of June 13, 2013.
This information is current as
of DNA Polymerase
Frequency in Complementary Mouse Models
Altered Ig Hypermutation Pattern and
and Marilyn Diaz
Madhumita Ray, W. Glenn McGregor, Thomas A. Kunkel
Kathleen Richter, Chuancang Jiang, Ming-Lang Zhao,
Janssen Daly, Katarzyna Bebenek, Danielle L. Watt,
2012; 188:5528-5537; Prepublished online 30
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The Journal of Immunology
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The Journal of Immunology
Altered Ig Hypermutation Pattern and Frequency in
Complementary Mouse Models of DNA Polymerase z Activity
Janssen Daly,* Katarzyna Bebenek,†Danielle L. Watt,†Kathleen Richter,*
Chuancang Jiang,* Ming-Lang Zhao,* Madhumita Ray,* W. Glenn McGregor,‡
Thomas A. Kunkel,†and Marilyn Diaz*
To test the hypothesis that DNA polymerase z participates in Ig hypermutation, we generated two mouse models of Pol z function:
a B cell-specific conditional knockout and a knock-in strain with a Pol z mutagenesis-enhancing mutation. Pol z-deficient B cells
had a reduction in mutation frequency at Ig loci in the spleen and in Peyer’s patches, whereas knock-in mice with a mutagenic Pol
z displayed a marked increase in mutation frequency in Peyer’s patches, revealing a pattern that was similar to mutations in yeast
strains with a homologous mutation in the gene encoding the catalytic subunit of Pol z. Combined, these data are best explained by
a direct role for DNA polymerase z in Ig hypermutation.The Journal of Immunology, 2012, 188: 5528–5537.
affinity of Abs to a specific Ag (1, 2). This mechanism, termed
somatic hypermutation (SHM), is triggered by the activation-
induced deaminase (AID), a molecule expressed when B lym-
phocytes are activated by foreign Ag (3, 4). AID deaminates
cytosines in the DNA encoding the Ig variable (V) regions (5).
Mice and humans defective in AID lack SHM and class switch
recombination (CSR), as AID is also required to generate switched
Abs such as IgG, IgA, and so on (3, 4). A subset of hyper IgM
syndrome patients are defective in AID; these patients lack CSR
and SHM, and they suffer from lymph node hyperplasia (6).
Despite the fact that AID is a cytosine deaminase, mice deficient
in AID lack mutations at both G-C and A:T bp (3). This paradox is
in part explained by the hypothesis that AID-mediated deamina-
tion of cytosines in Ig V regions triggers the recruitment of
translesion synthesis DNA polymerases to Ig loci (7). These DNA
polymerases have relaxed geometric requirements and thus are
prone to inserting incorrect bases during DNA synthesis (8). One
such polymerase is DNA polymerase h, which has been shown to
play a role in the misinsertion of bases at A:T sites during SHM (9,
10). Other DNA polymerases have also been implicated in SHM,
he genes encoding the Ag-interacting portions of Abs
and IgRs are subjected to a process of deliberate hyper-
mutation during immune responses leading to enhanced
but because they have important roles in other cellular functions
such as cell division and DNA repair, discerning whether they play
a direct or indirect role in SHM has been difficult (11–13).
The mutations made during SHM of Ig V genes are predomi-
nantly base substitutions. The pattern of hypermutation suggests
that a putative error-prone DNA polymerase must not only insert
the incorrect base but also extend from a mismatched terminus,
a very difficult task for most DNA polymerases. Confounding this
is the fact that some of the base substitutions in SHM occur in
tandem, suggesting multiple misinsertion and mismatch extension
events during a single DNA transaction (14). This led to the hy-
pothesis that DNA polymerase z plays a direct role in SHM, be-
cause it is a robust mismatch extender, alone and in conjunction
with other translesion synthesis DNA polymerases, including Pol h
(9, 14–16). However, demonstrating this has been difficult be-
cause mice deficient in DNA polymerase z are early embryonic
lethals (17–19). A mouse expressing antisense RNA against Rev3
(encoding the catalytic subunit of Pol z) experienced decreased
SHM frequency and severely impaired affinity maturation (20).
However, because all cells, not just B cells, expressed the anti-
sense transcript, it remained possible that the phenotype was due
to indirect effects, such as diminished T cell function. In addition,
SHM was reduced in human B cell lines in which the Rev3 gene
encoding the catalytic domain of DNA polymerase z was inhibited
by antisense oligonucleotides, suggesting a direct role for this
polymerase in an in vitro model of hypermutation (21). A con-
ditional knockout mouse model of Rev3 using the CD21 promoter
also resulted in a reduced SHM frequency that was difficult to
discern from a proliferation defect (22).
To circumvent the problem of embryonic lethality and non-
specific effects from Pol z deficiency in non-B cells, we generated
mice with B cell-specific deletion of Rev3. We also constructed
mice with a knocked-in leucine (L) to phenylalanine (F) mutation
at residue 2610 in Rev3 (Rev3L2610F mice). The homologous
change in Saccharomyces cerevisiae increases spontaneous and
ultraviolet light-induced mutagenesis and is associated with a
specific error signature (23, 24). We reasoned that if Pol z plays a
direct role in SHM, a more mutagenic variant would increase the
frequency of mutation at Ig loci and its error signature would be
accentuated. Indeed, we show in this article that Rev3 knockout
mice experienced a dramatic reduction in SHM frequency,
whereas Rev3L2610F mice showed a significant increase in SHM
*Somatic Hypermutation Group, Laboratory of Molecular Genetics, National Insti-
tute of Environmental Health Sciences, National Institutes of Health, Department of
Health and Human Services, Research Triangle Park, NC 27709;
Molecular Genetics and Laboratory of Structural Biology, National Institute of En-
vironmental Health Sciences, National Institutes of Health, Department of Health and
Human Services, Research Triangle Park, NC 27709; and‡Department of Pharma-
cology and Toxicology, James Graham Brown Cancer Center, University of Louis-
ville School of Medicine, Louisville, KY 40202
Received for publication September 13, 2011. Accepted for publication April 3,
This work was supported by the National Institutes of Health, National Institute of
Environmental Health Sciences, Division of Intramural Research, Project Z01
ES101603 (to M.D.) and Project Z01 ES065070 (to T.A.K.).
