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Mechanistic Aspects of Lymphoid Chromosomal Translocations

Mechanistic Aspects of Lymphoid Chromosomal Translocations Abstract Chromosomal translocations require double-strand breakage at two sites, followed by joining of the ends. The joining is usually done by nonhomologous DNA end-joining, though homologous recombination and single-strand annealing play roles in cases where there is homology. The mechanism of breakage can be more difficult to understand at sites other than the antigen receptor loci. Some breakage events in pre–B or pre–T cells are due to the RAG proteins recognizing heptamer/nonamer-like sequences, but most breaks are not. Rather, some of these breaks are due to RAG nicking at non–B DNA conformations. Translocation events in mature B cells, when RAGs are not expressed, may be due to the activation-induced deaminase (AID). But AID only acts on single-stranded DNA, and it is not yet clear how this single-stranded DNA arises at some transcribed sites and not others. During the physiologic process of class switch recombination, R-loops form at transcribed class switch regions, thereby accounting for how single strandedness is facilitated at these sites of AID action. The Joining of DNA Ends at Sites of Double-strand Breaks Nonhomologous DNA end-joining (NHEJ) is the most common mechanism for joining double-strand breaks (DSBs) in vertebrate cells. The other pathways for joining DSBs are homologous recombination (HR) and single-strand annealing (SSA). HR is limited to regions with substantial homology (hundreds of base pairs), and the efficiency of HR falls off markedly with increasing distance. Moreover, the DSB must be at the edge of one of the blocks of homology for maximum efficiency. These factors make HR a much less common basis for most translocations. SSA involving long regions of homology has some of the same constraints as HR. SSA involving two DNA ends that share six or more nucleotides of homology (micro-SSA) may sometimes join DNA ends when NHEJ does not. However, even six nucleotides of terminal homology would not be common at two random break sites. NHEJ can join DNA ends with zero nucleotide of terminal homology. When terminal microhomology is present, it can bias the outcome of rejoining to favor use of that microhomlogy (often two to four nucleotides). The use of microhomology in NHEJ does not seem to affect the efficiency of NHEJ. That is, DNA ends with no microhomology are joined as well as those with microhomology (1–3). The first protein to bind at a DSB is thought to be Ku. Ku is so abundant that a DSB at any random location will be no further than approximately four molecular diameters away from Ku. Ku only binds by threading onto a DNA end. Once bound at a DNA end, Ku changes conformation. The Ku:DNA complex binds DNA-PKcs with an affinity that is 100-fold tighter than that of DNA-PKcs binding to a DNA end lacking Ku. DNA-PKcs is a protein kinase that phosphorylates 11 serine and threonine sites in the C-terminal half of a protein called Artemis (4,5). This causes a change in Artemis conformation, thereby allowing it to function as an endonuclease. This activated form of Artemis can trim 5′ overhangs to a blunt configuration and can trim 3′ overhangs such that they have a short (one to four nucleotides) overhang. Activated Artemis can also open DNA hairpins. After DNA-PKcs phosphorylates Artemis, the two proteins bind quite tightly together. DNA-PKcs also phosphorylates itself, and this phosphorylation permits DNA-PKcs to bind less tightly to the DNA end (6). The Ku:DNA complex can not only recruit the nuclease complex for trimming the DNA ends but it can also increase the affinity of the ligase complex for the DNA ends (7,8). The ligase complex is XRCC4:DNA ligase IV. XLF (also called Cernunnos) is a XRCC4-like protein that may also be part of the ligase complex (9,10). The Ku:DNA complex can also recruit the POL X polymerases, pol mu, and pol lambda, via their BRCT domains (11–13). Hence, the Ku:DNA complex can recruit the nuclease, the polymerases, and the ligase to either DNA end for rejoining. The order with which Ku:DNA complexes recruit the components appears to vary, and this almost certainly contributes to the variation seen in the joining of DNA ends by NHEJ (13,14). Translocation junctions often have nucleotide additions (15,16). For translocations that occur in early lymphoid cells, some of these junctional additions can be due to TdT. Additional junctional addition may be due to fold-back, followed by synthesis or related flexibility of the POL X polymerases (14,16). This type of flexibility permits these polymerases to use either of the strands in either of two DNA ends as a template. Chromosomal translocation junctions show evidence of this type of addition, and it has been termed T-nucleotides, where T stands for templated. It is not yet clear if T-nucleotides occur during normal V(D)J recombination. The Role of the RAG Complex in Mediating Lymphoid Chromosomal Translocations The bcl-2 translocation in follicular lymphoma accounts for nearly half of non-Hogkins lymphomas. Most of the breaks at the bcl-2 gene occur within a 150-bp region called the major breakpoint region (Mbr) (17,18). Contemporaneously