Download Cutting Edge: DNA Polymerases and Are Dispensable for Ig Gene

Cutting Edge: DNA Polymerases µ and λ Are
Dispensable for Ig Gene Hypermutation
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of August 3, 2017.
Barbara Bertocci, Annie De Smet, Eric Flatter, Auriel Dahan,
Jean-Christophe Bories, Catherine Landreau, Jean-Claude Weill
and Claude-Agnès Reynaud
J Immunol 2002; 168:3702-3706; ;
doi: 10.4049/jimmunol.168.8.3702
http://www.jimmunol.org/content/168/8/3702
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References
●
Cutting Edge: DNA Polymerases ␮ and ␭ Are
Dispensable for Ig Gene Hypermutation1
Barbara Bertocci,* Annie De Smet,* Eric Flatter,* Auriel Dahan,*
Jean-Christophe Bories,† Catherine Landreau,‡ Jean-Claude Weill,2*
and Claude-Agnès Reynaud2,3*
T
hirty-five years after the initial proposition of Brenner and
Milstein (1), it is now generally considered that somatic
mutations introduced in Ig genes involve the participation
of an error-prone DNA polymerase (reviewed in Ref. 2). The specific characteristics of these mutations appear indeed poorly compatible with other types of DNA-modifying activities, as, for example, chemical alterations or base modifications. Among these
characteristics are a bias for transitions, a low frequency of deletions and duplications (but no insertions), and the occurrence of
slippage events at short repeated sequences (3).
Polymerase (pol)4 ␤ has long been the only known nonreplicative DNA polymerase whose intrinsic fidelity, in the 10⫺3 range,
would be compatible with the estimated mutation rate of Ig genes
(4). However, inactivation of this enzyme by gene targeting has
*Institut National de la Santé et de la Recherche Médicale, Unité 373, Faculté de
Médecine Necker-Enfants Malades, and †Institut National de la Santé et de la Recherche Médicale, Unité 462, Hôpital Saint-Louis, Center Hayem, Paris, France; and
‡
Service d’Expérimentation Animale et de Transgénèse du Centre National de la
Recherche Scientifique, Villejuif, France
Received for publication January 11, 2002. Accepted for publication February
21, 2002.
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 work has been supported by the APEX program of Institut National de la Santé
et de la Recherche Médicale, the Association pour la Recherche contre le Cancer, and
the Fondation Princesse Grace de Monaco.
2
J.-C.W. and C.-A.R. share equal senior authorship.
3
Address correspondence and reprint requests to Dr. Claude-Agnès Reynaud, Institut
National de la Santé et de la Recherche Médicale, Unité 373, Faculté de Médecine
Necker-Enfants Malades, 156 rue de Vaugirard, 75730 Paris Cedex 15, France. Email address: [email protected]
4
Abbreviations used in this paper: pol, polymerase; ES, embyronic stem; neoR, neomycin resistance gene; k.o., knockout; HhH, helix-hairpin-helix; PNA, peanut
agglutinin.
Copyright © 2002 by The American Association of Immunologists
●
excluded its participation in the mutation process (5). Over the past
4 years, nine additional DNA polymerases (if one includes Rev1,
a deoxycytidyl transferase) have been identified (reviewed in Ref.
2). Although many of these enzymes have bacterial or yeast homologs with known repair function, their precise role in mammalian cells remains hypothetical for most of them (3, 6).
Among the candidates for hypermutation is a group of DNA
polymerases described collectively as lesion bypass enzymes. The
participation of several of them has been proposed in hypermutation (7–10), but no direct evidence points so far to the implication
of a unique enzyme in this process.
We reported previously that the mutation pattern of microsatellite sequences embedded in the Ig locus share striking similarities
with the synthesis errors generated in vitro by pol ␤ (11). Therefore, we have searched for new DNA polymerases related to this
enzyme, which, together with Tdt, belongs to the polX family. We
have discovered two such enzymes, pol ␭ and pol ␮ (12), which
have been independently identified by other groups (13–15). Pol ␭
has 54% homology with pol ␤, while pol ␮ is closer to Tdt, an
enzyme whose only function is to contribute to the T and B cell
repertoire by diversification of V(D)J junctions during rearrangement of TCR and Ig genes. Its homology with a lymphoid-specific
enzyme and its higher expression in lymphoid tissues have made
pol ␮ a strong candidate for the Ig mutase (12, 13). For pol ␭, its
low ubiquitous expression and its lack of proofreading activity
(12) make it a possible candidate as well. We report in this work
the generation of gene-targeted mice for these two enzymes and
the lack of incidence of their inactivation on the Ig gene mutation
process.
