
coding joints (
31). Therefore,
nonoverlapping regions of
RAG-2 are responsible for the decreased
coding and signal joints
(residues 414 to 481) and the increased
signal ends (residues 517 to
527) caused by truncated RAG-2.
To map the regions of RAG-1 responsible for effects on recombination
intermediates (signal ends), we employed the series of
RAG-1 deletion
mutants diagrammed in Fig.
5A. A
construct containing
residues 1 to 1008 generated signal ends at levels
similar to
truncated RAG-1 (Fig.
5B, compare lanes 1 and 5), indicating
that
removal of the C-terminal "dispensable" region of RAG-1 is
sufficient
to produce this effect. However, removal of the N-terminal
"dispensable"
region of RAG-1 (amino acids 331 to 1040) is also
sufficient to
cause the high levels of signal ends seen with truncated
RAG-1
(Fig.
5B, compare lanes 6 and 9). As expected, signal ends were
increased when a construct with deletions at both ends (332 to
1008)
was used (Fig.
5B, lane 10). Therefore, removal of either
the
N-terminal 330 amino acids or the C-terminal 32 amino acids
of RAG-1 is
sufficient to reproduce the effect of truncated RAG-1
on levels of
signal ends.
View larger version (54K):
[in this window]
[in a new window]
|
FIG. 5.
Deletion analysis of regions of RAG-1 responsible for
increased levels of signal ends. (A) Schematic diagram of the RAG-1
constructs analyzed. Shaded boxes indicate regions that affect signal
end (SE) or signal joint (SJ) levels. (B) Representative Southern
blotting data that map the portions of RAG-1 responsible for increased
signal ends. Techniques were performed as in the experiment shown in
Fig. 2B. Signal end levels in the del1-1008 construct are unusually
high on this blot compared to those in other independent experiments
(data not shown).
|
|
We also analyzed the effects of the RAG-1 deletion mutants on
recombination products. Since coding joint levels were not affected
by
the use of truncated RAG-1, as shown above, only analysis of
signal
joint levels is discussed here. Removal of the N terminus
but not the C
terminus decreased signal joints to levels seen
with truncated RAG-1
(data not shown). In agreement with our findings,
others have also
mapped the region responsible for reduced signal
joints to the N
terminus of RAG-1 (
21,
26). Therefore, the
regions of RAG-1
responsible for the high levels of signal ends
seen with truncated
RAG-1 do not strictly correlate with the regions
responsible for low
levels of signal
joints.
Truncation increases levels of RAG proteins.
Previous work has
shown that levels of truncated RAG-1 protein are about 10-fold higher
than levels of a RAG-1 construct missing only the C-terminal 32 amino
acids (21). However, in that study, levels of RAG-2 were not
measured. We measured levels of the RAG proteins by Western blot
analysis with antibodies to c-myc epitope tags present on
both truncated proteins, full-length RAG-2, and a RAG-1 construct
missing only the C-terminal 32 amino acids (del1-1008) (30,
31). As shown in Fig. 6, truncated
RAG-1 and RAG-2 were expressed at approximately 10-fold-higher levels
than full-length RAG-2 protein and RAG-1 (del1-1008) (compare lane 1 to
lanes 3 and 4; lanes 6 and 7). Therefore, like RAG-1, RAG-2 protein is present at higher levels when truncated. When transfections contained full-length RAG-1 and truncated RAG-2 or vice versa (lanes 8 and 9),
levels of truncated proteins remained high and levels of full-length RAG-2 protein remained low. Thus, the intermediate effects of the
combinations of one truncated plus one full-length RAG protein on
signal ends are not simply caused by intermediate protein levels. These
experiments, which have been repeated three times with the same
results, also show that the truncated proteins do not influence the
levels of their full-length partners.
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 6.
Truncated RAG proteins are present at 10-fold-higher
levels than full-length RAG proteins. Western blot analysis of
transfections containing truncated or full-length RAG proteins.