Address correspondence and reprint requests to Dr. Marilyn Diaz, NIEHS/NIH, D3-
01, Building 101, 111 TW Alexander Drive, Durham, NC 27709. E-mail address:
The online version of this article contains supplemental material.
Abbreviations used in this article: AID, activation-induced deaminase; CGG, chicken
gamma globulin; CSR, class switch recombination; GC, germinal center; HC, H
chain; NP, 4-hydroxy-3-nitrophenylacetyl; SHM, somatic hypermutation; V, variable;
by guest on June 13, 2013
frequency and an altered SHM specificity. The results indicate
a direct role for DNA polymerase z in SHM.
Materials and Methods
Generation of B cell-specific Rev3 knockouts and Rev3L2610F
A linearized targeting vector containing loxP sites flanking exon 26 of Rev3
was generated and electroporated into embryonic stem cells from C57BL/6
mice (Supplemental Fig. 1A, 1B). Exon 26 of Rev3 encodes the metal
binding domain of DNA polymerase z and it is essential to its DNA
synthesis function (25, 26). Recombinants were electroporated with cre-
recombinase to eliminate the loxP site-flanked neo site. Clones still
retaining the loxP sites flanking exon 26 of Rev3 (Rev3-floxed mice) were
identified by Southern hybridization. Recombinant clones were confirmed
by PCR analysis using primers N17delckF: 59-GTTTGGGGCATTGGTTT-
ACAGGTG-39 and N17del3R: 59-CTCCTTACTGCTGGGGATACTCAT-
GTG-39. Several clones were selected for blastocyst injection resulting in
male chimeras. Chimeras were bred with C57BL/6 Taconic mice and het-
Rev3-floxed mice were crossed with the cre-recombinase transgenic line
C.Cg-Cd19tm1(cre)Cgn (BALB/c) (27), which we will refer to as CD19+,
and reporter mice B6.129 3 1-Gt(Rosa)26Sortm1(EYFP)Cos/J (C57BL/
6), which we will refer to as YFP+, strains purchased from The Jackson
Laboratory (Bar Harbor, ME). CD19+mice were crossed with YFP+mice
to give CD19+/2, YFP+/2; these mice were then backcrossed to the Rev3F/F
background (10 generations) to give CD19+/2, YFP+/2, Rev3F/F mice in
a C57BL/6 background. The resulting mice had B cell-specific deletion of
Rev3 exon 26, and the recombinant B cells can be collected by FACS by
virtue of their YFP expression (Supplemental Fig. 1B).
The targeted exon 26 of the Rev3 gene was examined by PCR with
primers N1753F (59-TTTCTATCAGCTTGTGCCCTTATCCTTACT-39) and
N17cKR (59-ACAAGATTTCTTTGTGTAACAGCCCTGG-39) for the 59
loxp site, giving WTand mutant products of 474 bp and 624 bp, respectively.
Genotyping of the whole exon 26 region, using N1753F and N17del3R,
resulted in a WT product of 1330 bp and a mutant product of 1630 bp
(Supplemental Fig. 1C). Upon cre-mediated deletion of the floxed region,
the above primers resulted in a product of 632 bp (Supplemental Fig. 1C).
To detect the CD19 cre-recombinase transgene primers, CD19 59 (59-
CTATCTGAAAAATATTTAACAGGTGCCAC-39), CD19 39 (59-CAC-
TATCCTCCACGTTCACTGTCCA-39), and CD19 cre (59-GGCAAATT-
TTGGTGTACGGTCAGTAA-39) were used, resulting in a WT product of
950 bp and a transgene product of 850 bp (Supplemental Fig. 1D). YFP
genotyping primers were 0IMR0316 (59-GGAGCGGGAGAAATGGATAT-
39), 0IMR0883 (59-AAAGTCGCTCTGAGTTGTTAT-39), and 0IMR4982
(59-AAGACCGCGAAGAGTTTGTC-39), which amplify a WT fragment
of 600 bp and a mutant fragment of 320 bp (Supplemental Fig. 1E).
To generate Rev3L2610F (mutator knock-ins), a second vector was
made, with a mutation replacing the leucine at position 2610 with phe-
nylalanine in exon 23 (Supplemental Fig. 2A). The homologousmutationin
yeast reveals a 2- to 3-fold increase in Rev3-mediated mutagenesis (23, 24).
Rev3L2610F cells with the L to F mutation that had undergone homolo-
gous recombination were identified by Southern hybridization and con-
firmed for the mutation by PCR, using primers N29B5PCRF: 59-CTTT-
CATGTTCTCCATCAGCGTTTCC-39 and N29B5PCRR: 59-GGTTAGCT-
GGGCTACATTCCAATTCATC-39, followed by sequencing with primer:
N29B5PCRR: 59-GGTTAGCTGGGCTACATTCCAATTCATC-39 (Sup-
plemental Fig. 2B, 2C).
