DNA Polymerase η Contributes to Strand
Bias of Mutations of A versus T in
Immunoglobulin Genes
This information is current as
of June 17, 2017.
Vladimir I. Mayorov, Igor B. Rogozin, Linda R. Adkison
and Patricia J. Gearhart
J Immunol 2005; 174:7781-7786; ;
doi: 10.4049/jimmunol.174.12.7781
http://www.jimmunol.org/content/174/12/7781
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References
The Journal of Immunology
DNA Polymerase ␩ Contributes to Strand Bias of Mutations of
A versus T in Immunoglobulin Genes1
Vladimir I. Mayorov,* Igor B. Rogozin,†‡ Linda R. Adkison,* and Patricia J. Gearhart2§
S
omatic hypermutation in variable and switch regions of Ig
genes produces a high frequency of mutations of all four
nucleotides. Hypermutation is initiated by the activationinduced cytidine deaminase (AID)3 protein (1), which deaminates
C to U in ssDNA (2– 8). The U lesion can generate mutation in two
phases, as initially described by Neuberger and colleagues (9). In
the first phase, U could remain in the DNA or be removed by
uracil-DNA glycosylase (UNG), and a DNA polymerase (pol)
would insert mutations opposite U or the UNG-generated abasic
site (9, 10). This would produce mutations of C bases or of G bases
if C is deaminated on the complementary strand. Mice and humans
deficient for UNG have fewer transversions of C:G bases, consistent with the absence of abasic sites (11, 12). In the second phase,
U could be recognized as a U:G mismatch by components of the
mismatch repair system (13, 14). This would generate a repair
patch, which then can be filled in by a low-fidelity pol to produce
mutations opposite neighboring A and T bases. Mice deficient for
mismatch repair proteins MSH2 (15–18), MSH6 (19 –21), and exonuclease 1 (22) have fewer mutations of A:T, indicating that they
are involved in error-prone patch repair.
A closer look at the frequency of mutations of all four nucleotides reveals an anomaly that has been a major question in the
*Department of Basic Medical Sciences, Mercer University School of Medicine, Macon, GA 31207; †National Center for Biotechnology Information, National Library of
Medicine, National Institutes of Health, Bethesda, MD 20894; ‡Institute of Cytology
and Genetics, Novosibirsk, Russia; and §Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224
Received for publication January 4, 2005. Accepted for publication March 22, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This research was supported by the National Institutes of Health Intramural Research Program, Russian Fund for Basic Research, and MedCen Foundation.
2
Address correspondence and reprint requests to Dr. Patricia J. Gearhart, Laboratory of
Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600
Nathan Shock Drive, Baltimore, MD 21224. E-mail address: [email protected]
3
Abbreviations used in this paper: AID, activation-induced cytidine deaminase;
UNG, uracil-DNA glycosylase; pol, DNA polymerase; XP-V, xeroderma pigmentosum variant.
Copyright © 2005 by The American Association of Immunologists, Inc.
hypermutation field for years. Humans and mice have ⬃25% mutations of C, 25% of G, 34% of A, and 16% of T in variable
regions, as recorded from the nontranscribed strand (23). The
equal frequency of C and G mutations suggests that C is deaminated on both strands during phase 1. However, the unequal frequency of A and T mutations suggests that there is a bias for
generating these mutations on only one strand during phase 2 (24,
25). One way to introduce strand polarity is during transcription,
when the nontranscribed strand is single stranded, and the transcribed strand is complexed to mRNA. Therefore, the transcription
complex may be involved in directing the phase 2 pathway to the
nontranscribed strand (26). If MSH2-MSH6 and exonuclease 1
initiate a repair patch on this strand, the types of mutations would
correspond to the specificity of the low-fidelity polymerase that
synthesizes in the patch.
The search to identify which low-fidelity polymerases generate
hypermutation has been explored intensely. Animals deficient for
polymerases ␤, ␩, ␫, ␬, ␭, and ␮ have been studied, and only pol
␩ has been shown to substantially alter the spectra of nucleotide
substitutions (27–31). We previously reported that humans with
xeroderma pigmentosum variant (XP-V) disease, who are deficient for pol ␩ (32), have fewer mutations of A:T bp in rearranged VH6 genes (28). However, Yavuz et al. (33) reported that
one XP-V patient did not have fewer mutations of A:T in sequences from other rearranged genes belonging to several VH
families. Recently, studies by Faili et al. (34) and Zeng et al.
(35) have confirmed the role of pol ␩ by showing a decrease of
mutations of A:T in JH4 introns and switch regions from several
XP-V patients.
To determine whether pol ␩ is also involved in generating the
strand bias of A vs T mutations, we sequenced introns downstream
of rearranged JH4 gene segments from three XP-V patients and
compared them to sequences from control individuals. The hypermutation spectrum significantly correlates with the enzymatic
specificity of pol ␩, indicating that pol ␩ contributes to somatic
hypermutation primarily through low-fidelity synthesis of the nontranscribed strand.