Truncated RAG-1 (TR1), truncated RAG-2 (TR2), full-length RAG-2 (FL2),
and a RAG-1 construct containing amino acids 1 to 1008 each have three
copies of the c-myc epitope on their carboxy termini and are
identified to the left and right of the blots. Full-length RAG-1 cannot
be detected, since it has no c-myc epitope tag. Two
independent experiments are shown. Blots were hybridized with
anti-human c-myc primary antibody. Lane 2, 10-fold dilution
of the sample in lane 1; lanes 5 and 10, no RAG proteins in
transfections.
|
|
Protein levels are not responsible for the effects of truncated RAG
proteins on signal ends or joints.
The results described above
suggest that the effects of truncated RAG proteins on signal ends and
recombination products may be secondary to the effects of these
truncations on protein levels. This hypothesis was tested in two ways:
by decreasing the levels of truncated RAG proteins and by increasing
the levels of full-length RAG proteins. As shown in Fig.
7A, transfections of 10-fold-less truncated RAG expression vector (0.1×) produced 10-fold-lower protein
levels than a standard (1×) transfection (compare lanes 2 and 4).
These reduced levels of truncated proteins were similar to standard
(1×) levels of full-length RAG proteins (compare lanes 4 and 5).
Similarly, transfecting fivefold-more (5×) full-length RAG expression
vector produced fivefold-higher protein levels (compare lanes 5 and 6).
The effects of altered RAG protein levels on V(D)J recombination were
assessed by Southern blotting (Fig. 7B). Levels of signal ends and
coding joints were comparable in transfections containing the standard
(1×) amounts of truncated RAG proteins and 10-fold-smaller (0.1×)
amounts (Fig. 7B, compare lanes 1 and 3). Transfections containing
either the standard amounts of full-length RAG proteins or
fivefold-larger (5×) amounts also generated similar levels of signal
ends and coding joints (compare lanes 4 and 5). Therefore, altered
protein levels are not responsible for the differences in levels of
signal ends and recombination products.
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 7.
Higher levels of truncated RAG proteins are not
responsible for the effects on signal end and coding joint levels. (A)
Western blot analysis showing that the amount of transfected expression
vector (indicated above each lane) closely correlates with levels of
protein. Different amounts of RAG expression vectors were transfected
(although the total amount of transfected DNA was held constant): 1×
denotes the standard amounts of RAG-1 and RAG-2 (see Materials and
Methods); 0.1× denotes transfections containing 10-fold-smaller
amounts of RAG constructs. Lane 1, recombination substrate but no RAG
constructs; lane 3, the same transfection sample as lane 2 but
10-fold-less sample was analyzed by electrophoresis; lane 7, the same
transfection sample as lane 6 but 10-fold-less sample was analyzed by
electrophoresis. Western blotting was performed as in the experiment
shown in Fig. 6. (B) Southern blot analysis of transfections identical
to those shown in panel A (performed simultaneously).
|
|
Additional evidence supporting this conclusion is provided by the RAG-1
construct 1-1008. This protein was expressed at levels
much lower than
truncated RAG-1 (Fig.
6, lane 4), comparable to
levels of full-length
RAG-2 (Fig.
6, lane 3). However, levels
of signal ends were similar to
levels produced by truncated RAG-1
(Fig.
5B, compare lanes 1 and 5).
Thus, increased levels of RAG
proteins are not responsible for the
increased levels of signal
ends seen with truncated RAG
proteins.
| |
DISCUSSION |
Here we report the first analysis of the effects of truncated and
full-length RAG proteins on formation of both the intermediates and the
products of V(D)J recombination. The ability to examine the effects of
these proteins on recombination intermediates allows us to more
precisely identify steps in the reaction pathway that are affected by
truncation of RAG-1 and RAG-2. Since the characterized functions of the
RAG proteins affect the cleavage step, we expected that the decreased
levels of coding and signal joints associated with truncated RAG
proteins would be a result of decreased cleavage. However, we did not
observe decreased levels of either signal or coding ends, indicating
that the truncations affect a step (or steps) subsequent to cleavage.