For Rev3L2610F mice, detection of the mutant allele was done by PCR
with primers N29BNeodelF: 59-GTTAAATCAGCTTCCGTTGCAGCA-
CT-39 and N29BNeodelR: 59-GCTTCCACAAGTGTTTCCTATGAGAG-
TTG-39. The presence of the mutation in mRNA encoding Rev3 in these
mice was confirmed using cDNA as template and using primers F59-
ATgAgAgCCCCACAgTgTgTT-39 and R59-CAACCCTAgCACCCCATTT-
CT-39 and the nested primers 59-gTCTCgTTTCTATAgCAACTCTg-39 and
59-CTggCTTTgTgAACAATgCTATC-39. The nested primers were also
used for sequencing (Supplemental Fig. 2D). All mice were kept at the
pathogen-specific–free animal facility at National Institute of Environ-
mental Health Sciences and were maintained in microisolator cages on
hardwood bedding, and provided with autoclaved food and reverse-
osmosis, deionized water.
Confirmation of Rev3 exon 26 deletion in YFP-expressing
B cells by PCR
For detection of WT transcripts, cDNA from Rev3-deleted B cells was
amplified using Taq DNA polymerase (Invitrogen) and using Rev3 junct
25/26 F (59-GCCGTGCATTGAGGTTGGTGATA-39) and Rev3 exon 27 R
(59-CTTCAGTTTCACTGGCCTAGGATTAGTA-39), resulting in a 229-bp
WT fragment. Mutant Rev3 transcripts were detected using Rev3 junct 25/
27 F (59-TGCCGTGCATTGAGTATGTTTGTACTACT-39) and Rev3 exon
30 R (59-CAGCGTTTCATACATGTAGCCCACAT-39), resulting in a 191-
bp mutant Rev3 transcript (Supplemental Fig. 1F). The RT-PCR of mouse
GAPDH was performed with GAPDH F primer (59-ACCACAGTCCAT-
GCCATCAC-39) and GAPDH R primer (59-TCCACCACCCTGTTGCT-
Mice 8–12 wk old were immunized by i.p. injection of 100 ml (0.1 ml of
1 mg/ml) of 4-hydroxy-3-nitrophenylacetyl (NP) hapten, or 2,4,6-trini-
trophenyl hapten, conjugated to chicken g-globulin (NP-chicken gamma
globulin [CGG], N-5055-5, and trinitrophenyl-CGG, T5052-1; Biosearch
Technologies, Novato, CA) prepared in an alum-precipitated suspension.
Mice were euthanized and their spleens and serum recovered for analysis
14–15 d post immunization.
Preparation of bone marrow B cells and of splenic and Peyer’s
patch germinal center B cells
Spleen and bone marrow cell suspensions were created as described in
Ref. 28. Peyer’s patches were collected from mice at 8, 12, and 26–52 wk
of age and cell suspensions generated as described previously (29).
Flow cytometry analysis
Bone marrow progenitor B cells were enriched using allophycocyanin-
labeled rat anti-mouse B220/CD45R (clone RA3-6B2). Combined stain-
ing with either PE-labeled rat anti-mouse CD117/c-kit (clone ack45),
CD25/IL-2Ra-chain (clone Pc61), or IgM (clone R6-60.2) isolated pre-BI,
pre-BII, or immature B cells, respectively. Splenic B cells were isolated
using PE-labeled rat anti-mouse CD19 (1D3) and IgM (clone R6-60.2), plus
allophycocyanin-labeled rat anti-mouse B220/CD45R (clone RA3-6B2),
all from Pharmingen (San Diego, CA). Activated splenic B cells from NP-
immunized mice were stained for allophycocyanin-labeled rat anti-mouse
B220/CD45R (clone RA3-6B2) (BD Pharmingen) and biotinylated rat anti-
mouse Ig l1, l2, and l3 L chain (clone R26-46) (BD Pharmingen). Bio-
tinylated Abs were revealed using streptavidin-PE (Southern Biotechnol-
ogy, Birmingham, AL), streptavidin-allophycocyanin (BD Pharmingen), or
streptavidin-tricolor (Caltag Laboratories, Burlingame, CA). Peyer’s patch
B cells were enriched by staining with PE-labeled rat anti-mouse B220/
CD45R (clone RA3-6B2) and allophycocyanin-labeled anti-mouse Ly-77
(clone GL7) from eBioscience (San Diego, CA).
Cell sorting was carried out using a Becton Dickinson FACSVantage SE
Flow Cytometer or FACSDiva (Franklin Lakes, NJ). Data were analyzed
using FlowJo (Ashland, OR).
PCR cloning and sequencing of the intronic region downstream
of rearranged V genes and of Vh186.2 regions
RNA from B220+YFP+l+sorted cells was prepared in TRIzol. One
microgram of RNAwas used as template for cDNA synthesis in the reverse
transcriptase reaction, using a SuperScript II First-Strand Synthesis Sys-
tem (RT-PCR; Invitrogen). cDNA (2 ml) was amplified using Phusion DNA
polymerase (New England BioLab, Ipswich, MA), with the VH186.2
specific primers VH186.2 F (59-AGCAGCCTGGGGGCTGAGCTT-39)
and IgH Cg1 R (59-CAGGGGCCAGTGGATAGACAGA-39) and the fol-
lowing PCR conditions: 94˚C for 2 min; 35 cycles of 94˚C for 1 min, 60˚C
for 45 s, and 72˚C for 45 s; and 72˚C for 5 min, resulting in an ∼400-bp
product. Sorting for l+cells and amplifying the VH186.2 region enriches
for Ig sequences that have experienced SHM during the NP response (30).
