Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination

Opinion

TRENDS in Genetics Vol.22 No.1 January 2006

Mismatch repair converts AID-instigated nicks to double-strand breaks for antibody class-switch recombination Janet Stavnezer and Carol E. Schrader Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655-0122, USA

Mismatch repair (MMR) proteins are important for antibody class-switch recombination (CSR), but their roles are unknown. We propose a model for the function of MMR in CSR in which MMR proteins convert singlestrand nicks instigated by activation-induced cytidine deaminase (AID) into the double-strand breaks (DSBs) that are required for CSR. This model does not invoke any novel functions for MMR but simply posits that, owing to numerous single-strand nicks in the switch (S) regions of both DNA strands, when MMR proteins are recruited by U:G mismatches, they excise one strand of DNA and soon reach a nick on the opposite strand. This halts excision activity and creates a DSB. This model explains why B cells that lack either Sm and MSH2 or UNG and MSH2 cannot undergo CSR. Antibody class switching occurs by a unique type of recombination Immunoglobulin class switching occurs after immunization or infection and enables B cells to switch the heavy chain constant (CH) regions of their expressed immunoglobulins (Igs), thereby switching from expressing IgM to expressing IgG, IgA or IgE. Different CH regions interact with different receptors and proteins (i.e. Fc receptors and complement proteins); therefore, class switching greatly increases the effectiveness of the humoral immune response. Class switching occurs by intrachromosomal deletion and DNA recombination in which the gene encoding the IgM constant region (Cm) is replaced by a gene that is further downstream (e.g. Cg, Ca, or C3; Figure 1). Class switch recombination (CSR) occurs within switch (S) regions located upstream of each C gene (except Cd) and is initiated by activation-induced cytidine deaminase (AID), which converts the cytosines in DNA to uracils [1–7]. Ig S-regions are 1–12 kb in length and consist of tandem repeats of 20–80 bp consensus sequences that differ in each Ig isotype. They contain numerous targets for AID including the hotspot motif WRC/GYW (W denotes A or T; R denotes A or G; Y denotes C or T) [6,8,9]. Only transcriptionally active S-regions undergo CSR and this is thought to be because the target for AID is single-stranded DNA (ssDNA), which can be Corresponding author: Stavnezer, J. ([email protected]). Available online 23 November 2005

generated during transcription [4,10]. In addition, RNA transcribed from S-regions hybridizes with the template strand to form R-loops with the transcribed DNA strand, generating long stretches of ssDNA in vivo [11]. The dU residue resulting from AID activity can be excised by a uracil DNA glycosylase (UNG), leaving an abasic site [3,12], and CSR is severely reduced in the absence of UNG [12,13]. Abasic sites are recognized by apurinic/apyrimidic (AP) endonucleases (APEX1 and APEX2) that nick the DNA backbone to create ssDNA breaks (SSBs) [14]. These endonucleases do not remove the deoxyribose phosphate (dRP) moiety remaining after UNG action, but leave this group attached to the 5 0 end at the break. During error-free base-excision repair, the lyase activity of DNA polymerase (Pol) b is required to excise the dRP group. It is unknown whether APEX1 and/ or APEX2 or DNA Pol b have a role in CSR. The current data support the conclusion that repair of the dU residues generates SSBs that are then, by unknown mechanisms, converted to the DSBs that are required for CSR. We propose here that mismatch repair (MMR) is an important part of the mechanism for conversion of SSBs to DSBs. Mismatch repair (MMR) proteins are involved in CSR A major function of MMR in all cells is to repair DNA mismatches caused by nucleotide substitutions or insertions/deletions that occur during DNA replication. This process involves recognition of the mismatch by a heterodimer of MSH2–MSH6 (for nucleotide substitutions and small loops) or MSH2–MSH3 (for larger loops), followed by recruitment of the MLH1–PMS2 heterodimer [15]. The combined heterotetramer recruits replication factor C (RFC), processivity factor PCNA and exonuclease 1 (EXO1) to a nearby nick, and together they excise the single-strand segment containing the mutated nucleotide [16,17]. The excised ssDNA patch can be hundreds of nucleotides in length in vitro, but its length in vivo is unknown. MMR specifically repairs the newly synthesized DNA strand, probably owing to its preference for excising and re-synthesizing the nicked DNA strand. B cells from mice that are deficient in MSH2, MSH6, MLH1, PMS2 or EXO1, and combinations thereof, have reduced (two-to-seven times) abilities to undergo CSR [18–24]. In addition, the S-S recombination junctions differ from those in wild-type cells. Msh2K/K and Exo1K/K B

www.sciencedirect.com 0168-9525/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2005.11.002