Instead, levels of signal ends are substantially increased in cells
transfected with truncated RAG proteins, while coding end levels are
not affected. It is surprising that these intermediates are so
differently affected by RAG truncation (discussed below). Regions of
RAG-2 responsible for decreased levels of joints and increased levels
of signal ends can be mapped to nonoverlapping regions within the C
terminus, indicating that these effects are not linked. These data
indicate that, in addition to performing DNA cleavage, the RAG proteins play previously unsuspected roles in the processing and joining of
V(D)J recombination intermediates. Interestingly, the C-terminal one-fourth of RAG-2 also appears to play a role in the regulation of
endogenous immunoglobulin gene rearrangement (11).
Possible mechanisms for the effects of truncated RAG proteins on
signal ends.
Several lines of evidence suggest that coding and
signal ends are processed along different pathways. For example, coding joints undergo loss and/or addition of nucleotides much more frequently than signal joints (16) and form with faster kinetics in
vivo than signal joints (24, 25). Furthermore, in cell-free
systems, signal joint formation is enhanced by removal of the RAG
proteins through deproteinization or heating before the addition of
joining factors, while coding joint formation is actually enhanced by the continued presence of RAG proteins during the joining phase of the
reaction (13, 23). In addition, the RAG proteins are more
stably associated with signal ends than coding ends in cell-free reactions (1, 9). These observations support a model in which signal ends remain in a stable two-ended RAG-DNA complex that
persists after coding ends are joined. For joining to occur, the RAG
proteins must be removed from the signal ends, a reaction which may be
mediated by specific disassembly or remodeling factors (1,
39).
Here we show, for the first time, that truncation of the RAG proteins
affects processing of coding and signal ends in different
ways. We
suggest that, in the absence of their "dispensable" termini,
truncated RAG-1 and RAG-2 stabilize the signal end intermediates
such
that postcleavage complexes containing signal ends are poor
substrates
for disassembly, resulting in their accumulation. Several
mechanisms
could be responsible for the effect of truncated RAG
proteins on signal
ends. Since truncation increases levels of
RAG proteins, one
possibility is that the 10-fold excess of truncated
RAG proteins
obstructs joining or disassembly simply by mass action.
However,
we have shown that decreasing truncated RAG protein levels
10-fold or increasing full-length RAG protein levels fivefold
does not
affect signal end levels. In addition, regions of the
RAG proteins
responsible for increased protein levels differ from
those responsible
for increased signal ends (for example, deletion
of the C-terminal 32 amino acids of RAG-1 reproduced the increased
signal ends but not the
increased protein levels seen with truncated
RAG-1). Thus, increased
levels of RAG proteins are not responsible
for increased levels of
signal
ends.
Since signal ends might also be generated by recleavage of signal
joints, we considered the possibility that more efficient
recleavage
might account for the increased levels of signal ends
observed with
truncated RAG proteins. This model predicts that
truncated RAG proteins
should not raise the levels of signal ends
in cells that are incapable
of forming signal joints. However,
we found that truncated RAG proteins
increased signal end levels
in xrs-6 cells, which are virtually
completely defective for signal
joint formation. These data argue
strongly against a model involving
recleavage of signal
joints.
Another possibility is that the termini of the RAG proteins could
facilitate disassembly of the postcleavage complex. For
example, the
"dispensable" regions of RAG-1 and RAG-2 may contain
protein
degradation signals or chaperone recognition sites that
aid in removal
of the RAG proteins from signal ends. A phosphorylation
site that
targets cell cycle-specific degradation (T490) has been
characterized
in the C terminus of RAG-2 (
17,
19); however,
this site
is not necessary for the effects reported here (Fig.
4 and data not
shown). A precedent for a chaperone recognition
site is provided by the
bacteriophage Mu transposition system.
The MuA transposase is
recognized by the chaperone ClpX to allow
removal of the transposase
from a stable transposition intermediate,
the strand transfer complex
(
14). A 10-amino-acid, positively
charged sequence at the C
terminus of MuA (LEQNRRKKAI) is sufficient
to direct ClpX
activity to MuA, indicating that the ClpX chaperone
recognizes a
discrete signal (
15). We have mapped the region
of RAG-2
responsible for increased signal end levels to the 10
C-terminal amino
acids:
KKS
FL
RRLFD. Seven of these
ten residues
are absolutely conserved among all six species examined
(underlined).
