Cutting Edge: Hypermutation in Ig V Genes
from Mice Deficient in the MLH1 Mismatch
Repair Protein
This information is current as
of June 15, 2017.
Quy H. Phung, David B. Winter, Rudaina Alrefai and Patricia
J. Gearhart
J Immunol 1999; 162:3121-3124; ;
http://www.jimmunol.org/content/162/6/3121
Subscription
Permissions
Email Alerts
This article cites 29 articles, 13 of which you can access for free at:
http://www.jimmunol.org/content/162/6/3121.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
References
●
Cutting Edge: Hypermutation in Ig V
Genes from Mice Deficient in the
MLH1 Mismatch Repair Protein
Quy H. Phung,*† David B. Winter,* Rudaina Alrefai,*
and Patricia J. Gearhart1*
T
wo types of somatic mutation mechanisms exist in B lymphocytes. One mechanism is spontaneous mutation,
which is common to all cells and is caused by the misincorporation of nucleotides or slippage of DNA polymerase during
DNA replication of chromosomes. Most of the replication errors
are then removed by the mismatch repair pathway to ensure high
fidelity of DNA replication. Thus, the frequency of spontaneous
mutation after correction is ;1028 mutations per base pair, and
this frequency increases several fold when mismatch repair proteins are deficient (1–5). The other mechanism is hypermutation,
which is unique to B cells and is caused by unknown enzymes. The
frequency of hypermutation is ;1022 mutations/base pair, and
mutation occurs in a 2-kb region around rearranged V genes (reviewed in Ref. 6).
*Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of
Health, Baltimore, MD 21224; and †Graduate Program in Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21205
Received for publication November 24, 1998. Accepted for publication January 8,
1999.
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
Address correspondence and reprint requests to Dr. Patricia J. Gearhart, Laboratory
of Molecular Genetics, Gerontology Research Center, National Institute on Aging,
National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224. Email address: [email protected]
Copyright © 1999 by The American Association of Immunologists
●
Recently, there has been a great deal of interest in studying
hypermutation in V genes from mice deficient in the mismatch
repair pathway (7–10). Studies from several labs have shown that
mice deficient for the MSH2 and PMS2 mismatch repair proteins
have hypermutation (11–15); this finding is in contrast to an earlier
report by Cascalho et al. (16). The frequency of mutation in the
repair-deficient mice was either the same or several fold lower than
in wild-type (wt)2 mice, depending upon the type of exposure to
Ag. After deliberate exposure by immunization with Ag, rearranged Vk and Vl genes from splenic B cells from MSH2- and
PMS2-deficient mice had the same frequencies of mutation as
repair-proficient mice (;1% mutations per base pair) (11–14).
After chronic exposure to environmental gut-associated Ags, rearranged VH genes from Peyer’s patch B cells from MSH2- and
PMS2-deficient mice had a three- to fivefold lower frequency of
mutation compared with wt mice (14, 15). The diminished response in the chronically stimulated cells may be due to early cell
death before the V genes can undergo many rounds of mutation
(14, 15). Thus, a lack of DNA repair allows spontaneous mutations
to persist in the overall genome; consequently, when such mutations occur in genes critical for cell survival, the cell dies.
The pattern of mutation from MSH2- and PMS2-deficient mice
has also been examined. V genes from Msh22/2 mice had a greatly
increased number of mutations at G and C nucleotides compared
with A and T nucleotides (11, 12, 14, 15). V genes from Pms22/2
mice had a greater number of tandem mutations, which was confirmed by the inability of Pms22/2 cell extracts to repair adjacent
mutations on artificial substrates (13). This altered spectrum of
mutation in the mismatch repair-deficient mice compared with wt
mice suggests that mismatch repair proteins remove some of the
mutations before DNA replication. However, this process is inefficient, perhaps because the mismatch repair pathway is unable to
deal with the excessively large number of mismatches generated
by the hypermutation mechanism.