0022-1767/05/$02.00
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DNA polymerase (pol) ␩ participates in hypermutation of A:T bases in Ig genes because humans deficient for the polymerase have
fewer substitutions of these bases. To determine whether polymerase ␩ is also responsible for the well-known preference for
mutations of A vs T on the nontranscribed strand, we sequenced variable regions from three patients with xeroderma pigmentosum variant (XP-V) disease, who lack polymerase ␩. The frequency of mutations in the intronic region downstream of rearranged JH4 gene segments was similar between XP-V and control clones; however, there were fewer mutations of A:T bases and
correspondingly more substitutions of C:G bases in the XP-V clones (p < 10ⴚ7). There was significantly less of a bias for mutations
of A compared with T nucleotides in the XP-V clones compared with control clones, whereas the frequencies for mutations of C
and G were identical in both groups. An analysis of mutations in the WA sequence motif suggests that polymerase ␩ generates
more mutations of A than T on the nontranscribed strand. This in vivo data from polymerase ␩-deficient B cells correlates well
with the in vitro specificity of the enzyme. Because polymerase ␩ inserts more mutations opposite template T than template A, it
would generate more substitutions of A on the newly synthesized strand. The Journal of Immunology, 2005, 174: 7781–7786.
DNA POLYMERASE ␩ CAUSES STRAND BIAS OF A:T HYPERMUTATION
7782
Materials and Methods
Results
Peripheral blood lymphocytes
Similar frequency and location of mutations in XP-V and
control clones
Cells were collected from three human patients, XP11BR, XP7BR, and
XP31BE, with mutations in the gene encoding pol ␩ on both alleles (28)
and three control donors. DNA was prepared as described previously (36).
Libraries of JH4 intronic regions
Statistical analyses
The Fisher exact test was used to compare frequencies of substitutions in A
and T sites. A Monte Carlo modification of the Pearson ␹2 test of spectra
homogeneity (37) and the Kendall’s ␶ correlation coefficient (38, 39) were
used to compare distribution of mutations along the intron sequence. Calculations were done using the programs CORR12 (38) and COLLAPSE (40).
Nucleotide sequence features can be correlated with a mutation spectrum, and the correlation can be tested for statistical significance. The significance of correlations between the distribution of mutable motifs and
mutations along a target sequence was measured by a Monte Carlo procedure (the CONSEN program) (41, 42). This approach takes into account
the frequencies of substitutions for each nucleotide, the possibility of multiple mutations in a site, and the context of the mutating sites. The Monte
Carlo simulation was run with weighted sites, with the weight of a site
defined as:
Wj ⫽
再
XP-V clones have fewer mutations of A:T base pairs than
control clones
We have shown previously that XP-V clones had a lower frequency of mutations of A:T base pairs in the coding sequence of
the VH6 gene from three patients, including XP31BE (28). Because
Yavuz et al. (33) reported that the XP31BE patient did not have
fewer mutations of A:T in sequences from other rearranged genes,
we re-examined the DNA from XP31BE and two other patients
and sequenced a different region of the H chain, e.g., the intron
region downstream of rearranged VH3-23 to JH4 genes. As shown
Mj if j is the hot spot site within a mutable motif
0 if j is not a mutable site
where Mj is the number of mutations in site j. Weights Wj were summed for
all sites in the analyzed sequence resulting in the total weight W. A distribution of total weights Wrandom was calculated for 10,000 target sequences with randomly shuffled mutation spectra. Each of the resulting
random mutation spectra contained the same number of mutations as the
observed spectrum with the same distribution of mutations over randomly
chosen sites. The distribution of Wrandom was used to calculate probability
PW ⱕWrandom. This probability is equal to the fraction of random spectra in
which Wrandom is the same or greater than W. Small probability values
(PW ⱕWrandom ⱕ 0.05) indicate a significant correlation between mutable
motif and mutation frequency (41, 42).
Mutation hot spots are defined using a threshold for the number of
mutations at a site. The threshold is established by analyzing the frequency
distribution derived from a mutation spectrum using the CLUSTERM program (具www.itb.cnr.it/webmutation/典) (39, 43). Briefly, this program decomposes a mutation spectrum into several homogeneous classes of sites,
with each class approximated by a Poisson distribution. Variations in mutation frequencies among sites of the same class are random by definition
(mutation probability is the same for all sites within a class), but differences
between classes are statistically significant. Each site has a probability P(C)
to be assigned to class C. A class with the highest mutation frequency is
called a hot spot class. Sites with P(Chot spot) ⱖ 0.95 are defined as hot spot
sites. This approach ensures that the assignment is statistically significant
and robust. See Ref. 39 for a detailed discussion of this approach and
problems associated with its application.
FIGURE 1. Similar frequency of mutations in clones of JH4 introns
from XP-V and control subjects. The total number of clones is shown in the
center of each circle, and the segments represent the proportion of clones
containing the specified number of mutations. Frequencies are given in
mutations per base.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
To amplify the intron downstream of rearranged JH4 gene segments, we
used 5⬘ primers for the third framework region of the VH3-23 gene segment
and 3⬘ primers for 320 nucleotides downstream of the JH4 gene. The following sets of nested primers were used: first set, forward, 5⬘-AGCCT
GAGAGCCGAGGACAC-3⬘; reverse, 5⬘-GTTGTCACATTGTGACA
ACA-3⬘; and second set, forward with XbaI addition in italics, 5⬘-AC
TCTAGACACGGCCCTATATTACTGTGC-3⬘; and reverse with EcoRI
addition in italics, 5⬘ACGAATTCAACAATGCCAGGACCCCAGG-3⬘.