Like the ClpX recognition site, which requires four basic
amino
acids for proper function (
15), four of the conserved
residues
are positively charged. Perhaps these 10 C-terminal amino
acids
of RAG-2 serve as a recognition site for a disassembly
factor.
A role for RAG proteins in joining.
We have shown that RAG-1
and RAG-2 contain regions important for efficient coding and signal
joint formation. According to our mapping data, a region within amino
acids 1 to 330 of RAG-1 is responsible for the decreased levels of
signal joints seen with truncated RAG-1, and a region within amino
acids 414 to 481 of RAG-2 is responsible for the decreased levels of
signal joints and coding joints seen with truncated RAG-2.
It may seem surprising that these regions that affect joining do not
also cause the increased levels of signal ends generated
by truncated
RAG proteins. Similarly, the modest decrease in coding
joint formation
is not accompanied by a discernible increase in
levels of coding ends.
However, in this complex in vivo system,
levels of ends reflect input
from several different factors, including
the efficiency of cleavage
and the use of alternative pathways
such as DNA degradation, in
addition to joining. A precedent for
this situation is seen in
Ku86-deficient mice, in which the abundance
of signal ends does not
differ from that in wild-type mice, despite
severely impaired formation
of signal joints (
39).
According to recent in vitro data, coding joint formation is stimulated
up to 50-fold in the presence of truncated RAG proteins
(
23), and the RAG proteins are capable of opening hairpin
coding
ends (
1a). Here we show that the "dispensable"
regions of the
RAG proteins also affect joining. It is unclear what
roles RAG-1
and RAG-2 may play in coding and signal joint formation.
Interestingly,
removal of the "dispensable" regions of RAG-1
only affects signal
joint formation, while removal of the
"dispensable" region of
RAG-2 affects levels of both signal and
coding joints, suggesting
that these regions have different functions.
Biochemical studies
of the full-length RAG proteins should further
illuminate the
roles of these proteins in the joining
reaction.
| |
ACKNOWLEDGMENTS |
We thank Mary Lowe for assistance in manuscript preparation and
Mary Purugganan and Tania Baker for helpful comments on the manuscript.
This work was supported in part by NIH grant AI-36420. S.S.
was supported in part by NIH Predoctoral Fellowship
T32-AI07495. D.B.R. is a Charles E. Culpeper Medical Research Scholar.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: HHMI/Baylor
College of Medicine, Immunology - M929/DeBakey Bldg., One Baylor
Plaza, Houston, TX 77030-3498. Phone: (713) 798-8145. Fax: (713)
798-3700. E-mail: davidbr{at}bcm.tmc.edu.
Present address: Department of Molecular Biology, Princeton
University, Princeton, NJ 08544.
| |
REFERENCES |
| 1.
|
Agrawal, A., and D. G. Schatz.
1997.
RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination.
Cell
89:43-53[Medline].
|
| 1a.
|
Besmer, E.,
J. Mansilla-Soto,
S. Cassard,
D. J. Sawchuk,
G. Brown,
M. Sadofsky,
S. M. Lewis,
M. C. Nussenzweig, and P. Cortes.
1998.
Hairpin coding end opening is mediated by RAG1 and RAG2 proteins.
Mol. Cell
2:817-828[Medline].
|
| 2.
|
Carlson, L. M.,
M. A. Oettinger,
D. G. Schatz,
E. L. Masteller,
E. A. Hurley,
W. T. McCormack,
D. Baltimore, and C. B. Thompson.
1991.
Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion.
Cell
64:201-208[Medline].
|
| 3.
|
Cuomo, C. A., and M. A. Oettinger.
1994.
Analysis of regions of RAG-2 important for V(D)J recombination.
Nucleic Acids Res.