In addition to MSH2 and PMS2, other proteins such as MSH6
and MLH1 participate in the mismatch repair pathway, as shown
in Fig. 1. Because the absence of MSH2 and PMS2 produced different mutational spectra in V genes, these other proteins may also
be involved in removing mismatches generated during hypermutation. For example, Mlh12/2, Pms22/2, and Msh22/2 mice each
have distinct phenotypes for reproduction and tumors (5, 17–21),
and they may possess different mutational patterns as well. Therefore, we examined the role of one of the proteins, MLH1, in hypermutation. MLH1, along with PMS2, binds to the MSH2-MSH6
2
Abbreviations used in this paper: wt, wild type; nt, nucleotide(s).
0022-1767/99/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the
DNA mismatch repair pathway. Previous studies have shown
that V genes from mice deficient for the MSH2 and PMS2
mismatch repair proteins have frequencies of mutation that
are comparable with those from wild-type (wt) mice; however,
the pattern of mutation is altered. Because the absence of
MSH2 and PMS2 produced different mutational spectra, we
examined the role of another protein involved in mismatch
repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone
Ag, and splenic B cells were analyzed for mutations in their
VkOx1 light chain genes. Although the frequency of mutation
in MLH1-deficient mice was twofold lower than in wt mice, the
pattern of mutation in Mlh12/2 clones was similar to wt clones.
These findings suggest that the MLH1 protein has no direct
effect on the mutational spectrum. The Journal of Immunology, 1999, 162: 3121–3124.
3122
CUTTING EDGE
Table I. Mutations in VkOx1 genes from MLH1-deficient micea
Clone
FIGURE 1. Mammalian DNA mismatch repair. A heterodimer of
MSH2 and MSH6 proteins recognizes base-base mismatches and extrahelical loops of 1 or 2 nt. The complex subsequently combines with a heterodimer of PMS2 and MLH1. Repair occurs after the newly synthesized
strand is nicked, exonuclease removes the mismatch, and DNA polymerase
fills in the gap.
Materials and Methods
Mice
MLH1-deficient mice (obtained from R. M. Liskay, Portland, OR) were
generated by insertion of an hprt minigene to replace a 2.5-kb fragment of
exon 4 in Mlh1 (17). Four Mlh12/2 mice that had been bred onto a
C57BL/6 and AB-129 background were given a primary i.p. injection of 50
mg of phenyloxazolone coupled to chicken serum albumin (a gift of C.
Milstein, Cambridge, U.K.) in CFA. After 1 mo, the mice were administered a secondary injection of 50 mg of Ag in IFA. Mice were sacrificed
after 4 days, and spleens were removed. B cells that bound to phycoerythrin-labeled B220 and fluorescein-labeled GL7 (22) (PharMingen, San
Diego, CA) as well as peanut agglutinin were isolated by flow cytometry.
5
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3
3
3
4
4
4
5
5
5
6
11
18
Positionb
T72G
C176T
TC-42p
A217T
C176T
A-120G
C265A
A217T
A92T
C78T
A27pp
G175A
G227A, G275C
A226C, C228A
C78T, C97A
G272A, G275A
C14A, C97A
G96A, C99T, G224A
T-85G, C97A, G275A
T-54G, C14A, C97A
T-158C, T-142A, T-22pp, G224A
A-13G, C60T, C97A, A104T
C97A, C99T, A104T, A117G
A-118G, T-51A, C-7T, T144C, G227A
A58T, C78T, A82C, C97A, A104T
C97A, C99T, A146T, C176T, G264A
G-80A, G-79C, G77C, A87T, G89A, C225T
C-156T, C-60G, T-51C, C-49G, G77C, G89C,
G108A, A113G, C149T, G244A, G264T
T-54C, C-44A, A-34T, T-24C, T29A, C30A,
A61T, A76T, G77C, C99G, A104T, A107G,
C157T, T163G, T198A, G227A, C251T,
C261T
a
Total 5 29 clones (13,514 bp sequenced); 93 mutations; 0.7% mutations per
base pair.
b
Nucleotide position is numbered according to Reference 13; negative numbers
represent mutations in the 59 intron. Tandem mutations are underlined. p, insertion;
pp, deletion.
c
Join is out of frame.