Twenty nanograms of genomic DNA were amplified with Platinum Pfx
polymerase and PCR enhancer (Invitrogen Life Technologies) in a 50-␮l
volume using the first set of primers for 30 cycles of 95°C for 30 s, 55°C
for 1 min, 68°C for 1 min, followed by a final incubation at 68°C for 10
min. Nested PCR was performed with 5 ␮l of the first reaction and the
second set of primers using the same conditions for another 30 cycles.
Products were digested and cloned into pBluescript vector (Stratagene).
High-efficiency JM109-competent cells (Promega) were used for transfection. The transfection mixture was spread onto antibiotic agar plates immediately after heat-shock to prevent multiplication of identical recombinant clones. Sequencing and analysis of DNA isolated from clones were
performed with the BigDye Terminator Cycle Sequencing kit v3.1 (Applied Biosystems) using T3 and T7 primers (Promega) and the Applied
Biosystems 310 Genetic Analyzer. The mutation data are available upon
request from I. Rogozin ([email protected]).
PBL were obtained from three patients with XP-V disease and
three control patients. To analyze mutations in unselected regions
near variable genes, we amplified the intron downstream of the
VH3-23 gene segment joined to a D gene segment and the JH4 gene
segment. Both of these V and J segments are commonly found in
human Igs: 10% of rearranged genes use VH3-23 (44) and 50% use
JH4 (45), so the libraries of intron sequences should be diverse.
The PCR primers annealed to the third framework region of
VH3-23 and to a sequence 320 bp downstream of JH4 to allow
sequencing of the VDJ junction as well. Only clones with unique
VDJ junctions were considered for analysis. The frequency of mutation in the 320-bp intron region is shown in Fig. 1; XP-V clones
had a slightly lower frequency than control clones, but the difference was not significant. Approximately 100 mutations from each
individual were identified. There was no difference in the number
of insertions and deletions between the XP-V and control groups.
In the XP-V clones, 96% of the mutations were nucleotide substitutions, 3% were deletions of 1–54 nt, and 1% were insertions of
1–25 nt. In the control clones, 96% of the mutations were substitutions, 3% were deletions of 1–24 nt, and 1% were insertions of
1–15 nt. The location of the substitutions is plotted in Fig. 2. Five
allelic nucleotides were identified at positions 71, 229, 248, 300,
and 309; mutations at these positions were excluded from additional consideration. There was no significant difference in the distribution of mutations from XP-V vs control clones.
The Journal of Immunology
7783
terparts and correspondingly more mutations of C:G pairs ( p ⬍
10⫺7). Individually, there were significantly fewer A:T mutations
compared with the controls for XP31BE ( p ⫽ 0.02, Fisher exact
two-tailed test), for XP11BR ( p ⫽ 0.0002), and for XP7BR ( p ⬍
in Table I, there were 46 A:T mutations and 250 C:G mutations for
all XP-V clones and 94 A:T mutations and 174 C:G mutations for
all control clones. Thus, clones from the XP-V patients clearly had
fewer mutations of A:T pairs compared with their control coun-
Table I. Substitutions in JH4 introns from XP-V and control subjectsa
Substitution
XP11BR
(84 mutb)
%
XP7BR
(107 mut)
%
XP31BE
(105 mut)
%
Control 1
(102 mut)
%
Control 2
(84 mut)
%
Control 3
(86 mut)
%
A:G
C
T
T:C
G
A
C:T
A
G
G:A
T
C
8
2
2
7
2
0
17
5
6
28
6
17
5
3
0
6
3
0
26
5
12
18
4
18
6
11
3
6
4
4
18
8
7
17
5
11
13
8
12
8
7
3
12
5
8
13
4
7
23
7
3
11
1
1
8
5
10
19
4
8
22
3
3
8
3
11
17
2
4
13
5
9
a
All mutations are shown from the nontranscribed strand. The sequence contains 17% A, 20% T, 31% C, and 32% G; values were corrected to
represent a sequence with equal amounts of the 4 bases. Substitutions from five allelic nucleotides have been excluded from the comparison.
b
mut, mutation.
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FIGURE 2. Distribution of mutations in the JH4 intron region. Mutations from XP-V and control clones are shown above and below the sequence,
respectively. Every 10th base is underlined. Five allelic nucleotides were found at positions 71 (C or G), 229 (G or A), 248 (T or C), 300 (T or G), and
309 (T or C).
DNA POLYMERASE ␩ CAUSES STRAND BIAS OF A:T HYPERMUTATION
7784
10⫺6). The experiments of Yavuz et al. (33) differed from ours in
that they combined sequences from many VH genes from one patient, whereas we examined a single region, such as the VH6 gene
or the JH4 intron, from three patients. Another difference is that
allelic variants of genes were not identified in Yavuz et al. (33),
whereas mutations at polymorphic positions were removed from
our data sets.