Materials and Methods
Construction of targeting vectors
␭ phages spanning 20 kb of both ␭ and ␮ genes were isolated from a mouse
129/SvJ genomic library (␭FixII; Stratagene, La Jolla, CA). One phage for
each gene was selected, and 5⬘ and 3⬘ fragments flanking the region to be
deleted were amplified. For pol ␮, a 3.9-kb 5⬘ fragment was amplified
using primers in exons 3 and 6, respectively; a 3.5-kb 3⬘ fragment spanned
from exon 10 up to the end of the phage insert. For pol ␭, a large part of
the genomic sequence was available (AC003694): a 5-kb 5⬘ fragment was
amplified with primers located, respectively, 1.8 kb upstream from the
ATG codon and at the end of the intron between exons 4 and 5; a 3.8-kb
3⬘ fragment spanned from the 5⬘ border of the intron between exons 7 and
8 up to the end of the phage insert, ⬃3 kb downstream from the stop codon.
Fragments were inserted either blunt or using restriction sites added in the
primers in the XhoI and SalI cloning sites flanking the neomycin resistance
gene of the pLNTK vector (a gift from F. W. Alt, Harvard Medical School,
Boston, MA) (16). Additional NotI and SfiI sites have been introduced in
this vector to be used for linearization.
0022-1767/02/$02.00
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Mutations arising in Ig V genes during an immune response
are most likely introduced by one or several error-prone DNA
polymerases. Many of the recently described nonreplicative
DNA polymerases have an intrinsic fidelity compatible with
such an activity, the strongest candidates being polymerase
(pol) ␩, pol ␫, pol ␨, and pol ␮. We report in this work that mice
inactivated for either of the two polymerases related to pol ␤
(i.e., pol ␮ and pol ␭) are viable and fertile and display a normal hypermutation pattern. The Journal of Immunology,
2002, 168: 3702–3706.
The Journal of Immunology
3703
Generation of gene-targeted mice
E14.1 embyronic stem (ES) cells (a gift from K. Rajewsky, Institute for
Genetics, Cologne, Germany) were transfected as described (17). G418-
FIGURE 2. Targeted disruption of the mouse pol ␮ gene. A, Schematic
representation of the targeted pol ␮ locus. Positions of the HindIII (H) sites
are indicated. B, Southern blot analysis of HindIII-digested spleen DNA
from wild-type, heterozygous, and homozygous pol ␮-deficient mice. Migration position of the 14-kb wild-type allele and of the 7-kb targeted allele
is indicated. C, Analysis of pol ␮ RNA expression in spleen PNAhigh B
cells from wild-type and pol ␮-deficient mice. RT-PCR amplification of the
complete pol ␮ coding sequence (1.5 kb) was performed. D, Representation of the protein domains deleted from the pol ␮ polymerase by gene
targeting. Other details are as described in Fig. 1
and ganciclovir-resistant clones were screened individually by PCR (30
cycles with the Long Expand PCR system according to the conditions of
the supplier; Boehringer Mannheim, Mannheim, Germany), using the following gene-specific primers, amplifying both the endogenous genes and
the construct if properly targeted (see location in Figs. 1 and 2): pol ␮ 5⬘
primer, GGGCAGAGTACATGCCAGTG, and 3⬘ primer, GCTGAAC
CGCCGTAGCTCCC; and pol ␭ 5⬘ primer, GCTCCATATGGTTGCT
GGGC, and 3⬘ primer, CAGAGCTGAGGAGGAAGGATG. One positive
Table I. Mutations in JH4 flanking sequences from PNAhigh Peyer’s patch B cells of DNA pol ␮- and ␭deficient mice
No. of
Littermates Mice Sequences
Mutated