DNA cloning and sequencing
DNA from ;50,000 cells was isolated by proteinase K digestion and phenol/chloroform extraction. The VkOx1 gene segment rearranged to the Jk5
gene segment was amplified through 30 rounds of PCR with Pfu polymerase (Stratagene, La Jolla, CA) using a primer specific for the leader sequence on the 59 side of the gene and a primer specific for the Jk5 gene
segment on the 39 side. Part of the reaction (1/25th) was then subjected to
another 30 rounds of PCR using nested primers with restriction sites for
cloning the amplified product into M13 bacteriophage. DNA containing the
ligated M13 vector was transformed into JM101 bacteria by electroporation and immediately poured onto agarose plates to obtain unique libraries.
M13 plaques were screened for inserts by hybridization to a VkOx1 probe,
and positive clones were sequenced.
Results and Discussion
Mlh12/2 V genes have a low frequency of mutation
Mutation was analyzed in a defined V gene, VkOx1 (23), from
splenic B cells from immunized mice. Some 52 clones were sequenced for 466 bp, which included 190 nucleotides (nt) of 59
intron DNA between the leader and V gene segment and 276 nt of
coding region DNA. Approximately 31 of 52 Mlh12/2 clones
(60%) had mutations. The same percentage of clones had mutations in Mlh11/1 C57BL/6 mice (13). Of the mutated clones, 29
were distinct in that they either had different sequences at the V-J
junction or had unique substitutions that were not shared by other
clones. These clones are listed in Table I in ascending order of
mutations per clone, ranging from 1 to 18. There were 90 base
substitutions and 3 single nt insertions and deletions, giving an
average frequency of 0.7% mutations/base pair. This frequency is
comparable with the 0.9% mutations/base pair observed in
Pms22/2 clones (Fig. 2) but is lower than the 1.4% mutations/base
pair in C57BL/6 clones and the 1.3% mutations/base pair in
Msh22/2 clones (12–14). The twofold lower frequency in the
Mlh12/2 clones was due to a predominance of sequences with less
than four mutations, as was the case for Pms22/2 clones (13). As
proposed by Frey et al. (14) and Rada et al. (15), mismatch repairdeficient mice likely have chromosomal alterations because of a
lack of repair of spontaneous mutations in all the genes. This
genomic instability becomes fatal when it affects those genes that
control growth and division, so that rapidly dividing B cells die
before an accumulation of large numbers of mutations in their V
genes. In support of this hypothesis, Vora et al. (24) have recently
reported that Msh22/2 mice have smaller germinal centers with
more apoptosis than wt mice. The lower frequencies of mutation in
V genes from immunized MLH1- and PMS2-deficient mice suggest that the absence of these two mismatch repair proteins is more
detrimental to B cell survival than the absence of MSH2.
In Fig. 2, the distribution of mutations in rearranged VkOx1
genes from Mlh12/2 mice is compared with the distribution seen
for C57BL/6, Msh22/2, and Pms22/2 mice (12, 13). There was an
accumulation of substitutions in the first complementarity-determining region in each group. This is expected, because mutations
in codons 34 and 36 have been shown to confer higher affinity for
oxazolone, and B cells expressing Abs with these mutations are
preferentially selected (25).
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
protein complex that recognizes base-base mismatches. MLH1deficient mice are infertile because of arrest at the pachytene stage
of meiosis, have microsatellite instability, and are susceptible to
adenocarcinomas (5, 17, 18). Mice were immunized, and mutations in V genes were analyzed.
73c
125c
134c
139c
140
141
148
151
184
219
236
258
217c
195
226
240
263
7
117
202
162
244
239
3c
211
269
4
24c
Number
The Journal of Immunology
3123
Table II. Types of substitutions in VkOx1 genesa
C57BL/6
92 mut %
Msh22/2
108 mut %
Pms22/2
76 mut %
Mlh12/2
74 mut %
A to: G
T
C
19
16
11
0
5
1
17
8
8
8
12
3
T to: C
A
G
9
2
1
1
1
1
7
4
5
7
5
5
C to: T
A
G
10
2
2
25
5
1
12
3
9
19
8
3
G to: A
T
C
17
4
7
44
5
11
19
1
7
20
2
8
Substitution
a
Mutations in codons 34 (nucleotides 97–99) and 36 (nucleotides 103–105) were
excluded because they encode amino acids that are highly selected for binding to
oxazolone. mut, mutation(s).