Pol ␩ generates mutations of A more frequently than T on the
nontranscribed strand
Strand bias for A mutations correlates with the specificity of pol ␩
Additional evidence for fewer mutations of A compared with T on
the nontranscribed strand in XP-V clones becomes apparent when
mutations at the four nucleotides are tabulated in Fig. 3. A significant excess of mutations in A vs T was found in the control spectra ( p ⫽ 0.003, the binomial test), whereas no difference was found
in the XP-V spectra ( p ⫽ 0.76). To determine which mutations of
A were affected in the absence of pol ␩, we examined the types of
mutations listed in Table I. The frequency of each type of A and
T mutation is plotted in Fig. 4; the category that decreased the most
in the XP-V clones compared with the control clones was the A to
G substitutions ( p ⫽ 0.004; the Bonferroni correction for multiple
binomial tests was repeated six times). The frequency of errors
generated by human pol ␩ copying an undamaged template in vitro
(47) is also shown in Fig. 4. The mispair frequency is highest for
A to G substitutions, which represents G being incorporated opposite template T. Thus, the data in vivo correlates with the specificity of pol ␩ in vitro (the linear correlation coefficient CC ⫽
FIGURE 3. Fewer mutations of A vs T in XP-V clones. The data in
Table I was grouped as mutations of each nucleotide. Error bars indicate
SEs calculated for XP-V and control subjects.
0.79, PCC ⫽ 0.02; the frequencies of 12 types of substitutions
generated by pol ␩ in vitro were correlated against differences
between the frequencies of 12 types of substitutions in XP-V and
control clones).
Discussion
Model for hypermutation
A basic model for hypermutation is proposed, based on genetic and
biochemical evidence, for involvement of the following proteins.
AID (1) is somehow targeted to Ig variable and switch regions
during transcription (48, 49), and it deaminates C to U (2– 8). UNG
glycosylase (9 –12) removes some uracils to produce abasic sites.
Other uracils remain as an U:G mispair and bind to the MSH2MSH6 heterodimer (13, 14, 20, 21), which then recruits an unspecified endonuclease to make a nick in the DNA. MSH2 also
attracts both exonuclease 1 (22) to produce a gap at the nicks and
DNA pol ␩ (28, 34, 35) to fill in the gap. MSH2-MSH6 then
stimulates the catalytic activity of pol ␩ (14), which allows it to
generate mutations opposite all four bases.
However, although mutations occur at all four nucleotides, the
frequency of mutation is skewed in variable genes so that there are
twice as many mutations of A compared with T as recorded from
the nontranscribed strand, whereas there are equal frequencies of
mutations of C and G. This strand bias for A vs T mutations but not
C vs G has been an enigma for many years and suggests that A:T
mutations occur preferentially on one strand, whereas C:G mutations occur on both strands. Because DNA pol ␩ participates in
mutations of A:T base pairs in Ig genes, we investigated if it is also
responsible for the A:T strand polarity.
Table II. Occurrence of mutations in different motifs
Increase in Mutationsb
Motifsa
WRC
GYW
WA
TW
XP-V
Control
3.4
4.6
1.2
0.8
3.1
4.4
2.4
1.3
a
Number of mutations in mutable motifs was calculated for the underlined bases.
The first and third lines list the motif on the nontranscribed strand, and the second and
fourth lines list the complementary sequence on the transcribed strand.
b
Values listed represent the fold increase in mutations at 22 WRC, 15 GYW, 13
WA, and 16 TW sites compared with mutations at 263 other sites. Bold italicized numbers
represent a significant increase in mutations at mutable motifs (PW ⱕ Wrandom ⱕ 0.05),
as revealed by using a Monte Carlo procedure (41, 42).
FIGURE 4. Specificity of pol ␩ correlates with change in mutations of
A to G from XP-V clones. The frequency of each possible mutation of A
and T to another base is shown for XP-V (u) and control clones (䡺). f,
The frequency of mispair events by human pol ␩ copying an undamaged
template in vitro (47). The asterisk denotes a significant change in mutation
frequency between XP-V and control clones (p ⫽ 0.004).
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Hypermutation frequently occurs in WRCY (W ⫽ A or T, R ⫽ A
or G, Y ⫽ C or T) and WA DNA motifs (42, 46), and the WRC
motif is targeted by AID in vitro (7, 8). The frequency of mutations
in these motifs and their complementary sequences is shown in
Table II. For the WRC motif, there was a 3- to 4-fold excess of
mutations in either C or G residues from both XP-V and control
clones, compared with SYC, WYC, and SRC motifs (S ⫽ C or G).
This suggests that C was targeted for mutation equally frequently
on both the nontranscribed (WRC, the mutable position is underlined) and transcribed (GYW) strands. However, for the WA motif, there was a significant 2-fold excess of mutations in WA vs SA
from control clones compared with XP-V clones but not for mutations in TW (Table II). This result can be explained using earlier
observations about the strand specificity of pol ␩ copying an undamaged template in vitro: WA and TW motifs were shown to be
targets of pol ␩ on the nontranscribed and transcribed strand, respectively (41, 47). Thus, the significant excess of mutations in
WA motifs indicates that A on the nontranscribed strand is targeted primarily for mutation by pol ␩ in vivo.