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Heavy chain genes in an IgM-expressing cell VDJ

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Heavy chain genes in an IgEexpressing cell VDJ

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Cα ε mRNA TRENDS in Genetics

Figure 1. Ig class-switch recombination (CSR) to IgE. The mouse Ig H locus in B cells expressing IgM and IgD (by alternative RNA transcription and processing) is shown in the upper panel. The Ig H locus following CSR to IgE is shown in the lower panel. During CSR, activation-induced cytidine deaminase (AID) deaminates dC residues in both strands of transcriptionally active S-regions (Sm and S3), initiating a process, described in the main text, that results in DSBs in both S-regions and CSR by intrachromosomal deletion. S-regions must be transcribed to undergo CSR [52]. RNA transcribed across S-regions stably associates with the transcribed DNA strand, apparently because of the G-rich nature of the RNA transcript, forming R-loops that leave the non-transcribed strand unpaired and vulnerable to attack by AID [11,53]. In addition, in vitro experiments indicate that when R-loops are removed by RNase H treatment, the S-regions reform duplex DNA, but they do not always reform perfect duplexes, probably owing to misalignment of the repeat units [11]. Such misaligned duplexes would form single-strand regions on both the transcribed and non-transcribed strands, which could provide single-strand targets for AID on the lower and upper strand.

cells have less junctional microhomology between the upstream and downstream S-regions, with increased frequency of apparent insertions of a few nucleotides at the junctions, whereas Mlh1K/K and Pms2K/K and also Msh2K/K Mlh1K/K double knockout mice have longer stretches of junctional microhomology [20,24–26]. In addition, mutations surrounding the S-S junctions are increased in MMR-deficient mice [26,27], consistent with the known role for MMR in identification and repair of DNA mismatches. These data suggest that MMR participates in the recombination process, perhaps by processing DNA ends, and that this processing affects mutations near the junctions and the nucleotide sequences at the junctions. In addition to being essential for MMR, Msh2 and Msh3 have been shown to participate in homologous recombination in Saccharomyces cerevisiae, recruiting the endonuclease Rad1–Rad10 [the yeast homolog of ERCC4–ERCC1, excision repair proteins; ERCC4 is more commonly known as xeroderma pigmentosum F (XPF)] to remove non-homologous DNA ends during DSB repair [28,29]. It is thought that Msh2–Msh3 recognizes recombination intermediates and that the presence of non-homologous DNA ends stimulates Msh2–Msh3 to recruit or activate Rad1–Rad10 for incision. Mlh1 and Pms2 are not required for this process. Although the available evidence suggests that one of the roles of MSH2 www.sciencedirect.com

in CSR is indeed to perform end-processing [18,25,30], it seems unlikely to use this mechanism. MSH3 does not appear to be involved in CSR [21,23], but MLH1 and PMS2 are involved. Also this role of Msh2–Msh3 has only been shown during homologous recombination, whereas CSR occurs by nonhomologous end-joining (NHEJ) [31,32]. Furthemore, we recently showed that B cells lacking either MSH2 or ERCC1 (the mammalian homolog of Rad10) have distinct CSR phenotypes [25,33]. Therefore, it does not seem likely that the role of MSH2 during CSR is to recruit the endonuclease ERCC1–XPF.

A model for the role of MMR in CSR We propose a model for the function of MMR proteins in CSR in which MMR functions to convert SSBs to the DSBs required for CSR (Figure 2). This model does not invoke any novel functions for MMR, but simply proposes that, because of the presence of multiple SSBs on both strands in S-regions, when MMR excises one strand of DNA it soon reaches a nick on the opposite strand, and this halts the excision activity and creates a DSB. The model suggests that, owing to the abundance of AID hotspot targets (WRC/GYW motifs) in S-regions, the high processivity of AID [6,9] and the likelihood of long stretches of ssDNA in S-regions, AID deaminates several dC residues within the S-region, generating several dU residues spread over a large region. UNG might not be able to excise them all

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TRENDS in Genetics Vol.22 No.1 January 2006

U AID deaminates dC

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UNG acts incompletely AP endo cuts at abasic sites