FIGURE 2. Distribution of mutations in the rearranged VkOx1 gene
from mismatch repair-proficient and -deficient mice. A diagram of the
flanking and coding regions that were sequenced is shown at the bottom of
the figure; complementarity-determining regions are indicated by striped
boxes. Numbers on the abscissa refer to the nucleotide distance, with “1”
representing the first base in the V gene and negative numbers representing
the 59 flanking region. Bars depict the frequency of mutation per 10-nt
increments. Data for C57BL/6, Msh22/2, and Pms22/2 clones were obtained from References 12 and 13.
Nucleotide substitutions in Mlh12/2 clones are similar to wt
clones
The types of substitutions in different mismatch repair-deficient
mice are compared in Table II. If hypermutation occurs nondiscriminatingly on each nucleotide, equal amounts of mutation
should occur at A:T pairs compared with G:C pairs. This was
generally the case for VkOx1 genes from C57BL/6, Pms22/2, and
Mlh12/2 mice. However, as reported previously (11, 12, 14, 15),
most of the mutations in Msh22/2 clones occurred at G and C
nucleotides. Thus, MSH2 behaves independently from PMS2 and
MLH1 in repairing mismatches generated by the hypermutation
mechanism.
Mlh12/2 clones do not have increased tandem mutations
Tandem mutations of two in a row were of particular interest,
because VkOx1 genes from Pms22/2 mice had a greatly increased
Independent roles for mismatch repair proteins
Although MSH2, PMS2, and MLH1 are all required for mismatch
repair, mice deficient in these proteins have distinct phenotypes for
several biological mechanisms, suggesting that they can function
independently. In the mechanisms of reproduction and recombination, MSH2-deficient male and female mice are fertile (19, 20),
PMS2-deficient males are sterile but females are fertile (21), and
MLH1-deficient males and females are sterile (17, 18). The yeast
equivalents of PMS2 and MSH2 also have different effects in suppressing meiotic and mitotic recombination (26, 27). Thus, these
three proteins have independent functions during the recombination of chromosomes. In the mechanism of tumor suppression,
Table III. Tandem mutationsa
Tandems
Mice
Mutations
(clones)
Observed
Expected
p Value
C57BL/6
Msh22/2
Pms22/2
Mlh12/2
106 (12)
135 (22)
128 (30)
81 (17)
4
3
11
3
2.46
1.84
1.19
1.22
0.48
0.56
,1026
0.25
a
p values refer to exact Poisson calculations regarding whether the observed and
expected values are equal. Data for C57BL/6, Msh22/2, and Pms22/2 clones were
obtained from References 12 and 13.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
frequency of adjacent mutations compared with those from
C57BL/6 and Msh22/2 mice (12, 13). Because the MLH1 protein
pairs with PMS2 at the same step in mismatch repair (Fig. 1),
Mlh12/2 clones may also have more tandem mutations. There
were three tandem mutations in the Mlh12/2 clones; these mutations are underlined in Table I. The observed numbers of tandems
in each of the repair-deficient strains are summarized in Table III
and compared with the expected numbers. Expected numbers were
calculated according to the probability that two mutations will randomly occur next to each other in clones with a length of 466 nt
(13). Only the Pms22/2 clones had a significant increase in tandem mutations ( p , 1026) as determined by exact Poisson calculations regarding whether the observed and expected values
were equal for each group. This observation suggests that the
PMS2 protein acts independently of MLH1 in correcting adjacent
mutations put in by the hypermutation mechanism.
3124
Acknowledgments
We thank R. Liskay for the Mlh12/2 mice, F. Chrest for flow cytometry,
and R. Tarone for statistical analyses. We also thank A. Walley,
J. Blumenthal, and L. Diamond for assistance in sequencing. We acknowledge V. Bohr for enthusiastic support and V. Bohr, R. Wood, and N.
Lipinski for critical comments on the manuscript.
References
1. Malkhosyan, S., A. McCarty, H. Sawai, and M. Perucho. 1996. Differences in the
spectrum of spontaneous mutations in the hprt gene between tumor cells of the
microsatellite mutator phenotype. Mutat. Res. 316:249.