22:1810-1814[Abstract/Free Full Text].
|
| 4.
|
Eastman, Q. M.,
T. M. J. Leu, and D. G. Schatz.
1996.
Initiation of V(D)J recombination in vitro obeying the 12/23 rule.
Nature
380:85-88[Medline].
|
| 5.
|
Fuschiotti, P.,
N. Harindranath,
R. G. Mage,
W. T. McCormack,
P. Dhanarajan, and K. H. Roux.
1998.
Recombination activating genes-1 and -2 of the rabbit: cloning and characterization of germline and expressed genes.
Mol. Immunol.
30:1021-1035.
|
| 6.
|
Greenhalgh, P.,
C. E. M. Olesen, and L. A. Steiner.
1998.
Characterization and expression of recombination activating genes (RAG-1 and RAG-2) in Xenopus laevis.
J. Immunol.
151:3100-3110[Abstract].
|
| 7.
|
Han, J.-O.,
S. B. Steen, and D. B. Roth.
1997.
Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products.
Mol. Cell. Biol.
17:2226-2234[Abstract].
|
| 8.
|
Hansen, J. D., and S. L. Kaattari.
1998.
The recombination activating gene 1 (RAG1) of rainbow trout (Oncorhynchus mykiss): cloning, expression, and phylogenetic analysis.
Immunogenetics
42:188-195.
|
| 9.
|
Hiom, K., and M. Gellert.
1998.
Assembly of a 12/23 paired signal complex: a critical control point in V(D)J recombination.
Mol. Cell
1:1011-1019[Medline].
|
| 10.
|
Ichihara, Y.,
M. Hirai, and Y. Kurosawa.
1992.
Sequence and chromosome assignment to 11p13-p12 of human RAG genes.
Immunol. Lett.
33:277-284[Medline].
|
| 11.
|
Kirch, S. A.,
G. Rathbun, and M. A. Oettinger.
1998.
Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly.
EMBO J.
17:4881-4886[Medline].
|
| 12.
|
Kirch, S. A.,
P. Sudarsanam, and M. A. Oettinger.
1996.
Regions of RAG1 protein critical for V(D)J recombination.
Eur. J. Immunol.
26:886-891[Medline].
|
| 13.
|
Leu, T. M.,
Q. M. Eastman, and D. G. Schatz.
1997.
Coding joint formation in a cell-free V(D)J recombination system.
Immunity
7:303-314[Medline].
|
| 14.
|
Levchenko, I.,
L. Luo, and T. A. Baker.
1995.
Disassembly of the Mu transposase tetramer by the ClpX chaperone.
Genes Dev.
9:2399-2408[Abstract/Free Full Text].
|
| 15.
|
Levchenko, I.,
M. Yamauchi, and T. A. Baker.
1997.
ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway.
Genes Dev.
11:1561-1572[Abstract/Free Full Text].
|
| 16.
|
Lewis, S. M.
1994.
The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses.
Adv. Immunol.
56:27-150[Medline].
|
| 17.
|
Li, Z.,
D. I. Dordai,
J. Lee, and S. Desiderio.
1996.
A conserved degradation signal regulates RAG-2 accumulation during cell division and links V(D)J recombination to the cell cycle.
Immunity
5:575-589[Medline].
|
| 18.
|
Lieber, M. R.,
J. E. Hesse,
S. Lewis,
G. C. Bosma,
N. Rosenberg,
K. Mizuuchi,
M. J. Bosma, and M. Gellert.
1988.
The defect in murine severe combined immune deficiency: joining of signal sequences but not coding segments in V(D)J recombination.
Cell
55:7-16[Medline].
|
| 19.
|
Lin, W.-C., and S. Desiderio.
1994.
Cell cycle regulation of V(D)J recombination-activating protein RAG-2.
Proc. Natl. Acad. Sci. USA
91:2733-2737[Abstract/Free Full Text].
|
| 20.
|
McBlane, J. F.,
D. C. van Gent,
D. A. Ramsden,
C. Romeo,
C. A. Cuomo,
M. Gellert, and M. A. Oettinger.
1995.
Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps.