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MSH2–MSH6 and MLH1–PMS2 bind to U:G, recruit EXO1 and helicase to a nearby nick. EXO1 excises in 5′ → 3′ or 3′ → 5′ direction

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This creates DSB when a nick is encountered on the other strand

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(iii) If the nearest nick is 5′ to U:G, it would generate a Upper strand: 5′ overhang, which could 3′ → 5′ be filled-in (error-prone owing to AID-initiated lesions)

ERCC1–XPF (e)

End-processing after formation of DSBs

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TRENDS in Genetics

Figure 2. A model for conversion of SSBs in Ig S-regions to DSBs by MMR. (a) AID is hypothesized to introduce several dU residues in S-regions during one cell cycle owing to numerous AID hotspot targets, present in long stretches of ssDNA substrate thought to be stabilized by R-loop formation [11]. (b) Some of the dU residues are excised by UNG, and some of the abasic sites are nicked by AP endonuclease. (c) The U:G mismatches that remain would be substrates for MSH2–MSH6 [37]. MSH2–MSH6 and MLH1–PMS2 recruit EXO1 (and helicase) to a nearby 3 0 nick, from where they begin to excise the ssDNA [17]. Alternatively, EXO1 and helicase could also be recruited to 5 0 nicks (not shown). (d) If EXO1 excised 3 0 /5 0 from the nick shown on the lower strand to the 3 0 side of the MMR complex, this would create a DSB with a single-strand tail, instead of a blunt DSB (d,i). If EXO1 binds to the upper strand to the 3 0 side of the MMR complex on the U:G mismatch, 3 0 /5 0 excision could continue until a nick on the opposite strand is reached, creating a blunt DS (d,ii). If there were a nick on the 5 0 side of the MMR complex (not shown), EXO1 activity (5 0 /3 0 ) could create a 3 0 single-strand tail (d,iii). The 5 0 overhang could be filled-in by DNA polymerase or removed by a 5 0 flap endonuclease (FEN1) or by EXO1. Fill-in DNA synthesis (perhaps performed by a combination of translesion polymerases, e.g. Pol i and Pol h [37], and error-free polymerases) could be highly error-prone owing to the dU residues and abasic sites remaining from AID and UNG activities. (e) If a 3 0 single-strand tail were produced as in part d,i, this tail could be excised by the structure-specific endonuclease ERCC1–XPF or by EXO1.

before the R-loop collapses to reform dsDNA (Figure 2a,b). We also hypothesize that APEX1 will nick most of the abasic sites, but perhaps not all, because it does not efficiently nick closely opposed abasic sites [34–36]. The U:G mismatches that remain when the S-region duplex DNA reforms would be substrates for MSH2–MSH6 [37]. Perhaps MSH2–MSH6 and UNG compete for recognition of dU residues. It is also possible that clusters of abasic sites form small loop-like structures that might be recognized by MSH2–MSH6. Subsequently, MLH1–PMS2 binds to the DNA, and EXO1 (and helicase) enters at either a 5 0 or 3 0 nick nearby and excises one strand [16,17] (Figure 2c). We hypothesize that excision continues until a nick on the opposite strand is reached, thereby creating a DSB (Figure 2d). Alternatively, if EXO1 enters at the nick shown on the lower strand to the left of the MMR complex, and excises 3 0 to 5 0 , it would create a DSB with a long 3 0 ssDNA tail, instead of a blunt DSB (Figure 2d). The ssDNA tail could be excised by the structure-specific endonuclease ERCC1–XPF or by EXO1. If there were a nick on the 5 0 side of the MMR complex, instead of the two nicks illustrated in Figure 2c, EXO1 activity could create a 5 0 ssDNA tail (Figure 2d). The 5 0 overhang could be filled-in by DNA polymerase or removed by a 5 0 flap www.sciencedirect.com

endonuclease (FEN1) or by EXO1. Fill-in DNA synthesis could be highly error-prone if there were dU residues and abasic sites in the 5 0 overhang, thus potentially explaining the presence of mutations surrounding S-S junctions [27,38]. MMR is not required for all CSR, presumably because some nicks on opposite strands will be sufficiently close to form a spontaneous DSB that could be joined by NHEJ, with or without further end-processing. Experimental support for the model Several experimental results provide important support for this model. B cells from mice that lack the Sm tandem repeats (SmKO) have surprisingly modest reductions in CSR (two-to-four times reduction) [39,40]. However, if SmKO B cells also lack MSH2, CSR is almost ablated [30]. This can be explained if in the absence of the numerous AID hotspot targets present in Sm, the SSBs are less frequent and therefore further apart from each other and, therefore, unlikely to form DSBs without MMR. The model hypothesizes involvement of both the MSH2–MSH6 and MLH1–PMS2 heterodimers in end-processing; therefore, we have also examined CSR in mice that lack both the Sm tandem repeats and MLH1. We found that CSR is almost ablated (%5% of that seen in wild-type mice),