Sequences (%)
No. of Mutations
Analyzed
Mutations/100 bp
(all sequences)
Mutations/100 bp
(mutated sequences)
pol ␮
1
2
3
All
18
15
17
50
61
53
64
60
43
39
63
145
0.9
1.0
1.4
1.1
1.5
1.8
2.2
1.8
pol ␮⫺/⫺
1
2
3
4
5
All
17
18
16
19
23
93
82
78
69
68
83
74
120
69
43
44
121
397
2.7
1.5
1.0
0.9
2.0
1.6
3.3
1.9
1.5
1.3
2.5
2.2
pol ␭
1
2
All
24
9
33
67
78
70
138
51
189
2.2
2.2
2.2
3.3
2.8
3.1
pol ␭⫺/⫺
1
2
All
15
16
31
47
68
58
35
59
94
0.9
1.4
1.2
1.9
2.1
2.0
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FIGURE 1. Targeted disruption of the mouse pol ␭ gene. A, Schematic
representation of the targeted pol ␭ locus. Filled boxes represent coding
exons. Primers used for the screening of targeted ES cells are represented
by horizontal arrows below the targeted allele. Positions of XmnI (X) sites
and of the probe used for Southern blot analysis are indicated. TK, Herpes
simplex thymidine kinase gene. B, Southern blot analysis of XmnI-digested
spleen DNA from wild-type, heterozygous, and homozygous pol ␭-deficient mice. Migration positions of the 13.5-kb wild-type allele and of the
6.5-kb targeted allele are indicated. C, Analysis of pol ␭ RNA expression
in spleen PNAhigh B cells from wild-type and pol ␭-deficient mice. RTPCR amplification of the complete pol ␭ coding sequence (1.7 kb) was
performed. D, Representation of the protein domains deleted from the
pol ␭ by gene targeting. The position of the breast cancer-associated
protein 1 C terminus, HhH, and polX domains relative to the pol ␭
exons is schematized, with their replacement by the neoR.
CUTTING EDGE: NO ROLE FOR pol ␭ AND pol ␮ IN HYPERMUTATION
3704
FIGURE 3. Accumulation of mutations among JH4 flanking sequences
of Peyer’s patch PNAhigh B cells from pol ␮- and pol ␭-deficient mice.
Sequences reported in Table I were analyzed according to the relative
distribution of sequences harboring a given number of mutations. Littermate controls of both k.o. mice were pooled.
Analysis of pol ␮ and pol ␭ expression
Spleen peanut agglutinin (PNA)high B cells from 2-mo-old animals were
isolated as described previously (18). RNA was extracted from 105 cells
using the RNeasy mini kit (Qiagen, Hilden, Germany) and cDNA was
synthesized using the ProSTAR First-Strand RT-PCR kit (Stratagene).
Primers amplifying the complete coding sequence of pol ␮ and pol ␭ were
used. PCR products were analyzed after gel transfer by hybridization with
a full-length ␮ or ␭ cDNA probe.
Analysis of V gene mutations
Peyer’s patch PNAhigh B cells were isolated as described previously (18).
Mutations were analyzed in a 260-bp segment downstream from rearranged JH4 sequences as described (18). Pol ␮⫺/⫺ mice were selected between 3 and 5 mo of age, and pol ␭⫺/⫺ mice were selected at 4 mo.
Wild-type littermates were taken as controls.
FIGURE 4. Distribution of mutations along JH4 flanking sequences of
Peyer’s patch PNAhigh B cells from pol ␮- and pol ␭-deficient mice. Mutations listed in Table I were analyzed for their distribution along JH4 flanking sequences. Hot spot positions are marked for control mice, with the
targeted nucleotide underlined in a 4-base context.
Flow cytometric analysis
Splenic cell suspensions from 4-wk-old mice were stained with FITCconjugated goat anti-mouse-IgM and PE-conjugated rat anti-mouse-IgD
Abs (Southern Biotechnology Associates, Birmingham, AL) and analyzed
with a FACStar apparatus (BD Biosciences, Mountain View, CA).