Cell
83:387-395[Medline].
|
| 21.
|
McMahan, C. J.,
J. Difilippantonio,
N. Rao,
E. Spanopoulou, and D. G. Schatz.
1997.
A basic motif in the N-terminal region of RAG1 enhances V(D)J recombination activity.
Mol. Cell. Biol.
17:4544-4552[Abstract].
|
| 22.
|
Oettinger, M. A.,
D. G. Schatz,
C. Gorka, and D. Baltimore.
1990.
RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination.
Science
248:1517-1523[Abstract/Free Full Text].
|
| 23.
|
Ramsden, D. A.,
T. T. Paull, and M. Gellert.
1997.
Cell-free V(D)J recombination.
Nature
388:488-491[Medline].
|
| 24.
|
Ramsden, D. A., and M. Gellert.
1995.
Formation and resolution of double-strand break intermediates in V(D)J rearrangement.
Genes Dev.
9:2409-2420[Abstract/Free Full Text].
|
| 25.
|
Roman, C. A. J., and D. Baltimore.
1996.
Genetic evidence that the RAG1 protein directly participates in V(D)J recombination through substrate recognition.
Proc. Natl. Acad. Sci. USA
93:2333-2338[Abstract/Free Full Text].
|
| 26.
|
Roman, C. A. J.,
S. R. Cherry, and D. Baltimore.
1997.
Complementation of V(D)J recombination deficiency in RAG-1 / B cells reveals a requirement for novel elements in the N-terminus of RAG-1.
Immunity
7:13-24[Medline].
|
| 27.
|
Roth, D. B.,
J. P. Menetski,
P. B. Nakajima,
M. J. Bosma, and M. Gellert.
1992.
V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes.
Cell
70:983-991[Medline].
|
| 28.
|
Roth, D. B.,
P. B. Nakajima,
J. P. Menetski,
M. J. Bosma, and M. Gellert.
1992.
V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor  rearrangement signals.
Cell
69:41-53[Medline].
|
| 29.
|
Roth, D. B.,
C. Zhu, and M. Gellert.
1993.
Characterization of broken DNA molecules associated with V(D)J recombination.
Proc. Natl. Acad. Sci. USA
90:10788-10792[Abstract/Free Full Text].
|
| 30.
|
Sadofsky, M. J.,
J. E. Hesse,
J. F. McBlane, and M. Gellert.
1993.
Expression and V(D)J recombination activity of mutated RAG-1 proteins.
Nucleic Acids Res.
21:5644-5650[Abstract/Free Full Text].
|
| 31.
|
Sadofsky, M. J.,
J. E. Hesse, and M. Gellert.
1994.
Definition of a core region of RAG-2 that is functional in V(D)J recombination.
Nucleic Acids Res.
22:1805-1809[Abstract/Free Full Text].
|
| 32.
|
Schatz, D. G.,
M. A. Oettinger, and D. Baltimore.
1989.
The V(D)J recombination activating gene, RAG-1.
Cell
59:1035-1048[Medline].
|
| 33.
|
Schlissel, M.,
A. Constantinescu,
T. Morrow,
M. Baxter, and A. Peng.
1993.
Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated.
Genes Dev.
7:2520-2532[Abstract/Free Full Text].
|
| 34.
|
Silver, D. P.,
E. Spanopoulou,
R. C. Mulligan, and D. Baltimore.
1993.
Dispensable sequence motifs in the RAG-1 and RAG-2 genes for plasmid V(D)J recombination.
Proc. Natl. Acad. Sci. USA
90:6100-6104[Abstract/Free Full Text].
|
| 35.
|
Steen, S. B.,
L. Gomelsky, and D. B. Roth.
1996.
The 12/23 rule is enforced at the cleavage step of V(D)J recombination in vivo.
Genes Cells
1:543-553[Abstract].
|
| 36.
|
Steen, S. B.,
L. Gomelsky,
S. L. Speidel, and D. B. Roth.
1997.
Initiation of V(D)J recombination in vivo: role of recombination signal sequences in formation of single and paired double-strand breaks.