with the generation of two DNA ends at the Mbr, a RAG complex–dependent D to J recombination event at the IgH locus begins, but the D and J coding ends fail to complete their normal joining. Rather, the two DNA ends at the Mbr break site join with the D and the J coding ends. We recently reproduced some aspects of the translocation on an episome in human 293 cells (and within a human pre–B cell line) (19). The episomes bear the bcl-2 Mbr region, a 12-RSS, and a 23-RSS, where the 12- and 23-RSS are the heptamer/nonamer signals adjacent to which the RAG complex normally cuts. The episomal translocation is RAG dependent within the human 293 cells, and the translocation junctions show T-nucleotides (see above) (19). The breaks at the bcl-2 Mbr on the episome are also RAG dependent. Why would the RAG complex, which is a sequence-specific endonuclease, act at the bcl-2 Mbr? We first considered the possibility that the Mbr might contain heptamer/nonamer-like sequences, sometimes called cryptic or pseudosignals or RSS-like sequences (19–21). Visual inspection did not support this possibility. More importantly, the patient translocations were distributed throughout the Mbr and not at specific locations (17). This lack of focusing was not consistent with the use of a specific heptamer/nonamer-like sequence because these typically show very precise cutting directly adjacent to the pseudosignal (21). Experimentally, we and others had shown that pseudosignals direct RAG cutting at a specific nucleotide directly adjacent to the “heptamer” (usually at least the CAC sequence) of the pseudosignal. Equally important, these pseudosignals can partner with a regular 12- or 23-RSS to carry out V(D)J recombination (20,21). When the 150-bp bcl-2 Mbr was tested for any latent ability to function like a pseudosignal, it exhibited none in our hands or when tested by others (19,20). If the RAG complex does not recognize the primary sequence of the bcl-2 Mbr as a pseudosignal, then why is the cutting at the bcl-2 Mbr RAG dependent? From a different line of work on the bcl-2 Mbr, we had determined that the Mbr frequently deviated from the double-helix conformation in a manner that gives it a substantial single-stranded character. This inference is based on reactivity of the DNA in vitro and in vivo using nucleophiles that can only attack C or T when these bases are unstacked, which means that the bases are not in the double helix (19). Defining conformations of non–B DNA structures is difficult because there are many possibilities. Hence, we are still trying to understand the configuration of these non–B DNA conformations and how long they last in the deviated form before collapsing back into B-form DNA. A triplex conformation is among the possibilities that we are considering, but some models based on slippage between the two strands are alternative possibilities (22). In another line of work on the bcl-2 Mbr, we found that the RAG complex could nick the bcl-2 Mbr (19). More generally, we found that the RAG complex could nick any bubble (heteroduplex) structure (23). This more general structure-specific nuclease action by the RAG complex was consistent with its more limited structure-specific nuclease action on 3′ flap structures or 3′ overhangs, which had been noted earlier (24,25). The ability of the RAG complex to nick bubble structures may explain how the RAG complex might catalyze certain lymphoid chromosomal translocations at regions that do not have RSS-like sequences. We conjecture that the two strands may slip so as to yield small heteroduplexes or possibly longer range non–B DNA conformations, which would then be nicked by the RAG complex (16,26). Why would the RAG complex be able to nick non–B DNA structures? The answer may relate to the normal functioning of the RAGs. The transition from the nicked intermediate to the hairpin product in normal V(D)J recombination almost certainly requires some distortion of the helix because the action requires a 3′-OH on one strand to attack the antiparallel strand, which is 20 angstroms away (27). Therefore, the RAG active site must be prepared to accommodate these deviations in the DNA. Therefore, it appears that the RAG complex can nick double- to single-strand transition regions within DNA, and this probably accounts for RAG action at the non–B DNA conformation at the bcl-2 Mbr. The recapitulation of the bcl-2 translocation on episomes in human cells permitted us to assess the mechanism by which the DNA ends are rejoined. We tested the translocation substrates in a human pre–B cell line that is wild type for NHEJ and in DNA ligase IV null version of the same line. We found that the translocation process is dependent on the presence of DNA ligase IV (28). We also tested whether signal ends and coding ends were equally capable of joining to the breaks at the bcl-2 Mbr (28). We found that signal ends are unable to join to the bcl-2 Mbr broken ends, where as either coding end can join with either end of the Mbr break site (Figure 1). Figure 1 Open in new tabDownload slide Model for the mechanism of the t(14;18) translocation. In the upper left corner, standard V(D)J recombination initiates double-strand breaks at the D and J segments of the IgH locus. The two