The reverse primer was anchored in the g constant domain to ensure
amplification of the Ig HC from B cells likely to have participated in the
germinal center (GC) reaction. The IgH intronic region downstream of
rearranged V genes was amplified by PCR, as previously described (28).
Spleen sectioning and immunohistochemistry
GC morphology was examined via biotin-labeled peanut agglutinin (Vector
from 8- to 12-wk-old mice were frozen in Tissue-Tek OCT (Sakura, Tor-
rance, CA) and sectioned on a Leica CM 3050 S cryostat (6 mm) and
affixed to slides with Rapid Fix solution (Shandon Lipshaw, Pittsburgh,
PA). Protein blocking was carried out with an avidin-biotin blocking kit
(Vector Laboratories). Incubation with peanut agglutinin was done at a 1/
1000 dilution and labeling with a Biogenex streptavidin label. The stain was
developed with diaminobenzidine chromogen (DakoCytomation, Glostrup,
The Journal of Immunology5529
by guest on June 13, 2013
Denmark), and the slides were counterstained with hematoxylin and visu-
alized with a fluorescence microscope.
Lymphocyte proliferation assay
For CFSE analysis, splenic B cells from 8- to 12-wk-old C57BL/6J, CD19+
(B6), Rev3L2610F, and Rev3-deleted mice were CD43 depleted using
CD43 MicroBeads and MACS cell separator magnetic columns, according
to the manufacturer’s instructions (Miltenyi Biotec, Bergisch Gladbach,
Germany). Cells were incubated with 5 nM CFSE for 15 min at 37˚C,
following the manufacturer’s instructions (Invitrogen, Molecular Probes,
Carlsbad, CA). Cells were washed twice with PBS and incubated in RPMI
1640 medium. Cells (106cells per milliliter) were then stimulated over 4 d,
using LPS alone (20 mg/ml; Sigma-Aldrich, St. Louis, MO) or together
with IL-4 (25 ng/ml; R&D Systems, Minneapolis, MN). All samples were
analyzed as duplicates. The absorbance (Ex. 492 nm/Em. 517 nm) was
measured using BD Biosciences FACSVantage SE Flow Cytometer. Ac-
tivated B cells were determined using flow cytometry with allophyco-
cyanin-Cy7–labeled rat anti-mouse B220/CD45R (clone RA3-6B2) and
PE-labeled IgM (clone R6-60.2) and IgG1 (clone 15H6), all from BD
Pharmingen. A total of 105cells were analyzed for each sample; five mice
per group were used.
The difference in the distribution of mutational classes (number of clones
with a particular number of mutations) among groups was examined for
significance using the Kolmogorv–Smirnov test. The differences in the
mutation frequency among genotypes in the spleen and Peyer’s patch were
examined using Mann–Whitney statistic and Kolmogov–Smirnov. The
difference in the proportion of insertions and deletions, of tandem multi-
ples, or of mutations from deoxythymidine among all mutations was tested
for significance among groups, using Fisher’s exact test or x2statistic. All
probability values were considered significant if ,0.05.
To test the hypothesis that DNA polymerase z directly induces
mutations at Ig loci during SHM, we generated mice with B cell-
specific deletion of Rev3 exon 26, which encodes the metal
binding domain, and mice with a mutation in Rev3 that, in yeast,
renders the polymerase more mutagenic (23, 24) (Supplemental
Figs. 1–3). Rev3 knockout mice were immunized with the NP
hapten, and mutations at the Vh186.2 H chain (HC) were analyzed
using cDNA from splenic B cells. To sort the cells, we used B220+,
l+, and YFP+B cells to make the cDNA and amplified rearranged
Vh186.2 regions that had switched to IgG. This regimen enriches
for NP-reactive, isotype-switched B cells. YFP is a marker for
expression of cre-recombinase activity and thus strongly corre-
lates with Rev3 deletion, as YFP+cells lacked the floxed exon
(Supplemental Figs. 1A, 1B, 1F). Vh186.2 is the Ig HC associated
with enhanced affinity to NP, and Igs bearing Vh186.2 HCs and l
light chains experience a 10-fold increase in affinity when tryp-
tophan at position 33 is mutated to leucine (30). Affinity matu-
ration to a specific Ag such as NP can profoundly influence the
mutational pattern and frequency at the relevant Ig loci. Therefore,
we also examined the accumulation of mutations in the intronic
region downstream of rearranged J558 Ig HC V genes in B cells
from the ileal Peyer’s patches of Rev3-altered mice. Because the
region analyzed is noncoding, mutations here are a more accurate
representation of the intrinsic SHM machinery in the absence of
B cell development in the bone marrow and the spleen appears
normal in Rev3-altered B cells
Impaired Pol z function did not impact B cell development in the
bone marrow or the spleen compared with CD19-driven cre-
recombinase transgenics and C57BL/6 controls (Fig. 1A, 1B).
More than 90% of B220+B cells were also YFP+, the marker for
Rev3 deletion (Fig. 1C, 1D). The number of GC B cells (GL7+) in
B cell-specific Rev3 knockout mice was similar to that in C57BL/6
controls. Although .90% of all splenic B cells were YFP+, the
percentage decreased to 56% among GL7+B cells (Fig. 1B). This
finding was accentuated dramatically in Peyer’s patches, where
only 3% of the GL7+B cells were YFP+(Fig. 1B). Splenic or
Peyer’s patch B cells from Rev3L2610F mice displayed no de-
fects in B cell development or in the number of GC cells (Fig.
1A; data not shown).
