Shifts in targeting of class switch recombination sites in mice that lack μ switch region tandem repeats or Msh2

Min, Irene M.; Rothlein, L.R.; Schrader, Carol E.; Stavnezer, Janet; Selsing, Erik

doi:10.1084/jem.20042491

Cited by 31 publications

(33 citation statements)

References 28 publications

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“…In fact, the level of residual switching observed in APE1-deficient cells in this study is remarkably similar to that observed in cells deficient in UNG2. MMR has been shown to be particularly important when core switch repeats were deleted and overlapping AID hot spots were scarce (10,32,33). Among the MMR factors, the MutL␣ (MLH1/ PMS2) complex has been reported to possess a latent endonuclease activity that is activated upon mismatch recognition (34).…”

Section: Discussionmentioning

confidence: 99%

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Masani

Han

2013

Molecular and Cellular Biology

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Immunoglobulin (Ig) class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID) that catalyzes numerous DNA cytosine deaminations within switch regions. The resulting uracils are processed by uracil base excision and/or mismatch repair enzymes that ultimately generate switch region DNA double-strand breaks (DSBs). Uracil glycosylase 2 (UNG2) is required for CSR, most likely by removing uracils to generate abasic sites. Although it is presumed that the apurinic/ apyrimidinic endonuclease 1 (APE1) generates DNA strand incisions (a prerequisite for CSR) at these abasic sites, a direct test of the requirement for APE1 in CSR has been difficult because of the embryonic lethality of APE1 ablation in mice. Here, we report the successful deletion of the APE1 gene in a mouse B cell line (CH12F3) capable of robust CSR in vitro. In contrast to the general assumption that APE1 is essential for cellular viability, deletion of APE1 in CH12F3 cells has no apparent effect on cell viability or growth. Moreover, CSR in APE1-null CH12F3 cells is drastically reduced, providing direct evidence for an essential role for APE1 in switch region cleavage and CSR. Finally, deletion of AP endonuclease 2 (APE2) has no effect on CSR in either APE1-proficient or -deficient cells. In antigen-stimulated B cells, somatic hypermutation (SHM) diversifies the variable regions of immunoglobulin (Ig) heavy (H) and light (L) chain genes, whereas class switch recombination (CSR) provides a mechanism to diversify the constant region of the Ig heavy chain (1, 2). SHM introduces point mutations into the Ig variable (V) regions, which is the mechanistic basis for Ig affinity maturation. CSR introduces DNA double-strand breaks (DSBs) into donor and acceptor switch (S) regions; subsequently a "cut-and-paste" mechanism results in intrachromosomal deletion. Thus, CSR juxtaposes a previously distal constant region to the V region, allowing a "switch" of the class (or isotype) of the expressed immunoglobulin without altering its antigen specificity (1, 2).Both SHM and CSR require activation-induced cytidine deaminase (AID) (3, 4), which deaminates DNA cytosines to uracils at transcribed V and S regions (1, 2, 5). Uracils in DNA can be detected and repaired by either the uracil glycosylase (UNG2)-dependent base excision repair (BER) pathway or by the MSH2-dependent mismatch repair (MMR) pathway. Indeed, deletion of either UNG2 or MSH2 reduces CSR efficiency (6, 7) and deletion of both completely abolishes CSR (8). However, in contrast to the normal high-fidelity repair that restores DNA to its original state, processing of AID-generated uracils leads to mutations in the V region during SHM and DNA double-strand breaks in switch regions during CSR. It is therefore unclear whether the BER and MMR factors that are involved in SHM and CSR have been usurped to function differently (without their characteristic high fidelity) in germinal center B cells or whether perhaps only some of the components in either pathway are utilized.A notic...