EMBO J.
16:2656-2664[Medline].
|
| 37.
|
van Gent, D. C.,
D. A. Ramsden, and M. Gellert.
1996.
The RAG1 and RAG2 proteins establish the 12/23 rule in V(D)J recombination.
Cell
85:107-113[Medline].
|
| 38.
|
van Gent, D. C.,
J. F. McBlane,
D. A. Ramsden,
M. J. Sadofsky,
J. E. Hesse, and M. Gellert.
1995.
Initiation of V(D)J recombination in a cell-free system.
Cell
81:925-934[Medline].
|
| 39.
|
Zhu, C.,
M. A. Bogue,
D.-S. Lim,
P. Hasty, and D. B. Roth.
1996.
Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates.
Cell
86:379-389[Medline].
|
| 40.
|
Zhu, C., and D. B. Roth.
1995.
Characterization of coding ends in thymocytes of scid mice: implications for the mechanism of V(D)J recombination.
Immunity
2:101-112[Medline].
|
Molecular and Cellular Biology, April 1999, p. 3010-3017, Vol. 19, No. 4
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Simkus, C., Anand, P., Bhattacharyya, A., Jones, J. M.
(2007). Biochemical and Folding Defects in a RAG1 Variant Associated with Omenn Syndrome. J. Immunol.
179: 8332-8340
[Abstract]
[Full Text]
-
Chatterji, M., Tsai, C.-L., Schatz, D. G.
(2006). Mobilization of RAG-Generated Signal Ends by Transposition and Insertion In Vivo. Mol. Cell. Biol.
26: 1558-1568
[Abstract]
[Full Text]
-
Elkin, S. K., Ivanov, D., Ewalt, M., Ferguson, C. G., Hyberts, S. G., Sun, Z.-Y. J., Prestwich, G. D., Yuan, J., Wagner, G., Oettinger, M. A., Gozani, O. P.
(2005). A PHD Finger Motif in the C Terminus of RAG2 Modulates Recombination Activity. J. Biol. Chem.
280: 28701-28710
[Abstract]
[Full Text]
-
Talukder, S. R., Dudley, D. D., Alt, F. W., Takahama, Y., Akamatsu, Y.
(2004). Increased frequency of aberrant V(D)J recombination products in core RAG-expressing mice. Nucleic Acids Res
32: 4539-4549
[Abstract]
[Full Text]
-
Repasky, J. A. E., Corbett, E., Boboila, C., Schatz, D. G.
(2004). Mutational Analysis of Terminal Deoxynucleotidyltransferase- Mediated N-Nucleotide Addition in V(D)J Recombination. J. Immunol.
172: 5478-5488
[Abstract]
[Full Text]
-
Jiang, H., Ross, A. E., Desiderio, S.
(2004). Cell Cycle-dependent Accumulation in Vivo of Transposition-competent Complexes between Recombination Signal Ends and Full-length RAG Proteins. J. Biol. Chem.
279: 8478-8486
[Abstract]
[Full Text]
-
Swanson, P. C., Volkmer, D., Wang, L.
(2004). Full-length RAG-2, and Not Full-length RAG-1, Specifically Suppresses RAG-mediated Transposition but Not Hybrid Joint Formation or Disintegration. J. Biol. Chem.
279: 4034-4044
[Abstract]
[Full Text]
-
Jones, J. M., Gellert, M.
(2003). Autoubiquitylation of the V(D)J recombinase protein RAG1. Proc. Natl. Acad. Sci. USA
100: 15446-15451
[Abstract]
[Full Text]
-
Dudley, D. D., Sekiguchi, J., Zhu, C., Sadofsky, M. J., Whitlow, S., DeVido, J., Monroe, R. J., Bassing, C. H., Alt, F. W.
(2003). Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice. JEM
198: 1439-1450
[Abstract]
[Full Text]
-
Ross, A. E., Vuica, M., Desiderio, S.
(2003). Overlapping Signals for Protein Degradation and Nuclear Localization Define a Role for Intrinsic RAG-2 Nuclear Uptake in Dividing Cells. Mol. Cell. Biol.