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identical to the SmKO Msh2K/K mice (C.E. Schrader et al., unpublished). This model is also consistent with published data showing that in Msh2K/K B cells, a greater proportion of the Sm–Sg3 and Sm–Sa recombination junctions occur within the Sm tandem repeats, rather than both within and upstream of the Sm repeats, as they do in wild-type B cells [18,41]. AID targets are further apart in this upstream region, suggesting that DSBs cannot form upstream of Sm in the absence of MSH2 or MLH1. This model is also supported by recent studies in which ligation-mediated (LM)-PCR was used to detect the sites of AID-dependent DNA breaks induced in vivo during CSR [42–44]. The DNA deamination model for AID predicts that ssDNA lesions that initiate CSR occur at C residues, and that the resulting DNA breaks should be staggered with their ends also at C residues. LM-PCR analyses of DNA breaks in splenic B cells induced to undergo CSR show that most of the breaks in the Sm region are indeed staggered [43,44]. Furthermore, cloning and sequencing the staggered DSBs showed they occur at C residues on both the strands in proportion to the relative frequency of C residues [44]. Although analysis of AID activity in vitro on kanamycin-resistance genes in plasmids suggested that it acts almost exclusively on the non-transcribed DNA strand [4,10], both strands are targeted in S-regions in vivo [44]. It is likely that the staggered DSBs at C residues are generated by SSBs that occur sufficiently close to each other on opposite strands to not require further end-processing before forming a DSB. If, however, SSBs on opposite strands are further apart than a few base pairs, they would need to be end-processed to create a DSB; we hypothesize that this involves MMR and produces blunt DSBs. This is consistent with the detection of AID-dependent blunt DSBs in Sm and Sg3 regions during CSR [13,42–44], and with the fact that the blunt breaks occur preferentially at G and C bases in WRC/GYW AID hotspots [42–44] (Table 1). This finding is consistent with the hypothesis that the blunt DSBs are generated by end-processing from an SSB at a C residue on one strand until a nick is reached at a former C residue on the opposite strand, as shown in Figure 2.

Why are UngK/KMsh2K/K B cells unable to undergo CSR? UngK/K B cells in mice and humans undergo low levels of CSR, w5% of that in wild-type mouse splenic B cells [12,13,44], and have few S-region DSBs [13,44]. As discussed earlier, Msh2K/K B cells switch w15-50% as efficiently as wild-type cells. However, when both Ung and Msh2 genes are deleted, CSR is entirely ablated [45]. These results can be explained by the model shown in Figure 2. In the absence of UNG, lesions instigated by AID would lead to few SSBs. MSH2–MSH6 would bind to the numerous U:G mismatches remaining, recruit MLH1–PMS2 and EXO1 and convert the SSBs to DSBs. Thus, we propose that the requirement of MSH2 for CSR in UngK/K B cells is because of its involvement in the creation of DSBs from the few SSBs that are produced in the absence of UNG. How could S-region breaks be introduced in UngK/K cells? In the absence of UNG, it is possible that S-region breaks are introduced as a result of the activity of an alternative uracil DNA glycosylase [e.g. single-strand selective monofunctional uracil DNA glycosylase (SMUG1), methyl-CpG binding domain protein 4 (MBD4) or thymine DNA glycosylase (TDG)]. However, it has been shown that SMUG1 cannot compensate for UNG-deficiency [45], and deletion of MBD4 does not reduce CSR [45,46]. The mammalian uracil DNA glycosylase, TDG, preferentially recognizes dU residues derived from CpG dinucleotides, which are extremely rare in S-regions [14]. Furthermore, if another UDG were involved, one would predict that the DNA breaks in S-regions in UNG-deficient B cells would occur preferentially at G or C bases in AID hotspots, whereas we have found that they do not [44]. Therefore, we hypothesize that another repair pathway serves as a bypass pathway to create SSBs in the absence of UNG. MMR is not an attractive candidate because no endonuclease is known to associate with MSH2–MSH6 or MLH1–PMS2, although extensive searches have been performed. By contrast, the nucleotide excision-repair pathway is a form of long-patch repair in which a nick is