2. Glaab, W. E., and K. R. Tindall. 1997. Mutation rate at the hprt locus in human
cancer cell lines with specific mismatch repair-gene defects. Carcinogenesis
18:1.
3. Narayanan, L., J. A. Fritzell, S. M. Baker, R. M. Liskay, and P. M. Glazer. 1997.
Elevated levels of mutation in multiple tissues of mice deficient in the DNA
mismatch repair gene Pms2. Proc. Natl. Acad. Sci. USA 94:3122.
4. Andrew, S. E., M. McKinnon, B. S. Cheng, A. Francis, J. Penney, A. H. Reitmair,
T. W. Mak, and F. R. Jirik. 1998. Tissues of MSH2-deficient mice demonstrate
hypermutability on exposure to a DNA methylating agent. Proc. Natl. Acad. Sci.
USA 95:1126.
5. Prolla, T. A., S. M. Baker, A. C. Harris, J.-L. Tsao, X. Yao, C. E. Bronner,
B. Zheng, M. Gordon, J. Reneker, N. Arnheim, et al. 1998. Tumour susceptibility
and spontaneous mutation in mice deficient in Mlh1, Pms1, and Pms2 DNA
mismatch repair. Nat. Genet. 18:276.
6. Somatic hypermutation of immunoglobulin genes. 1998. Immunol. Rev. 162.
7. Kelsoe, G. 1998. V(D)J hypermutation and DNA mismatch repair: vexed by
fixation. Proc. Natl. Acad. Sci. USA 95:6576.
8. Kim, N., and U. Storb. 1998. The role of DNA repair in somatic hypermutation
of immunoglobulin genes. J. Exp. Med. 187:1729.
9. Wiesendanger, M., M. D. Scharff, and W. Edelmann. 1998. Somatic hypermutation, transcription, and DNA mismatch repair. Cell 94:415.
10. Wood, R. D. 1998. Knockouts still mutating after first round. Curr. Biol. 8:R757.
11. Jacobs, H., Y. Fukita, G. T. J. van der Horst, J. de Boer, G. Weeda, J. Essers,
N. de Wind, B. P. Engelward, L. Samson, S. Verbeek, et al. 1998. Hypermutation
of immunoglobulin genes in memory B cells of DNA repair-deficient mice.
J. Exp. Med. 187:1735.
12. 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.
13. Winter, D. B., Q. H. Phung, A. Umar, S. M. Baker, R. E. Tarone, K. Tanaka,
R. M. Liskay, T. A. Kunkel, V. A. Bohr, and P. J. Gearhart. 1998. Altered spectra
of hypermutation in antibodies from mice deficient for the DNA mismatch repair
protein PMS2. Proc. Natl. Acad. Sci. USA 95:6953.
14. 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.
15. 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.
16. Cascalho, M., J. Wong, C. Steinberg, and M. Wabl. 1998. Mismatch repair coopted by hypermutation. Science 279:1207.
17. Baker, S. M., A. W. Plug, T. A. Prolla, C. E. Bronner, A. C. Harris, X. Yao,
D. Christie, C. Monell, N. Arnheim, A. Bradley, T. Ashley, and R. M. Liskay.
1996. Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing
over. Nat. Genet. 13:336.
18. Edelmann, W., P. E. Cohen, M. Kane, K. Lau, B. Morrow, S. Bennett, A. Umar,
T. Kunkel, G. Cattoretti, R. Chaganti, et al. 1996. Meiotic pachytene arrest in
MLH1-deficient mice. Cell 85:1125.
19. De Wind, N., M. Dekker, A. Berns, M. Radman, and H. te Riele. 1995. Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation
tolerance, hyperrecombination, and predisposition to cancer. Cell 82:321.
20. Reitmair, A. H., R. Schmits, A. Ewel, B. Bapat, M. Redston, A. Mitri,
P. Waterhouse, H.-W. Mittrucker, A. Wakeham, B. Liu, et al. 1995. MSH2 deficient mice are viable and susceptible to lymphoid tumours. Nat. Genet. 11:64.