signal ends are joined, but the two coding ends fail to join. A RAG-dependent break at the bcl-2 major breakpoint region generates two DNA ends that are arbitrarily labeled “a” and “b.” Either the “a” or the “b” end can join to the D or the J coding end. In the chromosome, only the “a” end can join to the J end, and only the “b” end can join to the D end because only this combination provides one centromere for each derivative chromosome. Figure 1 Open in new tabDownload slide Model for the mechanism of the t(14;18) translocation. In the upper left corner, standard V(D)J recombination initiates double-strand breaks at the D and J segments of the IgH locus. The two signal ends are joined, but the two coding ends fail to join. A RAG-dependent break at the bcl-2 major breakpoint region generates two DNA ends that are arbitrarily labeled “a” and “b.” Either the “a” or the “b” end can join to the D or the J coding end. In the chromosome, only the “a” end can join to the J end, and only the “b” end can join to the D end because only this combination provides one centromere for each derivative chromosome. The Role of Activation-Induced Deaminase in Chromosomal Translocations Some chromosomal translocations do not involve the V, D, or J segments but instead involve the class switch recombination (CSR) regions. For example, in sporadic Burkitts lymphoma, the c-myc gene breaks in exon 1 or intron 1, and the two DNA ends are joined to the two DNA ends of a break at the immunoglobulin Sμ switch region (29). This chromosomal translocation occurs in both mice and humans, and it occurs much more efficiently in wild-type mice than in mice whose B cells lack expression of a cytidine deaminase called activation-induced deaminase (AID) (30). AID is a lymphoid-specific cytidine deaminase that is only expressed in activated B cells that already have surface Ig (31). AID is normally required for somatic hypermutation (SHM) and CSR (32). AID only acts on cytidines that are in single-stranded DNA, and it does not act on cytidines in duplex DNA (33–36). A fundamental question concerns how the DNA at these locations becomes single stranded. It is not yet clear how single-stranded regions are exposed in the Ig variable domains to permit SHM. One could argue that all transcription causes transient single strandedness. But this line of reasoning would predict that all transcribed genes in activated B cells would undergo SHM, and this is clearly not the case. For CSR, we have shown that the unique sequence properties of class switch regions permit the formation of R-loops in vivo, and these provide substantial lengths of single strandedness at which AID can act (33,37). We have demonstrated this for chromosomal switch regions at Sγ3 and Sγ2b, and we are in the process of extending these studies to other murine switch regions (38). We have also demonstrated R-loops for mammalian switch regions present on stable episomes maintained in human cells (33). These in vivo findings correlate well with our in vitro findings when we use phage RNA polymerases to transcribe class switch regions on purified DNA templates (33,39,40). In summary, class switch regions in vivo form R-loops when transcription occurs through these regions. For activated B cells, the transcription begins from sterile transcript promoters. If R-loops are responsible for targeting CSR at switch regions, could R-loop formation be occurring at the c-myc exon 1/intron 1 region? There is one study reporting that in vitro transcription of c-myc results in R-loop formation. However, no one has yet reported evidence of R-loops in chromosomal DNA in vivo. It will be interesting to see if there is a structural reason for breakage of the c-myc gene in this region. " This chapter was originally presented in Oct. 2006 at an NCI Workshop. Research in the authors laboratory is supported by NIH. " Present address: Department of Microbiology and Molecular Genetics, Michigan State University, 5175 Biomedical Physical Sciences, East Lansing, MI 48824 (K. Yu). References 1. Gerstein RM , Lieber MR . 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The downstream boundary of chromosomal R-loops at murine switch regions: implications for the mechanism of class switch recombination , Proc Natl Acad Sci , 2006 , vol. 103 (pg. 5030 - 5035 ) Google Scholar Crossref Search ADS PubMed WorldCat 39. Yu K , Roy D , Bayramyan M , Haworth IS , Lieber MR . Fine-structure analysis of activation-induced deaminase accessibility to class switch region R-loops , Mol Cell Biol. , 2005 , vol. 25 (pg. 1730 - 1736 ) Google Scholar Crossref Search ADS PubMed WorldCat 40. Daniels GA , Lieber MR . RNA:DNA complex formation upon transcription of immunoglobulin switch regions: implications for the mechanism and regulation of class switch recombination , Nucl Acids Res. , 1995 , vol. 23 (pg. 5006 - 5011 ) Google Scholar Crossref Search ADS WorldCat Published by Oxford University Press 2008. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JNCI Monographs Oxford University Press

Mechanistic Aspects of Lymphoid Chromosomal Translocations

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Oxford University Press
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Published by Oxford University Press 2008.