Table 1. Blunt DSBs in both mouse and human Sm and mouse Sg3 regions induced during CSR occur preferentially at G:C base pairs in WRC/GYW AID hotspots Nucleotide at the breaka G C A T Total GCC P-valued Hotspots P-valued

Mouse Ref. [44]

Mouse Ref. [43]

83.9% (47)b 5.4% (3) 7.1% (4) 3.6% (2) 56 breaks 89.3% !0.001 41.1% 0.049

33% (3)b 44% (4) 11% (1) 11% (1) 9 breaks 78% NSe 44% NSe

Sm Sequencec (mouse) 40.7% 16.1% 21.4% 21.9% 2000 nts 56.8% 23.2%

Human Ref. [42] 73% (11)b 13% (2) 13% (2) 0% 15 breaks 87% NSe 67.7%% 0.004

Sequencec (human) 41.87% 22.1% 17.1% 19.0% 1000 nts 63.9% 24.8%

Sg3 Mouse Ref. [44] Sequencec 84% (21)b 4.0% (1) 8.0% (2) 4.0% (1) 25 breaks 88.8% 0.039 40.0% !0.001

48.7% 14.7% 18.3% 18.4% 1460 nts 63.4% 14.8%

The nucleotide immediately 3 0 to the cloned break-site fragments on the upper strand (i.e. the nucleotide missing from the cloned fragments, which were derived by amplification using gene-specific primers at the 5 0 end of the S-regions in conjunction with the linker primer). This represents the nucleotide attacked by AID in wild-type cells. The high G:C ratio is introduced during end-processing, as the staggered breaks occur on both strands approximately in proportion to their G:C ratio [44]. b The number of breaks analyzed. The number for each region analyzed is shown in parenthesis. c The distribution of nucleotides in the genomic sequence analyzed. d P-values were determined by Fisher’s exact test. e Not significant (NS), P O0.05. Although most of the data from Rush et al. [43] and Catalan et al. [42] are not significantly different from random, this is clearly a result of insufficient data, because the trends indicate a preference for breaks at G and C bases in AID hotspots. a

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TRENDS in Genetics Vol.22 No.1 January 2006

introduced on either side of a duplex-distorting lesion by two associated endonucleases (ERCC1–XPF and ERCC5, also known as XPG), and the damaged patch is then excised and re-synthesized [47]. If nucleotide excision repair were involved in creation of DNA breaks, they would be introduced at random nucleotides. Although nucleotide-excision repair does not recognize single U:G mismatches or abasic residues [47], it is possible that the presence of multiple lesions, owing to the absence of UNG, results in a substrate that is recognized by nucleotideexcision repair. Does this model explain the altered S–S junctions observed during CSR in the absence of an MMR protein? We can only speculate about the cause of the altered S–S junctions in MMR-deficient B cells. If SSBs occurring more than a few base pairs apart cannot be converted to DSBs owing to the lack of MMR, then all S-junctions in MMR-deficient cells will involve DNA ends with short single-strand tails, although at least some of these tails are likely to be further processed by either fill-in synthesis, to create blunt ends, or an endonuclease (e.g. ERCC1–XPF) that can recognize single-strand 3 0 tails in the absence of MMR [48]. However, if end-processing only occurs across a few base pairs, then the AID-instigated mutations will remain in the recombining S-regions, possibly explaining the increased mutation frequency surrounding the S–S junctions in MMR-deficient cells [27]. This model does not seem to explain why the lengths of short microhomologies between the upstream and downstream S-regions found at S–S junctions in Mlh1K/K and Pms2K/K cells are longer than those found in Msh2K/K cells. This has led to the suggestion that MSH2–MSH6 or MLH1–PMS2 might have an additional function(s) in CSR independent of the other heterodimer [22,26,49]. Support for this hypothesis was reported recently; it was shown in vitro that EXO1 performs more extensive excision in the absence of MLH1–PMS2, creating longer single-strand regions of DNA [50]. It has also been shown that EXO1 can only excise in the 5 0 /3 0 direction in the absence of MLH1–PMS2, which would result in ends having 5 0 but not 3 0 single-strand tails (17). Perhaps these two effects of MLH1–PMS2 explain the greater lengths of microhomology at S–S junctions in Mlh1K/K and Pms2K/K cells than in Msh2K/K or wildtype cells. Predictions of the model We believe this model for the role of MMR in CSR is in agreement with all available data. However, additional, direct support is required. The hypothesis that MMR converts DNA nicks to DSBs predicts that in the absence of MSH2, MLH1 or EXO1, there should be a reduction in blunt DSBs as assayed by LM-PCR during CSR, but no decrease in SSBs. In addition, the blunt DSBs should be mostly confined to the tandem repeat S-regions in MMRdeficient cells, whereas they are found both upstream and within the tandem repeats of Sm in wild-type cells [44]. The model also predicts that mice deficient in both the Sm tandem repeats and Exo1 would have greatly reduced CSR. Although it has been shown that MSH2–MSH6 www.sciencedirect.com