Results and Discussion
DNA polymerases ␮ and ␭ share a similar organization comprising
a DNA polymerase domain, two HhH (helix-hairpin-helix) domains, and, unlike pol ␤, an N-terminal breast cancer-associated
protein 1 C terminus domain that could mediate protein-protein
Table II. Percentage of nucleotide substitution in JH4 flanking sequences from PNAhigh Peyer’s patch B
cells of DNA pol ␭- and ␮-deficient micea
To
From
A
A
G
C
T
12.5–16.0
14.3
3.2–7.8
6.1
7.2–5.0
3.9
G
C
T
Total
17.4–13.8
18.5
6.4–8.3
5.6
10.7–8.1
7.0
8.7–10.0
9.1
6.8–4.7
2.3
11.2–7.0
12.9
32.5–32.1
33.2
30.0–28.8
23.6
17.6–21.8
26.3
20.0–17.2
16.9
3.2–7.0
7.3
2.8–3.2
5.4
10.0–9.0
7.6
a
Mutations described in Table I are analyzed for nucleotide substitutions, in boldface for polymerase-deficient mice (first
figure, pol ␭; second figure, pol ␮) and in plain for pooled littermate controls. Pol ␭, 94 mutations analyzed; pol ␮, 347 mutations;
controls, 334 mutations. Percentages of substitutions are corrected for the base composition of the JH4 flanking sequence: A,
25%; G, 28%; C, 17%; T, 30%. Percent transition was 51.1– 45.8 for polymerase-deficient mice and 53.3 for littermate controls.
Percent transversion was 48.9 –54.2 for polymerase-deficient mice and 46.7 for littermate controls.
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clone among 363 was obtained for pol ␮, and two among 370 were obtained for pol ␭. Homologous recombination was confirmed by Southern
blot analysis of DNA from ES clones and from spleen of gene-targeted
mice (see Figs. 1B and 2B). Injection of ES cells into C57BL/6 blastocysts
was performed as described (17), and chimeric mice were bred into the
C57BL/6 background. Genotyping of mice was performed by PCR, with
simultaneous amplification of the wild-type allele (250 bp) and the genetargeted allele (500 bp) for both pol ␮ and pol ␭. Primers were as follows:
pol ␮ 5⬘ primer, GGGTTGGGGTGAAGACTGC, 3⬘ wild-type-specific
primer, CTCATGGCCAACCCTGGGTC, and 3⬘ neomycin resistance
gene (neoR) primer, CATAGCGTTGGCTACCCGTG; pol ␭ 5⬘ primer,
GCTCCATATGGTTGCTGGGC, 3⬘ wild-type-specific primer, CAGCTC
CCCAGATGTTGGAG, and 3⬘-neoR primer as above.
The Journal of Immunology
3705
FIGURE 5. Flow cytometric analysis of the splenic B cell compartment of pol ␮- and pol ␭-deficient mice. Spleen cells from pol ␮- and pol ␭-deficient
mice, gated on lymphoid cells, were stained with anti-IgM and anti-IgD Abs, and the fraction of IgD⫹IgM⫹ cells is indicated. Pol ␮⫺/⫺ (2) mouse has a
marked B cell deficit, whereas the value for pol ␮⫺/⫺ (1) still matches the lower edge of control values.
pol ␭-deficient mice over several generations argues that this enzyme is dispensable for mouse development. Most repair polymerases are highly expressed in testis, as, for example, ␤, ␨, ␬, or
␫; therefore, there might be some redundancy between them, at
least over the time scale of a few generations. Moreover, whatever
its contribution to base excision repair might be, pol ␭ is unable to
substitute for the repair function of pol ␤ during early mouse development, the inactivation of the latter being lethal at birth due to
massive embryonic neuronal apoptosis (22). However, the lack of
phenotype of pol ␭ deficiency is quite unexpected, considering the
strong interspecies conservation of this enzyme, which has a putative ortholog in plants (14).