21. Baker, S. M., C. E. Bronner, L. Zhang, A. W. Plug, M. Robatzek, G. Warren,
E. A. Elliott, J. Yu, T. Ashley, N. Arnheim, R. A. Flavell, and R. M. Liskay.
1995. Male mice defective in the DNA mismatch repair gene PMS2 exhibit abnormal chromosome synapsis in meiosis. Cell 82:309.
22. Laszlo, G., K. S. Hathcock, H. B. Dickler, and R. J. Hodes. 1993. Characterization of a novel cell-surface molecule expressed on subpopulations of activated T
and B cells. J. Immunol. 150:5252.
23. Kaartinen M., G. M. Griffiths, A. F. Markham, and C. Milstein. 1983. mRNA
sequences define an unusually restricted IgG response to 2-phenyloxazolone and
its early diversification. Nature 304:320.
24. Vora, K. A., K. M. Tumas-Brundage, V. M. Lentz, A. Cranston, R. Fishel, and
T. Manser. 1999. Severe attenuation of the B cell immune response in Msh2deficient mice. J. Exp. Med. 189:471.
25. Alzari, P. M., S. Spinelli, R. A. Mariuzza, G. Boulot, R. J. Poljak, J. M. Jarvis,
and C. Milstein. 1990. Three-dimensional structure determination of an anti-2phenyloxazolone antibody: the role of somatic mutation and heavy/light chain
pairing in the maturation of an immune response. EMBO J. 9:3807.
26. Hunter, N., S. R. Chambers, E. J. Lewis, and R. H. Borts. 1996. The mismatch
repair system contributes to meiotic sterility in an interspecific yeast hybrid.
EMBO J. 15:1726.
27. Datta, A., A. Adjiri, L. New, G. F. Crouse, and S. Jinks-Robertson. 1996. Mitotic
crossovers between diverged sequences are regulated by mismatch repair proteins
in Saccaromyces cerevisiae. Mol. Cell. Biol. 16:1085.
28. Liu, B., R. Parsons, N. Papadopoulos, N. C. Nicolaides, H. T. Lynch, P. Watson,
J. R. Jass, M. Dunlop, A. Wyllie, P. Peltomaki, et al. 1996. Analysis of mismatch
repair genes in hereditary non-polyposis colorectal cancer patients. Nat. Med.
2:169.
29. Leadon, S. A., and A. V. Avrutskaya. 1997. Differential involvement of the human mismatch repair proteins hMLH1 and hMSH2, in transcription-coupled repair. Cancer Res. 57:3784.
30. Bertocci, B., L. Quint, F. Delbos, C. Garcia, C.-A. Reynaud, and J.-C. Weill.
1998. Probing immunoglobulin gene hypermutation with microsatellites suggests
a nonreplicative short patch DNA synthesis process. Immunity 9:257.
Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017
PMS2- and MSH2-deficient mice predominantly have lymphomas,
whereas MLH1-deficient mice mostly have intestinal adenomas
and adenocarcinomas (5, 19 –21). Furthermore, humans with hereditary nonpolyposis colorectal cancer have mutations predominantly in Msh2 and Mlh1 genes and rarely in Pms2 genes (28).
These different tumor spectra suggest that PMS2 and MLH1 have
overlapping but nonidentical functions. In the mechanism of DNA
repair, MSH2-deficient human cells cannot remove oxidative damage from the transcribed strand of DNA, whereas MLH1-deficient
cells can remove the damage; this observation suggests a differential involvement of the two proteins in transcription-coupled
repair (29).
In the mechanism of hypermutation, we propose that MSH2,
PMS2, and MLH1 proteins, although not required to generate or
fix hypermutation in V genes, have independent functions for removing a portion of the mismatches. The altered spectrum of mutations in V genes from repair-deficient mice suggests that MSH2
removes mismatches at G and C nucleotides, PMS2 removes tandem mismatches, and MLH1 has no discernible effect. This different pattern of mutation suggests that the hypermutation mechanism frequently generates substitutions opposite G and C
nucleotides and produces tandem mutations, which may occur during short-patch repair (30).
CUTTING EDGE