The Journal of Immunology
Pol ␩ generates significant mutations of A:T bases
The involvement of pol ␩ in hypermutation was confirmed by
sequencing clones containing the intronic region downstream of
rearranged VH3-23 to JH4 gene segments in DNA from three XP-V
patients who were deficient for pol ␩. Around 300 mutations were
compared with a similar number obtained from three control individuals. Although the frequencies of mutation were similar between the two groups, there were significantly fewer mutations of
A:T base pairs in the XP-V clones compared with control clones
and correspondingly more mutations of C:G. This is consistent
with previous data from several XP-V patients showing a decrease
in A:T mutations in the coding sequence of VH6 genes (28), in JH4
introns from several rearranged VH genes (34), and in the ␮-␥
switch regions (34, 35).
Pol ␩ synthesizes primarily on the nontranscribed strand
Specificity of pol ␩ correlates with A:T asymmetry
Unequal frequencies of A and T mutations suggest that either base
is preferentially mutated or repaired on one DNA strand. This
would occur as error-prone synthesis extends past the U lesion into
neighboring A and T nucleotides. As noted above, the analysis of
mutations in the WA/TW motif suggests that synthesis occurs on
the nontranscribed strand (Table II and Ref. 53). Recruitment to U
on the nontranscribed strand may occur via the multiprotein transcription complex, TFIIH, which directs RNA polymerase II to the
transcribed strand by an unknown process. This asymmetry may
also deposit the mismatch repair proteins and pol ␩ on the opposite
nontranscribed strand.
During gap filling or strand displacement of the repair patch, pol
␩ would then synthesize DNA on the nontranscribed strand, using
the transcribed strand as a template. In this case, pol ␩ should have
a strong tendency to insert a mismatched base opposite template T
but not opposite templates A, G, or C. This is exactly the specificity of human and mouse pol ␩ in vitro when copying undamaged
templates (41, 47, 53). Specifically, there is a 5-fold increase in
incorporation of G opposite T, which would represent an A to G
substitution, compared with the other 11 possibilities (Fig. 4 and
Ref. 47). In XP-V clones, the frequency of A to G mutations declined the most significantly in the mutational spectra, which is
consistent with the mutational pattern of pol ␩. If the template is
the transcribed strand, then pol ␩ would generate A to G substitutions in excess when synthesizing a repair patch on the nontranscribed strand. Although other roles for pol ␩ to generate A:T
mutations have been suggested (54, 55), the ability of the enzyme
to catalyze error-prone synthesis on DNA is the most logical explanation for its function in hypermutation.
Which polymerases produce the remaining mutations?
In the absence of pol ␩, the residual A:T mutations could be generated by other low-fidelity polymerases that bypass an abasic site
generated by UNG glycosylase at deaminated C bases. This would
produce mutations of C and G and, perhaps less efficiently, mutations of adjacent A and T through strand displacement. There
would be no A:T bias if this occurred on both strands. As noted
earlier, mice deficient for several other error-prone polymerases
had normal frequencies of mutation of all four nucleotides, which
has confounded the problem of identifying another polymerase.
Because pol ␩ appears to be a major player in hypermutation, it
may compensate in the absence of other polymerases and synthesize all the mutations. When pol ␩-deficient mice become available, it will be possible to study mice doubly deficient for pol ␩
and other candidate polymerases to identify which one also participates in bypassing abasic sites to generate the C:G and residual
A:T mutations. Significantly, although there are some A:T mutations in mice deficient for either UNG or MSH2, there are no A:T
mutations in mice deficient for both UNG and MSH2 (56). This
indicates that A:T mutations can occur during both the phase 1
pathway caused by bypass of UNG-generated abasic sites and the
phase 2 pathway caused by repair of MSH2-generated patches. It
will be interesting to see how pol ␩ participates in each pathway by
creating mice doubly deficient for pol ␩ and UNG vs pol ␩
and MSH2.
Acknowledgments
We thank William Yang and Elena Vasunina for technical assistance, and
Stella Martomo and Michael Seidman for thoughtful comments.
Disclosures
The authors have no financial conflict of interest.
References
1. Muramatsu, M., K. Kinoshita, S. Faragasan, S. Yamada, Y. Shinkai, and
T. Honjo. 2000. Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell
102: 553–563.
2. Bransteitter, R., P. Pham, M. D. Scharff, and M. F. Goodman. 2003. Activationinduced cytidine deaminase deaminates deoxycytidine on single-stranded DNA
but requires the action of RNase. Proc. Natl. Acad. Sci. USA 100: 4102– 4107.
3. Chaudhuri, J., M. Tian, C. Khuong, K. Chua, E. Pinaud, and F. W. Alt. 2003.
Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422: 726 –730.
4. Ramiro, A. R., P. Stavropoulos, M. Jankovic, and M. C. Nussenzweig. 2003.
Transcription enhances AID-mediated cytidine deamination by exposing singlestranded DNA on the nontemplate strand. Nat. Immunol. 4: 452– 456.