Rearranged Vh186.2 HCs from Rev3-deleted splenic B cells
displayed a reduction in mutation frequency
Following immunization with NP-CGG in alum, YFP+, Rev3-de-
leted B cells from Rev3 conditional knockouts that had been
crossed to CD19 promoter-driven cre-recombinase transgenics
were examined for mutations at the rearranged Vh186.2 locus.
More than 40% of the clones from these mice had either zero or
one mutation compared with ,15% of the clones from either
C57BL/6 controls or CD19 cre-recombinase transgenics without
the floxed Rev3 gene (Fig. 2A, 2B, Supplemental Fig. 4). This
distribution of mutational frequencies was significantly different
from that in controls (Mann–Whitney and Kolmogorov–Smirnov
test, p = 0.00041 and p = 0.001, respectively). In addition, among
VH186.2 clones with at least one mutation, 46% had the affinity-
enhancing mutation W to L at amino acid 33 (Fig. 2B). Re-
markably, in some clones this was the only mutation. GCs from
these mice were indistinguishable from those of control mice (Fig.
1B), in terms of both morphology and abundance, 15 d following
immunization. The fact that GC morphology was intact and that
the same proportion of clones from these mice acquired the NP
affinity-enhancing mutations as in controls suggests that the de-
crease in the mutation frequency is not due to a defect in prolif-
eration in these cells (see below).
Altered immune responses to NP in Rev3L2610F mice
Following immunization, most of the unique clones derived from
Rev3L2610F mice B cells contained slightly fewer mutations than
did C57BL/6 controls (Fig. 2C). However, the fraction of clones
containing $10 mutations increased, with a few clones harboring
.15 in the rearranged V region (Fig. 2C). Although significance
at 0.05 was not achieved, a trend was noted for an increase in the
fraction of Vh186.2 clones with the characteristic W to L change
at amino acid 33 that is associated with increased affinity to NP
(.60% of the clones, Fig. 2C). These data may be explained by an
increased rate of SHM due to an enhanced mutagenic potential or
activity of DNA polymerase z. For example, B cells from these
mice may acquire the affinity-enhancing mutations in VH186.2
more efficiently, and thus terminate SHM early in the GC reaction.
The accumulation of mutations in conditions in which the impact
of selection is minimized (see below) helps clarify these data.
Proliferation among Rev3-altered B cells
Given that only 3% of GL7+B cells in Peyer’s patches and 56% of
GL7+B cells from the spleen were YFP+(compared with .90%
of all B cells), and to rule out a defect in proliferation from Rev3
deficiency, we measured proliferation in Rev3-deleted B cells.
Following activation with LPS, Rev3-deleted B cells consistently
exhibited a significant defect in proliferation compared with CD19
transgenic WT controls (Fig. 3A), as LPS only-treated Rev3-de-
leted B cells appeared to have an excess of cells in earlier stages of
division. This defect was less obvious in LPS plus IL-4–treated
cells when compared with CD19 transgenics with normal Rev3
function (Fig. 3B). Rev3L2610F mice activated with LPS and IL-4
displayed normal proliferation (Fig. 3B). As CSR is tied to pro-
liferation, we examined the switch to IgG1 following activation
with LPS and IL-4 and found that Rev3 knockout cells displayed
5530Rev3 IN Ig HYPERMUTATION
by guest on June 13, 2013
decreased numbers of IgG1+cells when compared with CD19
transgenic controls, whereas Rev3L2610F had levels that were
similar to those in C57BL/6 controls (Fig. 3C).
Mutation accumulation at the intron downstream of rearranged
V genes in Peyer’s patch B cells of Rev3-deleted B cells is
Given the profound impact of Ag-driven selection on the frequency
and pattern of SHM, we examined SHM in Rev3-altered mice in
the intronic region downstream of rearranged VDJ segments from
Peyer’s patch B cells. This region has been characterized as a
target of the SHM machinery, with a reported peak mutation
frequency of 0.01–0.15 mutation per base pair (31, 32). Peyer’s
patch B cells are chronically stimulated and accumulate mutations
over time, but by 5 mo of age most mice reach their peak mutation
frequency (33). All but one of the clones from mice with B cell-
specific deletion of Rev3 were not mutated, even at 6 mo of age
(Fig. 4A). In contrast, .75% of the clones from control mice had
at least one mutation by 6 mo of age. Because only 3% of GL7+
B cells in Peyer’s patches were YFP+in the Rev3 knockout mice,
it is possible that the low mutation frequency of Rev3-deleted
B cells places them at a selective disadvantage in the GC envi-
ronment of Peyer’s patches, especially given the pressure exerted
by microbial flora in the small intestine. Alternatively, a prolifer-
ation defect may place these cells at a competitive disadvantage.
Mutation accumulation at the intronic region downstream of
rearranged V genes in Peyer’s patch B cells of Rev3L2610F
mice is increased and the pattern is significantly altered
Rev3L2610F mice were also examined for mutations at the in-
tronic region downstream of rearranged V genes. A highly signif-
icant increase in the mutation frequency in GC B cells from
FACS. Development of Rev3 knockout and Rev3L2610FB (Rev3L2F) cells in bone marrow stage pre-BII (left, B220+CD25+) and in the immature B cell
stage (middle, B220+IgM+) is comparable to CD19 promoter-driven cre-recombinase transgenics (heterozygotes) and C57BL/6 controls (not shown). As
the B cells mature and migrate to the spleen (right, B220+IgM+), normal numbers of mature/resting splenic B cell populations are observed in both the
Rev3 knockout and Rev3L2610F. (B) Unimmunized 12-wk-old mice were NP-CGG injected and their B cells examined 15 d later. The activated splenic GC
B cell population in Rev3 knockout mice undergoing SHM (B220+GL7+) appears normal in numbers, with 42–56% of this gated population being YFP+.