23: 5308-5319
[Abstract]
[Full Text]
-
Yurchenko, V., Xue, Z., Sadofsky, M.
(2003). The RAG1 N-terminal domain is an E3 ubiquitin ligase. Genes Dev.
17: 581-585
[Abstract]
[Full Text]
-
Akamatsu, Y., Monroe, R., Dudley, D. D., Elkin, S. K., Gartner, F., Talukder, S. R., Takahama, Y., Alt, F. W., Bassing, C. H., Oettinger, M. A.
(2003). Deletion of the RAG2 C terminus leads to impaired lymphoid development in mice. Proc. Natl. Acad. Sci. USA
100: 1209-1214
[Abstract]
[Full Text]
-
Noordzij, J. G., de Bruin-Versteeg, S., Verkaik, N. S., Vossen, J. M. J. J., de Groot, R., Bernatowska, E., Langerak, A. W., van Gent, D. C., van Dongen, J. J. M.
(2002). The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins. Blood
100: 2145-2152
[Abstract]
[Full Text]
-
Tsai, C.-L., Drejer, A. H., Schatz, D. G.
(2002). Evidence of a critical architectural function for the RAG proteins in end processing, protection, and joining in V(D)J recombination. Genes Dev.
16: 1934-1949
[Abstract]
[Full Text]
-
Jones, J. M., Gellert, M.
(2001). Intermediates in V(D)J recombination: A stable RAG1/2 complex sequesters cleaved RSS ends. Proc. Natl. Acad. Sci. USA
10.1073/pnas.221471198v1
[Abstract]
[Full Text]
-
Sadofsky, M. J.
(2001). The RAG proteins in V(D)J recombination: more than just a nuclease. Nucleic Acids Res
29: 1399-1409
[Abstract]
[Full Text]
-
Kale, S. B., Landree, M. A., Roth, D. B.
(2001). Conditional RAG-1 Mutants Block the Hairpin Formation Step of V(D)J Recombination. Mol. Cell. Biol.
21: 459-466
[Abstract]
[Full Text]
-
Villa, A., Sobacchi, C., Notarangelo, L. D., Bozzi, F., Abinun, M., Abrahamsen, T. G., Arkwright, P. D., Baniyash, M., Brooks, E. G., Conley, M. E., Cortes, P., Duse, M., Fasth, A., Filipovich, A. M., Infante, A. J., Jones, A., Mazzolari, E., Muller, S. M., Pasic, S., Rechavi, G., Sacco, M. G., Santagata, S., Schroeder, M. L., Seger, R., Strina, D., Ugazio, A., Valiaho, J., Vihinen, M., Vogler, L. B., Ochs, H., Vezzoni, P., Friedrich, W., Schwarz, K.
(2001). V(D)J recombination defects in lymphocytes due to RAG mutations: severe immunodeficiency with a spectrum of clinical presentations. Blood
97: 81-88
[Abstract]
[Full Text]
-
Agard, E. A., Lewis, S. M.
(2000). Postcleavage Sequence Specificity in V(D)J Recombination. Mol. Cell. Biol.
20: 5032-5040
[Abstract]
[Full Text]
-
Noordzij, J. G., Verkaik, N. S., Hartwig, N. G., de Groot, R., van Gent, D. C., van Dongen, J. J. M.
(2000). N-terminal truncated human RAG1 proteins can direct T-cell receptor but not immunoglobulin gene rearrangements. Blood
96: 203-209
[Abstract]
[Full Text]
-
HUYE, L.E., HAN, J.-O., ROTH, D.B.
(1999). What Prevents Intermolecular V(D)J Recombination?. Cold Spring Harb Symp Quant Biol
64: 191-196
[Abstract]
-
Jones, J. M., Gellert, M.
(2001). Intermediates in V(D)J recombination: A stable RAG1/2 complex sequesters cleaved RSS ends. Proc. Natl. Acad. Sci. USA
98: 12926-12931
[Abstract]
[Full Text]
Top
Abstract
Introduction