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Section: Discussionmentioning

confidence: 99%

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Masani

Han

2013

Molecular and Cellular Biology

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“…AID only deaminates C's when these are located in single-stranded DNA (4,25,44). CSR at the downstream switch regions occurs within the switch repetitive regions, and recom-bination at S can sometimes occur upstream (35%) or downstream (8%) of the S switch repeats (8,(21)(22)(23). Given that AID requires single-stranded DNA, a key question concerns how any single strandedness is exposed within the switch regions (33).…”

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confidence: 99%

Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences

Roy

Lieber

2008

Molecular and Cellular Biology

134

136

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R-loops have been described in vivo at the immunoglobulin class switch sequences and at prokaryotic and mitochondrial origins of replication. However, the biochemical mechanism and determinants of R-loop formation are unclear. We find that R-loop formation is nearly eliminated when RNase T 1 is added during transcription but not when it is added afterward. Hence, rather than forming simply as an extension of the RNA-DNA hybrid of normal transcription, the RNA must exit the RNA polymerase and compete with the nontemplate DNA strand for an R-loop to form. R-loops persist even when transcription is done in Li ؉ or Cs ؉ , which do not support G-quartet formation. Hence, R-loop formation does not rely on G-quartet formation. R-loop formation efficiency decreases as the number of switch repeats is decreased, although a very low level of R-loop formation occurs at even one 49-bp switch repeat. R-loop formation decreases sharply as G clustering is reduced, even when G density is kept constant. The critical level for R-loop formation is approximately the same point to which evolution drove the G clustering and G density on the nontemplate strand of mammalian switch regions. This provides an independent basis for concluding that the primary function of G clustering, in the context of high G density, is R-loop formation.R-loops are nucleic acid structures in which an RNA strand displaces one strand of DNA for a limited length in an otherwise duplex DNA molecule. R-loops were named by analogy to D-loops, which is where all three strands are DNA. R-loops form in vivo at sequences that generate a G-rich transcript at the prokaryotic origins of replication (20), mitochondrial origins of replication (18), and mammalian immunoglobulin (Ig) class switch sequences (reviewed in reference 45).

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“…S tandem repeats that are rich in RGYW/WRCY undergo breakage and mediate CSR independently of Msh2. In contrast, DNA breakage in sequences flanking the S tandem repeats requires Msh2 to allow recombinational joining and CSR to unfold (68,69), implicating a more significant role of MMR proteins in S-S DNA recombination events involving sequences with scarcity of RGYW/ WRCY (68). Thus, Mlh3 deficiency shifts the S␥ breakpoints of S-S␥ to RGYW/WRCY, implicating a significantly role of Mlh3 in facilitating S-S recombination events outside RGYW/WRCY.…”

Section: Discussionmentioning

confidence: 95%

A Role for the MutL Mismatch Repair Mlh3 Protein in Immunoglobulin Class Switch DNA Recombination and Somatic Hypermutation

Tsai

Patam

et al. 2006

The Journal of Immunology

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Class switch DNA recombination (CSR) and somatic hypermutation (SHM) are central to the maturation of the Ab response. Both processes involve DNA mismatch repair (MMR). MMR proteins are recruited to dU:dG mispairs generated by activation-induced cytidine deaminase-mediated deamination of dC residues, thereby promoting S-S region synapses and introduction of mismatches (mutations). The MutL homolog Mlh3 is the last complement of the mammalian set of MMR proteins. It is highly conserved in evolution and is essential to meiosis and microsatellite stability. We used the recently generated knockout mlh3−/− mice to address the role of Mlh3 in CSR and SHM. We found that Mlh3 deficiency alters both CSR and SHM. mlh3−/− B cells switched in vitro to IgG and IgA but displayed preferential targeting of the RGYW/WRCY (R = A or G, Y = C or T, W = A or T) motif by Sγ1 and Sγ3 breakpoints and introduced more insertions and fewer donor/acceptor microhomologies in Sμ-Sγ1 and Sμ-Sγ3 DNA junctions, as compared with mlh3+/+ B cells. mlh3−/− mice showed only a slight decrease in the frequency of mutations in the intronic DNA downstream of the rearranged JH4 gene. However, the residual mutations were altered in spectrum. They comprised a decreased proportion of mutations at dA/dT and showed preferential RGYW/WRCY targeting by mutations at dC/dG. Thus, the MMR Mlh3 protein plays a role in both CSR and SHM.

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Shifts in targeting of class switch recombination sites in mice that lack μ switch region tandem repeats or Msh2

Cited by 31 publications

References 28 publications

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences

A Role for the MutL Mismatch Repair Mlh3 Protein in Immunoglobulin Class Switch DNA Recombination and Somatic Hypermutation

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