However, although the percentage of GL7+B cells in Peyer’s patches appears normal, only a tiny fraction of those (3%) were YFP+. (C) Enrichment for
activated splenic B cells from conditional Rev3 knockout, 15 d after immunization with 150 mg NP-CGG in alum. The postsort (B220+YFP+l+) GC
splenic B cell population is greatly enriched for the YFP (marker for Rev3 deletion). The l L chain was selected because it is associated with the Vh186.2
HC for the high-affinity response to the NP hapten. This pure Rev3-null population was used for subsequent RT-PCR and mutation analysis. (D) More than
90% of B220+B cells in the spleen were YFP+in Rev3 knockout mice.
Normal B cell development in Rev3 knockout and Rev3L2610F mice. (A) B cells from 8- to 12-wk-old mice were sorted and examined by
The Journal of Immunology 5531
by guest on June 13, 2013
Peyer’s patches of these mice was noted (Fig. 4A). The number
of mutations per base pair in Rev3 knock-in B cells was ∼0.035,
which represents nearly a 2-fold increase over the WT controls.
Because of the elevated mutation frequency, for analysis, we di-
vided the 1.2-kb region into proximal and distal regions, as defined
by the distance from the rearranged V region, with the idea that the
distal region may be less saturated and a more accurate assess-
ment of the mutational frequency and pattern will be possible. This
analysis revealed that the distal region from Rev3L2610F mice
had experienced a 3-fold increase in mutation frequency (0.014)
over the same region in the WT controls (0.004) that was also
statistically significant (Fig. 4; Supplemental Fig. 3D). Strikingly,
40% of the clones derived from Rev3L2610F mice had accumu-
lated .15 independent mutations, with many of those having 30–
40 mutations in the 420 bp of the proximal region alone (Fig. 4B).
Highly significant changes in the pattern of mutation in B cells
derived from Rev3L2610F mice were also observed. Tandem
mutations, such as adjacent doublets and triplets, were increased
3- to 4-fold, and insertions or deletions (mostly +1 or –1 bp) were
5-fold higher than in controls (Fig. 4C; Fig. 5). Finally, exami-
nation of the nonsaturated sequences (distal region or mutations
from 2-mo-old mice) showed that mutations at thymidine bases
were moderately increased in these cells (Fig. 6), suggesting
strand bias by Pol z in SHM at A:T bp.
In this study, we tested the hypothesis that DNA polymerase z
plays a direct role in SHM, using mice with altered Rev3 function,
the gene encoding the catalytic subunit of DNA polymerase z.
Mice with a B cell-specific deletion of Rev3 displayed a significant
reduction in the accumulation of mutations at the VH186.2 locus
during the immune response to the NP hapten, and Peyer’s patch
B cells from these mice displayed a dramatic reduction in muta-
tion frequency at the intronic region downstream of rearranged V
frequency of mutations at Ig V regions during the immune response to NP-CGG, compared with those in WT mice and CD19 transgenics. Close to 40% of
the clones in the Rev3 knockout had zero or one mutation, compared with ,10% in the controls. Spleens from Rev3 knockout mice displayed normal GC as
shown by peanut agglutinin staining (white arrows), with 46% of the clones achieving the affinity-enhancing W to L mutation at position 33 of Vh186.2. (C)
Rev3 knock-in mice (Rev3L2610F) had a similar overall distribution of mutational classes to controls but had a higher percentage of clones with more than
nine mutations. These mice displayed normal GC, but also showed an increase in the fraction of Vh186.2 clones with the affinity-enhancing W to L change
(pie chart). The following mice were used for these experiments: C57BL/6 and CD19 promoter-driven cre-recombinase transgenics in the C57BL/6
background, n = 6 mice and 75 unique clones; Rev3 knockout, n = 6 mice and 59 unique clones; Rev3L2610F clones, n = 4 mice and 50 unique clones.
Unique clones were determined by the CDR3 amino acid sequence.
The immune response in mice with altered DNA polymerase z function. (A and B) Rev3 knockout B cells experienced a decrease in the
5532Rev3 IN Ig HYPERMUTATION
by guest on June 13, 2013
genes. Indirect effects on SHM, a possibility plaguing previous
studies, are minimized in this study, as B cell-specific deletion of
Rev3 eliminated the impact of Rev3 deletion in other cells, such as
Th cells or APCs. Furthermore, in our model, Rev3-deleted B cells
could be specifically recovered because cre-recombinase deletion
of Rev3 (under control of the CD19 promoter) coincides with YFP
expression (resulting from cre-recombinase–mediated deletion of
a floxed stop codon upstream of the YFP gene). Thus, Rev3-de-
leted cells could be isolated based on YFP expression. This iso-
lation minimized the impact of incomplete Rev3 deletion, particu-
larly in the highly competitive GC environment. Indeed, although
.90% of the splenic B cells were YFP+, only 56% of GL7+B
cells, a GC marker, were YFP+. Peyer’s patch B cells with Rev3
deletion displayed almost no mutation accumulation, even at 6 mo
of age. B cells from Rev3 knockout mice formed morphologically
normal GCs, yet displayed a proliferation defect following acti-
vation with LPS. Among those GC B cells that were mutated, a
similar proportion had acquired the affinity-enhancing tryptophan
to leucine change at amino acid 33 of VH186.2 following immu-
nization with NP, suggesting that eventual acquisition of high-
affinity Abs was not significantly altered in these cells, although
it is possible that the affinity maturation rate may have been al-
tered. These results are similar to those of a previous study using
conditional Rev3 knockout mice, in terms of reduced mutation
frequency, but differ in that smaller GCs from Rev3 knockout mice
were reported in the previous study, whereas GCs appeared nor-
mal in our study (22). This discrepancy may be due to the different
cre-recombinase transgenic strains used in the studies (CD19
versus CD21). For instance, with earlier Rev3 deletion (as the
CD19 promoter is active earlier than the CD21 promoter), it is
possible Rev3-deleted cells may have partially compensated, in
terms of proliferative capacity from Rev3 deficiency, because this
polymerase is not essential for DNA replication. To circumvent
the proliferation issue, and to determine if the SHM frequency
reduction observed in both studies is the result of DNA poly-
merase z participating in the SHM process rather than entirely
explained by a defect in proliferation, we examined mutations at
Ig loci in Rev3 knock-in mice with a hypermutagenic polymerase
z and an altered mutational spectra. An alteration in the pattern
of mutations, consistent with the observed error signature for the
Rev3Ki polymerase, is strong evidence for a direct involvement
and unlikely to be explained by a defect in proliferation.