5. Dickerson, S. K., E. Market, E. Besmer, and F. N. Papavasiliou. 2003. AID
mediates hypermutation by deaminating single-stranded DNA. J. Exp. Med. 197:
1291–1296.
6. Sohail, A., J. Klapacz, M. Samaranayake, A. Ullah, and A. S. Bhagwat. 2003.
Human activation-induced cytidine deaminase causes transcription-dependent,
strand-biased C to U deaminations. Nucleic Acids Res. 31: 2990 –2994.
7. Pham, P., R. Bransteitter, J. Petruska, and M. F. Goodman. 2003. Processive
AID-catalysed cytosine deamination on single-stranded DNA simulates somatic
hypermutation. Nature 424: 103–107.
8. Yu, K., F. T. Huang, and M. R. Lieber. 2004. DNA substrate length and surrounding sequence affect the activation induced deaminase activity at cytidine.
J. Biol. Chem. 279: 6496 – 6500.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
Mutations of A:T in Ig genes occur preferentially in the WA/TW
sequence motif (41, 47). In the control clones, mutations were
overrepresented in WA on the nontranscribed strand by 2-fold
compared with random sequences, whereas no increase was found
in the XP-V clones. However, there was no increase in the corresponding TW motif from both groups, which would represent WA
on the complementary strand. A decline in substitutions of A relative to T can also be seen in the data from the ␮-␥ switch regions
from these patients (35) and in the JH4 intron and ␮ switch region
from two other XP-V patients (34). This suggests that pol ␩ inserts
substitutions preferentially on the nontranscribed strand.
In contrast, in the WRC/GYW motif, there was a 3- to 4-fold
increase in mutations of both C and G in control and XP-V clones
(Table II). This suggests that mutations of C can occur on both
strands in the presence or absence or pol ␩. Equal frequencies of
C and G mutations in variable regions suggest that AID deaminates C to U on both DNA strands (50). This may occur during
transcription when both strands are single stranded at the trailing
edge of the transcription bubble or possibly if they are supercoiled
(51). However in switch regions, there is a preference for C mutations on the nontranscribed strand compared with G mutations
(35), which may reflect the formation of stable R-loops in switch
DNA (52) to expose the nontranscribed strand for deamination.
The two regions also differ in that AID deamination of variable
regions requires replication protein A (49), which may stabilize
ssDNA, whereas deamination of switch regions does not require
the cofactor (3).
7785
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DNA POLYMERASE ␩ CAUSES STRAND BIAS OF A:T HYPERMUTATION
32. Masutani, C., R. Kusumoto, A. Yamada, N. Dohmae, M. Yokoi, M. Yuasa,
M. Araki, S. Iwai, K. Takio, and F. Hanaoka. 1999. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase ␩. Nature 399:
700 –704.
33. Yavuz, S., A. S. Yavuz, K. H. Kraemer, and P. E. Lipsky. 2002. The role of
polymerase ␩ in somatic hypermutation determined by analysis of mutations in
a patient with xeroderma pigmentosum variant. J. Immunol. 169: 3825–3830.
34. Faili, A., S. Aoufouchi, S. Weller, F. Vuillier, A. Stary, A. Sarasin,
C. A. Reynaud, and J. C. Weill. 2004. DNA polymerase ␩ is involved in hypermutation occurring during immunoglobulin class switch recombination. J. Exp.
Med. 199: 265–270.
35. Zeng, X., G. A. Negrete, C. Kasmer, W. W. Yang, and P. J. Gearhart. 2004.
Absence of DNA polymerase ␩ reveals targeting of C mutations on the nontranscribed strand in immunoglobulin switch regions. J. Exp. Med. 199: 917–924.
36. Rosner, K., D. B. Winter, C. Kasmer, G. L. Skovgaard, R. E. Tarone, V. A. Bohr,
and P. J. Gearhart. 2001. Impact of age on hypermutation of immunoglobulin
variable genes in humans. J. Clin. Immunol. 21: 102–115.
37. Adams, W. T., and T. R. Skopek. 1987. Statistical test for the comparison of
samples from mutational spectra. J. Mol. Biol. 194: 391–396.
38. Babenko, V. N., and I. B. Rogozin. 1999. Use of a rank correlation coefficient for
comparing mutational spectra. Biofizika 44: 632– 638.
39. Rogozin, I. B., F. A. Kondrashov, and G. V. Glazko. 2001. Use of mutation
spectra analysis software. Hum. Mutat. 17: 83–102.
40. Khromov-Borisov, N. N., I. B. Rogozin, J. A. Pegas Henriques, and
F. J. De Serres. 1999. Similarity pattern analysis in mutational distributions.
Mutat. Res. 430: 55–74.
41. Rogozin, I. B., Y. I. Pavlov, K. Bebenek, T. Matsuda, and T. A. Kunkel. 2001.
Somatic mutation hotspots correlate with DNA polymerase ␩ error spectrum.
Nat. Immunol. 2: 530 –536.