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Abstract

Abstract Chromosomal translocations require double-strand breakage at two sites, followed by joining of the ends. The joining is usually done by nonhomologous DNA end-joining, though homologous recombination and single-strand annealing play roles in cases where there is homology. The mechanism of breakage can be more difficult to understand at sites other than the antigen receptor loci. Some breakage events in pre–B or pre–T cells are due to the RAG proteins recognizing heptamer/nonamer-like sequences, but most breaks are not. Rather, some of these breaks are due to RAG nicking at non–B DNA conformations. Translocation events in mature B cells, when RAGs are not expressed, may be due to the activation-induced deaminase (AID). But AID only acts on single-stranded DNA, and it is not yet clear how this single-stranded DNA arises at some transcribed sites and not others. During the physiologic process of class switch recombination, R-loops form at transcribed class switch regions, thereby accounting for how single strandedness is facilitated at these sites of AID action. The Joining of DNA Ends at Sites of Double-strand Breaks Nonhomologous DNA end-joining (NHEJ) is the most common mechanism for joining double-strand breaks (DSBs) in vertebrate cells. The other pathways for joining DSBs are homologous recombination (HR) and single-strand annealing (SSA). HR is limited to regions with substantial homology (hundreds of base pairs), and the efficiency of HR falls off markedly with increasing distance. Moreover, the DSB must be at the edge of one of the blocks of homology for maximum efficiency. These factors make HR a much less common basis for most translocations. SSA involving long regions of homology has some of the same constraints as HR. SSA involving two DNA ends that share six or more nucleotides of homology (micro-SSA) may sometimes join DNA ends when NHEJ does not. However, even six nucleotides of terminal homology would not be common at two random break sites. NHEJ can join DNA ends with zero nucleotide of terminal homology. When terminal microhomology is present, it can bias the outcome of rejoining to favor use of that microhomlogy (often two to four nucleotides). The use of microhomology in NHEJ does not seem to affect the efficiency of NHEJ. That is, DNA ends with no microhomology are joined as well as those with microhomology (1–3). The first protein to bind at a DSB is thought to be Ku. Ku is so abundant that a DSB at any random location will be no further than approximately four molecular diameters away from Ku. Ku only binds by threading onto a DNA end. Once bound at a DNA end, Ku changes conformation. The Ku:DNA complex binds DNA-PKcs with an affinity that is 100-fold tighter than that of DNA-PKcs binding to a DNA end lacking Ku. DNA-PKcs is a protein kinase that phosphorylates 11 serine and threonine sites in the C-terminal half of a protein called Artemis (4,5). This causes a change in Artemis conformation, thereby allowing it to function as an endonuclease. This activated form of Artemis can trim 5′ overhangs to a blunt configuration and can trim 3′ overhangs such that they have a short (one to four nucleotides) overhang. Activated Artemis can also open DNA hairpins. After DNA-PKcs phosphorylates Artemis, the two proteins bind quite tightly together. DNA-PKcs also phosphorylates itself, and this phosphorylation permits DNA-PKcs to bind less tightly to the DNA end (6). The Ku:DNA complex can not only recruit the nuclease complex for trimming the DNA ends but it can also increase the affinity of the ligase complex for the DNA ends (7,8). The ligase complex is XRCC4:DNA ligase IV. XLF (also called Cernunnos) is a XRCC4-like protein that may also be part of the ligase complex (9,10). The Ku:DNA complex can also recruit the POL X polymerases, pol mu, and pol lambda, via their BRCT domains (11–13). Hence, the Ku:DNA complex can recruit the nuclease, the polymerases, and the ligase to either DNA end for rejoining. The order with which Ku:DNA complexes recruit the components appears to vary, and this almost certainly contributes to the variation seen in the joining of DNA ends by NHEJ (13,14). Translocation junctions often have nucleotide additions (15,16). For translocations that occur in early lymphoid cells, some of these junctional additions can be due to TdT. Additional junctional addition may be due to fold-back, followed by synthesis or related flexibility of the POL X polymerases (14,16). This type of flexibility permits these