In the Rev3Ki mice, the Rev3L2610F mice, a highly significant
increase in mutation frequency at the intronic region downstream
of rearranged V genes was seen in Peyer’s patch B cells. In yeast,
the equivalent leucine to phenylalanine replacement in the Rev3
gene increases the error rate of DNA polymerase z ∼2- to 3-fold
without significantly altering its catalytic activity (23, 24). It is
also associated with an increase in complex mutations (more than
one mutation within a short patch presumed to occur in a single
event) and with an increase in insertions and deletions. Given the
high mutation frequency in the Rev3L2610F clones, it was diffi-
cult to ascertain which complex mutations originated from an
accumulation of single mutational events or from a single DNA
polymerase transaction that yielded complex mutational changes.
However, limiting the analysis to tandem doublets or triplets
revealed a 3-to 4-fold increase in these events. Noted also was a 5-
fold increase in insertions and deletions, mostly adding or deleting
a single nucleotide. These are similar to the mutational pattern
observed in yeast (23, 24). Interestingly, an increase in mutations
from thymine was also observed, suggesting Pol z strand bias
during SHM. This is reminiscent of strand bias at A:T bp in Ig
templates from DNA polymerase h-deficient patients and may
imply complementary roles for Pol z and Pol h in SHM. Together,
isolated and incubated with 5 nM CFSE in PBS to measure cell proliferation. B cells (106cells/ml) were stimulated over 4 d, using LPS alone (20 mg/ml)
(A) or together with IL-4 (25 ng/ml) (B), to stimulate CSR. In vitro stimulation of B cells was determined at day 0 and day 4 of stimulation. Flow cytometry
was used to determine the B cell proliferation following in vitro stimulation, as depicted by histograms. The numbered gates represent the percentage of B
cells within sequential rounds of cell division. These proliferation peaks were based on 105events, duplicate samples, and a total of five mice per group. (C)
Percentage of IgG1+B cells following 4 d of activation with LPS and IL-4.
B cell proliferation in Rev3 knockout (Rev3KO) and Rev3L2610F mice. CD43-depleted splenic B cells from 8- to 12-wk-old mice were
The Journal of Immunology5533
by guest on June 13, 2013
the results from both mouse models can best be explained by a
direct role for DNA polymerase z in SHM.
Although significant changes in both the frequency and the
pattern of SHM in mice with altered Rev3 function were observed,
no obvious difference between mutations at G:C and A:T bp was
noted in either strain: All base pairs experienced either a decrease
in Rev3-deleted B cells or an increase in Rev3L2610F B cells
(with the exception of a modest increase in mutations from T
but not from A in the Rev3L2610F mice). The mutation frequency
for Rev3 knockout B cells and the lack of G:C bias are consistent
with data from previously described models of Rev3 deletion,
including mice expressing antisense transcripts to Rev3 (20, 22).
These results are different from those with human B cells deficient
in DNA polymerase h, in which the loss of mutations was pri-
marily at A:T bp (10), and suggest that DNA polymerase z par-
ticipates in both the G:C and the A:T phase of SHM.
On the surface, it appears difficult to reconcile the fact that Pol z
is directly involved in introducing mutations at both G:C and A:T
The IgH intronic region downstream of rearranged V genes of GC B cells was assessed for accumulation of mutations. Peyer’s patch B220+GL7+(and
YFP+for Rev3 knockout) B lymphocytes were isolated from mice aged 12 or 36 wk: C57BL/6, n = 20 mice and 76 unique clones (with 53% of the mutated
clones from 36-wk-old mice); Rev3L2610F, n = 8 mice and 84 unique clones (with 58% of the mutated clones from 36-wk-old mice); Rev3 knockout, n = 4
mice and 18 unique clones (with 100% of the clones from 36-wk-old mice). Because the rearranged CDR3 was included in the PCR fragment, unique
clones could be assessed, and those differing by more than five mutations were considered different clones, although shared mutations were counted only
once. (A) Mutations per nucleotide among mutated clones were calculated for both the proximal (∼430 bp, beginning immediately downstream of JH4) and
the distal ends of the IgH intronic region downstream of rearranged V genes (region was the last 590 bp of the 1.2-kb PCR fragment). The regions were
analyzed separately to examine mutation pattern and frequency in less saturated regions. In both the proximal and the distal regions, the mutation rate in the
Rev3L2610 F mice is at least twice that observed in controls, and this difference was highly significant. Rev3 knockout clones had an extremely low
mutation frequency that was highly significantly different from controls. All except one of the unique Rev3 knockout clones had no mutation; thus, all were
included in the analysis. (B) The increase in mutation frequency among mutated clones in Rev3L2610F mice was the result of clones with a large number of
mutations: A dramatic increase was noted in the fraction of Rev3L2610F clones with .15 mutations, some having $30 more in the proximal region alone
(p = 0.037). Of the Rev3 knockout clones, 98% were unmutated, even in 6 mo-old-mice, compared with 33 and 57% for the control and the Rev3L2610F
clones, respectively. (C) Tandem doublets and triplets and insertions and deletions were significantly increased in Rev3L2610F mice. The difference in
mutation frequencies was tested for significance using the Mann–Whitney U test, the difference in the distribution of mutations among clones was tested for
significance using the Kolmogorov–Smirnov test, and the difference in the frequency of tandems and indels among all mutations was analyzed using the
Fisher exact test (two-sided p values are given).