42. Rogozin, I. B., and N. A. Kolchanov. 1992. Somatic hypermutagenesis in immunoglobulin genes. II. Influence of neighbouring base sequences on mutagenesis. Biochim. Biophys. Acta 1171: 11–18.
43. Glazko, G. V., L. Milanesi, and I. B. Rogozin. 1998. The subclass approach for
mutational spectrum analysis: application of the SEN algorithm. J. Theor. Biol.
192: 475– 487.
44. Stewart, A. K., C. Huang, B. D. Stollar, and R. S. Schwartz. 1993. High-frequency representation of a single VH gene in the expressed human B cell repertoire. [Published erratum in 1993 J. Exp. Med. 177: 1227.] J. Exp. Med. 177:
409 – 418.
45. Yamada, M., R. Wasserman, B. A. Reichard, S. Shane, A. J. Caton, and
G. Rovera. 1991. Preferential utilization of specific immunoglobulin heavy chain
diversity and joining segments in adult human peripheral blood B lymphocytes.
J. Exp. Med. 173: 395– 407.
46. Rogozin, I. B., and M. Diaz. 2004. Cutting edge: DGYW/WRCH is a better
predictor of mutability at G:C bases in Ig hypermutation than the widely accepted
RGYW/WRCY motif and probably reflects a two-step activation-induced cytidine deaminase-triggered process. J. Immunol. 172: 3382–3384.
47. Matsuda, T., K. Bebenek, C. Masutani, I. B. Rogozin, F. Hanaoka, and
T. A. Kunkel. 2001. Error rate and specificity of human and murine DNA polymerase ␩. J. Mol. Biol. 312: 335–346.
48. Nambu, Y., M. Sugai, H. Gonda, C. G. Lee, T. Katakai, Y. Agata, Y. Yokota, and
A. Shimizu. 2003. Transcription-coupled events associating with immunoglobulin switch region chromatin. Science 302: 2137–2140.
49. Chaudhuri, J., C. Khuong, and F. W. Alt. 2004. Replication protein A interacts
with AID to promote deamination of somatic hypermutation targets. Nature 430:
992–998.
50. Boursier, L., W. Su, and J. Spencer. 2004. Analysis of strand biased ‘G’.C hypermutation in human immunoglobulin V␭ gene segments suggests that both
DNA strands are targets for deamination by activation-induced cytidine deaminase. Mol. Immunol. 40: 1273–1278.
51. Shen, H. M., and U. Storb. 2004. Activation-induced cytidine deaminase (AID)
can target both DNA strands when the DNA is supercoiled. Proc. Natl. Acad. Sci.
USA 101: 12997–13002.
52. Yu, K., F. Chedin, C. L. Hsieh, T. E. Wilson, and M. R. Lieber. 2003. R-loops
at immunoglobulin class switch regions in the chromosomes of stimulated B
cells. Nat. Immunol. 4: 442– 451.
53. Pavlov, Y. I., I. B. Rogozin, A. P. Galkin, A. Y. Aksenova, F. Hanaoka, C. Rada,
and T. A. Kunkel. 2002. Correlation of somatic hypermutation specificity and
A-T base pair substitution errors by DNA polymerase ␩ during copying of a
mouse immunoglobulin ␬ light chain transgene. Proc. Natl. Acad. Sci. USA 99:
9954 –9959.
54. Steele, E. J. 2004. DNA polymerase ␩ as a reverse transcriptase: implications for
the mechanisms of hypermutation in innate anti-retroviral defenses and antibody
SHM systems. DNA Repair 3: 687– 692.
55. Neuberger, M. S., J. M. Di Noia, R. C. L. Beale, T. T. Williams, Z. Yang, and
C. Rada. 2005. Somatic hypermutation of A 䡠 T pairs: polymerase error versus
dUTP incorporation. Nat. Rev. Immunol. 5: 171–178.
56. Rada, C., J. M. Di Noia, and M. S. Neuberger. 2004. Mismatch recognition and
uracil-excision provide complementary paths to both Ig switching and the A/Tfocused phase of somatic mutation. Mol. Cell 16: 163–171.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
9. Petersen-Mahrt, S. K., R. S. Harris, and M. S. Neuberger. 2002. AID mutates E.
coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418: 99 –103.
10. Di Noia, J., and M. S. Neuberger. 2002. Altering the pathway of immunoglobulin
hypermutation by inhibiting uracil-DNA glycosylase. Nature 419: 43– 48.
11. Rada, C., G. T. Williams, H. Nilsen, D. E. Barnes, T. Lindahl, and
M. S. Neuberger. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12:
1748 –1755.
12. Imai, K., G. Slupphaug, W. I. Lee, P. Revy, S. Nonoyama, N. Catalan, L. Yel,
M. Forveille, B. Kavli, H. E. Krokan, et al. 2003. Human uracil-DNA glycosylase
deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat. Immunol. 4: 1023–1028.
13. Gu, L., J. Wu, L. Qiu, C. D. Jennings, and G. M. Li. 2002. Involvement of DNA
mismatch repair in folate deficiency-induced apoptosis. J. Nutr. Biochem. 13:
355–363.