polymerases to use either of the strands in either of two DNA ends as a template. Chromosomal translocation junctions show evidence of this type of addition, and it has been termed T-nucleotides, where T stands for templated. It is not yet clear if T-nucleotides occur during normal V(D)J recombination. The Role of the RAG Complex in Mediating Lymphoid Chromosomal Translocations The bcl-2 translocation in follicular lymphoma accounts for nearly half of non-Hogkins lymphomas. Most of the breaks at the bcl-2 gene occur within a 150-bp region called the major breakpoint region (Mbr) (17,18). Contemporaneously with the generation of two DNA ends at the Mbr, a RAG complex–dependent D to J recombination event at the IgH locus begins, but the D and J coding ends fail to complete their normal joining. Rather, the two DNA ends at the Mbr break site join with the D and the J coding ends. We recently reproduced some aspects of the translocation on an episome in human 293 cells (and within a human pre–B cell line) (19). The episomes bear the bcl-2 Mbr region, a 12-RSS, and a 23-RSS, where the 12- and 23-RSS are the heptamer/nonamer signals adjacent to which the RAG complex normally cuts. The episomal translocation is RAG dependent within the human 293 cells, and the translocation junctions show T-nucleotides (see above) (19). The breaks at the bcl-2 Mbr on the episome are also RAG dependent. Why would the RAG complex, which is a sequence-specific endonuclease, act at the bcl-2 Mbr? We first considered the possibility that the Mbr might contain heptamer/nonamer-like sequences, sometimes called cryptic or pseudosignals or RSS-like sequences (19–21). Visual inspection did not support this possibility. More importantly, the patient translocations were distributed throughout the Mbr and not at specific locations (17). This lack of focusing was not consistent with the use of a specific heptamer/nonamer-like sequence because these typically show very precise cutting directly adjacent to the pseudosignal (21). Experimentally, we and others had shown that pseudosignals direct RAG cutting at a specific nucleotide directly adjacent to the “heptamer” (usually at least the CAC sequence) of the pseudosignal. Equally important, these pseudosignals can partner with a regular 12- or 23-RSS to carry out V(D)J recombination (20,21). When the 150-bp bcl-2 Mbr was tested for any latent ability to function like a pseudosignal, it exhibited none in our hands or when tested by others (19,20). If the RAG complex does not recognize the primary sequence of the bcl-2 Mbr as a pseudosignal, then why is the cutting at the bcl-2 Mbr RAG dependent? From a different line of work on the bcl-2 Mbr, we had determined that the Mbr frequently deviated from the double-helix conformation in a manner that gives it a substantial single-stranded character. This inference is based on reactivity of the DNA in vitro and in vivo using nucleophiles that can only attack C or T when these bases are unstacked, which means that the bases are not in the double helix (19). Defining conformations of non–B DNA structures is difficult because there are many possibilities. Hence, we are still trying to understand the configuration of these non–B DNA conformations and how long they last in the deviated form before collapsing back into B-form DNA. A triplex conformation is among the possibilities that we are considering, but some models based on slippage between the two strands are alternative possibilities (22). In another line of work on the bcl-2 Mbr, we found that the RAG complex could nick the bcl-2 Mbr (19). More generally, we found that the RAG complex could nick any bubble (heteroduplex) structure (23). This more general structure-specific nuclease action by the RAG complex was consistent with its more limited structure-specific nuclease action on 3′ flap structures or 3′ overhangs, which had been noted earlier (24,25). The ability of the RAG complex to nick bubble structures may explain how the RAG complex might catalyze certain lymphoid chromosomal translocations at regions that do not have RSS-like sequences. We conjecture that the two strands may slip so as to yield small heteroduplexes or possibly longer range non–B DNA conformations, which would then be nicked by the RAG complex (16,26). Why would the RAG complex be able to nick non–B DNA structures? The answer may relate to the normal functioning of the RAGs. The transition from the nicked intermediate to the hairpin product in normal V(D)J recombination almost certainly requires some distortion of the helix because the