Mutations at the intronic region downstream of rearranged V genes in Peyer’s patch B cells in mice with altered DNA polymerase z function.
5534 Rev3 IN Ig HYPERMUTATION
by guest on June 13, 2013
bp with the fact that AID triggers SHM by deaminating cytosines
in both strands. One possibility, however, is that Pol z is involved
in the mutagenic bypass of the abasic sites generated by the re-
moval of AID-generated uracils in the DNA of Ig V regions.
Continued synthesis past the abasic site would also result in
mutations at A:T bp, perhaps in complex with Pol h, and this
would generate mutations at all base pairs. Pol z is uniquely ef-
ficient at extending synthesis past a variety of DNA lesions, in-
cluding abasic sites, alone or in complex with REV1, a cofactor
shown to contribute to mutations at AID-mediated abasic sites
in a cell line (34, 35). Furthermore, it has been shown to extend
mismatches generated by other TLS polymerases (36, 37). In Pol
z-deficient B cells, AID-generated abasic sites may be repaired in
an error-free fashion, reducing mutations at all base pairs, not just
G:C bp. Inefficient recruitment of error-prone DNA polymerases
to compensate for Pol z ablation may occur, explaining why 15%
of Rev3-deleted clones derived from splenic B cells had more than
six mutations. Alternatively, Pol z may assemble a targeted
tandem (squares) and indel mutations (+ or 4) when compared with controls (41 unique clones). A similar pattern is seen in the distal region of the
sequences (data not shown). Shared mutations from oligoclonality and single base substitutions are not shown.
Tandem and indel mutations in the proximal HC intronic region. B cells from Rev3L2610F mice (33 unique clones) reveal an increase in
mice reveals a moderate increase in mutations at T
bases, indicating Rev3L2610F-mediated strand
bias. The mutation pattern at the IgH intronic
proximal regions of B cells from young mice is
depicted for controls (A) and Rev3L2610F (B)
mice. The IgH intronic distal region of all controls
(C) and all Rev3L2610F (D) mice B cells are also
shown. The combined increase at thymidine was
statistically significant (x2test, p = 0.04). The base
composition in the proximal region is 57% A:T
and 43% G:C; for the distal region, it is 61% A:T
and 39% G:C.
Mutational pattern of Rev3L2610F
The Journal of Immunology5535
by guest on June 13, 2013
mutasome complex to Ig loci that include AID. This complex
would explain an impact on mutation at all base pairs, but it
appears less likely because it would predict loss of all mutations,
not just a reduction in the mutation frequency, with Rev3 deletion.
In addition, the results using Rev3L2610F mice indicate a role for
Pol z as a DNA polymerase, not just as a targeting complex.
It is interesting that Rev3-deleted B cells appear to be at a dis-
advantage in the Peyer’s patch environment, compared with WT
B cells within the same animal. Although close to 90% of B220+
B cells were YFP+ and therefore Rev3 deleted, only 3% of GC
B cells in Peyer’s patches expressed YFP. In the spleen, the re-
duction was less marked (56% of GL7+B cells) and is more in
agreement with other models of CD19 promoter-driven cre-
recombinase deletion (38). This defect may be due to impaired
proliferation, as seen with splenic cells, or the result of intense
competition in the GC environment for Ag binding and increased
affinity through SHM. A reduced mutation frequency may impact
the rate at which affinity-enhancing mutations are acquired, and
this may place Rev3-deleted B cells in a competitive disadvantage
in the ileum, which is chronically stimulated by microbial flora.
Interestingly, B cells from Rev3L2610F mice in the spleen were
more likely to have affinity-enhancing mutations to NP hapten (60
versus 40% of controls). This fact raises the possibility that the
converse is true: A higher rate of SHM with a mutagenic Pol z is
associated with an enhanced affinity maturation rate for those
cells. Having these novel strains that differ in the ability of their
B cells to undergo SHM will be useful in examining the impact of
SHM rate on the rate of affinity maturation to certain pathogens,
especially those associated with specific Abs bearing a large
number of mutations, as appears to be the case for HIV (39) and
We thank John W. Drake and Dmitry Gordenin for critical reading of the
manuscript, Natasha Clayton and Tiwanda Masinde for assistance with im-
munohistochemistry, Carl Bortner and Maria Sifre for assistance with flow
cytometry, Yingbin Ouyang and Leanne Zhu from Xenogen and Manas Ray
from National Institute of Environmental Health Sciences for chimera gen-
eration, and Scott Jenkins for generation of the Rev3-deleted yeast strain.
The authors have no financial conflicts of interest.
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