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binds to U:G mismatches in vitro [37], it will be important to demonstrate this in vivo by showing that the association of MSH2–MSH6 with S-regions increases in UngK/K cells. Finally, the model predicts that U:G mismatches will occur in S-region DNA. One way this could occur is if AID has a greater rate of catalysis than UNG in vivo. Although the catalytic rate in vitro for UNG has been shown to be high (ten dU excisions per second) [51], the catalytic rate for AID has not been determined. Acknowledgements We thank Amy L. Kenter for helpful criticisms. We acknowledge support from the National Institutes of Health, NIAID to J.S. (R01 AI 23283 and AI 63026) and to C.E.S. (R01 AI 65639).

References 1 Muramatsu, M. et al. (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563 2 Revy, P. et al. (2000) Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 102, 565–575 3 Petersen-Mahrt, S.K. et al. (2002) AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–104 4 Chaudhuri, J. et al. (2003) Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature 422, 726–730 5 Dickerson, S.K. et al. (2003) AID mediates hypermutation by deaminating single stranded DNA. J. Exp. Med. 197, 1291–1296 6 Pham, P. et al. (2003) Processive AID-catalysed cytosine deamination on single-stranded DNA simulates somatic hypermutation. Nature 424, 103–107 7 Bransteitter, R. et al. (2003) Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc. Natl. Acad. Sci. U. S. A. 100, 4102–4107 8 Yu, K. et al. (2004) DNA substrate length and surrounding sequence affect the activation-induced deaminase activity at cytidine. J. Biol. Chem. 279, 6496–6500 9 Bransteitter, R. et al. (2004) Biochemical analysis of hypermutational targeting by wild type and mutant activation-induced cytidine deaminase. J. Biol. Chem. 279, 51612–51621 10 Ramiro, A.R. et al. (2003) Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat. Immunol. 4, 452–456 11 Yu, K. et al. (2003) R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells. Nat. Immunol. 4, 442–451 12 Rada, C. et al. (2002) Immunoglobulin isotype switching Is inhibited and somatic hypermutation perturbed in UNG-deficient mice. Curr. Biol. 12, 1748–1755 13 Imai, K. et al. (2003) Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat. Immunol. 4, 1023–1028 14 Krokan, H.E. et al. (2002) Uracil in DNA–occurrence, consequences and repair. Oncogene 21, 8935–8948 15 Kunkel, T. and Erie, D. (2005) DNA mismatch repair. Annu. Rev. Biochem. 74, 681–710 16 Genschel, J. et al. (2002) Human exonuclease I is required for 5 0 and 3 0 mismatch repair. J. Biol. Chem. 277, 13302–13311 17 Genschel, J. and Modrich, P. (2003) Mechanism of 5 0 -directed excision in human mismatch repair. Mol. Cell 12, 1077–1086 18 Ehrenstein, M.R. and Neuberger, M.S. (1999) Deficiency in Msh2 affects the efficiency and local sequence specificity of immunoglobulin class-switch recombination: parallels with somatic hypermutation. EMBO J. 18, 3484–3490 19 Schrader, C.E. et al. (1999) Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes. J. Exp. Med. 190, 323–330 20 Ehrenstein, M.R. et al. (2001) Switch junction sequences in PMS2deficient mice reveal a microhomology- mediated mechanism of Ig class switch recombination. Proc. Natl. Acad. Sci. U. S. A. 98, 14553–14558

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