action requires a 3′-OH on one strand to attack the antiparallel strand, which is 20 angstroms away (27). Therefore, the RAG active site must be prepared to accommodate these deviations in the DNA. Therefore, it appears that the RAG complex can nick double- to single-strand transition regions within DNA, and this probably accounts for RAG action at the non–B DNA conformation at the bcl-2 Mbr. The recapitulation of the bcl-2 translocation on episomes in human cells permitted us to assess the mechanism by which the DNA ends are rejoined. We tested the translocation substrates in a human pre–B cell line that is wild type for NHEJ and in DNA ligase IV null version of the same line. We found that the translocation process is dependent on the presence of DNA ligase IV (28). We also tested whether signal ends and coding ends were equally capable of joining to the breaks at the bcl-2 Mbr (28). We found that signal ends are unable to join to the bcl-2 Mbr broken ends, where as either coding end can join with either end of the Mbr break site (Figure 1). Figure 1 Open in new tabDownload slide Model for the mechanism of the t(14;18) translocation. In the upper left corner, standard V(D)J recombination initiates double-strand breaks at the D and J segments of the IgH locus. The two signal ends are joined, but the two coding ends fail to join. A RAG-dependent break at the bcl-2 major breakpoint region generates two DNA ends that are arbitrarily labeled “a” and “b.” Either the “a” or the “b” end can join to the D or the J coding end. In the chromosome, only the “a” end can join to the J end, and only the “b” end can join to the D end because only this combination provides one centromere for each derivative chromosome. Figure 1 Open in new tabDownload slide Model for the mechanism of the t(14;18) translocation. In the upper left corner, standard V(D)J recombination initiates double-strand breaks at the D and J segments of the IgH locus. The two signal ends are joined, but the two coding ends fail to join. A RAG-dependent break at the bcl-2 major breakpoint region generates two DNA ends that are arbitrarily labeled “a” and “b.” Either the “a” or the “b” end can join to the D or the J coding end. In the chromosome, only the “a” end can join to the J end, and only the “b” end can join to the D end because only this combination provides one centromere for each derivative chromosome. The Role of Activation-Induced Deaminase in Chromosomal Translocations Some chromosomal translocations do not involve the V, D, or J segments but instead involve the class switch recombination (CSR) regions. For example, in sporadic Burkitts lymphoma, the c-myc gene breaks in exon 1 or intron 1, and the two DNA ends are joined to the two DNA ends of a break at the immunoglobulin Sμ switch region (29). This chromosomal translocation occurs in both mice and humans, and it occurs much more efficiently in wild-type mice than in mice whose B cells lack expression of a cytidine deaminase called activation-induced deaminase (AID) (30). AID is a lymphoid-specific cytidine deaminase that is only expressed in activated B cells that already have surface Ig (31). AID is normally required for somatic hypermutation (SHM) and CSR (32). AID only acts on cytidines that are in single-stranded DNA, and it does not act on cytidines in duplex DNA (33–36). A fundamental question concerns how the DNA at these locations becomes single stranded. It is not yet clear how single-stranded regions are exposed in the Ig variable domains to permit SHM. One could argue that all transcription causes transient single strandedness. But this line of reasoning would predict that all transcribed genes in activated B cells would undergo SHM, and this is clearly not the case. For CSR, we have shown that the unique sequence properties of class switch regions permit the formation of R-loops in vivo, and these provide substantial lengths of single strandedness at which AID can act (33,37). We have demonstrated this for chromosomal switch regions at Sγ3 and Sγ2b, and we are in the process of extending these studies to other murine switch regions (38). We have also demonstrated R-loops for mammalian switch regions present on stable episomes maintained in human cells (33). These in vivo findings correlate well with our in vitro findings when we use phage RNA polymerases to transcribe class switch regions on purified DNA templates (33,39,40). In summary, class switch regions in vivo form R-loops when transcription occurs through these regions. 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