Pol ␮ has been considered a good candidate for the elusive Ig
gene mutase according to several criteria (12, 13): 1) its very
strong homology with Tdt, a strictly lymphoid-specific enzyme
whose contribution is crucial to diversify the third complementarity-determining region of Ig and TCR genes during V(D)J rearrangement; 2) despite a ubiquitous expression, a higher level of
transcription in lymphoid tissues, in particular B cells from tonsils,
but also, and obviously not in favor of an Ig mutase, in thymus; 3)
its rather high level of infidelity during DNA synthesis, which
classified it as one of the most error-prone polymerases described
so far (13). However, the last point was challenged recently, this
enzyme being more prone to generate frameshifts based on misalignments of primer template in vitro than to perform strict base
misincorporations (23). No specific defect was detected by histology in nonlymphoid tissues of pol ␮-deficient mice (data not
shown). In contrast, about half of the mice have a marked depletion of B cells in peripheral lymphoid organs. Indeed, flow cytometric analysis of splenic lymphoid cells showed that, among 20
mice between 4 and 16 wk of age, 10 have a splenic B:T cell ratio
between 18 and 28%, while the others have values between 33 and
59%, i.e., within the normal variation range. Nevertheless, the B
cell maturation monitored by the progressive diminution in surface
IgM with concomitant increase in IgD appears unaffected. Representative cases are shown in Fig. 5. We are currently investigating
what type of lymphoid-specific DNA transaction involving pol ␮
could generate such a B cell deficit, with these rather surprising
individual variations.
It has been suggested that several of the translesional DNA
polymerases may cooperate in the hypermutation process, and,
more precisely, that G/C and A/T targeted mutations could be generated by two different steps and/or enzymes (Ref. 24; discussed in
Ref. 3). In any event, considering the complete neutral effect of pol
␮ deletion on the mutation profile of Ig genes, it appears unlikely
that this enzyme is involved at any step of the process.
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interactions (named after the prototypal C-terminal domain of the
breast cancer protein 1) (19). Because multiple splicing variants
have been described for both polymerases (12), we chose not to
delete their first exon, but rather their catalytic domain. Exons 5–7
were deleted in the pol ␭-targeted allele, thus removing not only
the DNA polymerase catalytic site but also the first HhH domain
corresponding to the 5⬘-deoxyribose phosphate lyase activity (20)
(Fig. 1). For pol ␮, exons 7–9 were deleted and exons 6 and 10
were truncated, resulting in the deletion of most of the polX domain (Fig. 2). The residues characterized in pol ␤ and pol ␭ as
critical for the 5⬘-deoxyribose phosphate lyase activity are conserved in the first HhH domain of neither pol ␮ nor Tdt, making it
unlikely that these enzymes possess an enzymatic activity other
than their nucleotidyl transferase function (20, 21). Homologous
recombination was confirmed by Southern blot on both the ES
clones chosen for injection and spleen DNA from the resulting
heterozygous and homozygous mice (Figs. 1B and 2B). No expression was detected in either case upon amplification of the
complete coding sequence (Figs. 1C and 2C).
Homozygous knockout (k.o.) mice were viable and were obtained in both cases with normal mendelian segregation. Despite
the strong expression seen in testis for pol ␭, the male k.o. mice
were fertile, and homozygous breeding has been performed up to
the third generation without noticeable problem. Pol ␮-deficient
mice were also bred as homozygous mutants.
To assess the possible contribution of these polymerases to the
Ig gene hypermutation process, Ig gene mutations from chronically
stimulated B cells were analyzed. PNAhigh B cells were sorted
from Peyer’s patches of 3- to 5-mo-old animals, and mutations
occurring within 260 bp flanking rearranged JH4 segments, i.e.,
within nonselected sequences, were studied.
Mutations obtained from both types of k.o. mice appear normal
in every aspect, compared with littermate control animals. They
are quantitatively similar, expressed either as average mutation
frequency, the differences observed being within the range of the
normal individual variations (Table I), or as distribution of mutations per sequence (Fig. 3). Qualitatively, the relative mutation
frequency of the four nucleotides, as well as the transition:
transversion ratio, is comparable (Table II). Distribution of mutations along the JH4 intronic sequence is also similar between
controls and k.o. mice, with a similar targeting of hot spot positions (Fig. 4).
A role of pol ␭ in meiosis and in base excision repair has been
proposed (14, 20). An overall histological examination of various
tissues from pol ␭-deficient mice has been performed without revealing any defect (data not shown). Nevertheless, apoptosis
within specific tissues, in particular during embryonic development, remains to be assessed. The perfect viability and fertility of
3706
CUTTING EDGE: NO ROLE FOR pol ␭ AND pol ␮ IN HYPERMUTATION
Acknowledgments
We thank C. Garcia and F. Delbos for performing cell sorting and Ana
Cumano for help with the analysis of lymphoid development of pol ␮- and
pol ␭-deficient mice.
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