14. Wilson, T. M., A. Vaisman, S. A. Martomo, P. Sullivan, L. Lan, F. Hanaoka,
A. Yasui, R. Woodgate, and P. J. Gearhart. 2005. MSH2-MSH6 stimulates DNA
polymerase ␩, suggesting a role for A:T mutations in antibody genes. J. Exp.
Med. 201: 637– 645.
15. Phung, Q. H., D. B. Winter, A. Cranston, R. E. Tarone, V. A. Bohr, R. Fishel, and
P. J. Gearhart. 1998. Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair
protein. J. Exp. Med. 187: 1745–1751.
16. Frey, S., B. Bertocci, F. Delbos, L. Quint, J. C. Weill, and C. A. Reynaud. 1998.
Mismatch repair deficiency interferes with the accumulation of mutations in
chronically stimulated B cells and not with the hypermutation process. Immunity
9: 127–134.
17. Rada, C., M. R. Ehrenstein, M. S. Neuberger, and C. Milstein. 1998. Hot spot
focusing of somatic hypermutation in MSH2-deficient mice suggests two stages
of mutational targeting. Immunity 9: 135–141.
18. Martin, A., Z. Li, D. P. Lin, P. D. Bardwell, M. D. Iglesias-Ussel, W. Edelmann,
and M. D. Scharff. 2003. Msh2 ATPase activity is essential for somatic hypermutation at A-T base pairs and for efficient class switch recombination. J. Exp.
Med. 198: 1171–1178.
19. Wiesendanger, M., B. Kneitz, W. Edelmann, and M. D. Scharff. 2000. Somatic
hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the
base substitution pattern. J. Exp. Med. 191: 579 –584.
20. Li, Z., S. J. Scherer, D. Ronai, M. D. Iglesias-Ussel, J. U. Peled, P. D. Bardwell,
M. Zhuang, K. Lee, A. Martin, W. Edelmann, and M. D. Scharff. 2004. Examination of Msh6- and Msh3-deficient mice in class switching reveals overlapping
and distinct roles of MutS homologues in antibody diversification. J. Exp. Med.
200: 47–59.
21. Martomo, S. A., W. W. Yang, and P. J. Gearhart. 2004. A role for Msh6 but not
Msh3 in somatic hypermutation and class switch recombination. J. Exp. Med.
200: 61– 68.
22. Bardwell, P. D., C. J. Woo, K. Wei, Z. Li, A. Martin, S. Z. Sack, T. Parris,
W. Edelmann, and M. D. Scharff. 2004. Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice. Nat. Immunol.
5: 224 –229.
23. Smith, D. S., G. Creadon, P. K. Jena, J. P. Portanova, B. L. Kotzin, and
L. J. Wysocki. 1996. Di- and trinucleotide target preferences of somatic mutagenesis in normal and autoreactive B cells. J. Immunol. 156: 2642–2652.
24. Lebecque, S., and P. J. Gearhart. 1991. Boundaries of somatic mutation in rearranged immunoglobulin genes: 5⬘ boundary is near the promoter and 3⬘ boundary
is ⬃1 kb from V(D)J gene. J. Exp. Med. 172: 1717–1727.
25. Spencer, J., M. Dunn, and D. K. Dunn-Walters. 1999. Characteristics of sequences around individual nucleotide substitutions in Ig VH genes suggest different GC and AT mutators. J. Immunol. 162: 6596 – 6601.
26. Peters, A., and U. Storb. 1996. Somatic hypermutation of immunoglobulin genes
is linked to transcription initiation. Immunity 4: 57– 65.
27. Esposito, G., G. Texido, U. A. Betz, H. Gu, W. Müller, U. Klein, and
K. Rajewsky. 2000. Mice reconstituted with DNA polymerase ␤-deficient fetal
liver cells are able to mount a T cell-dependent immune response and mutate their
Ig genes normally. Proc. Natl. Acad. Sci. USA 97: 1166 –1171.
28. Zeng, X., D. B. Winter, C. Kasmer, K. H. Kraemer, A. R. Lehmann, and
P. J. Gearhart. 2001. DNA polymerase ␩ is an A-T mutator in somatic hypermutation of immunoglobulin variable genes. Nat. Immunol. 2: 537–541.
29. McDonald, J. P., E. G. Frank, B. S. Plosky, I. B. Rogozin, C. Masutani,
F. Hanaoka, R. Woodgate, and P. J. Gearhart. 2003. 129-derived strains of mice
are deficient in DNA polymerase ␫ and have normal immunoglobulin hypermutation. J. Exp. Med. 198: 635– 643.
30. Schenten, D., V. L. Gerlach, C. Guo, S. Velasco-Miguel, C. L. Hladik,
C. L. White, E. C. Friedberg, K. Rajewsky, and G. Esposito. 2002. DNA polymerase ␬ deficiency does not affect somatic hypermutation in mice. Eur. J. Immunol. 32: 3152–3160.
31. Bertocci, B., A. de Smet, E. Flatter, A. Dahan, J. C. Bories, C. Landreau,
J. C. Weill, and C. A. Reynaud. 2002. DNA polymerases ␭ and ␮ are dispensable
for Ig gene hypermutation. J. Immunol. 168: 3702–3706.