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Alain Fischer - One of the best experts on this subject based on the ideXlab platform.
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reduced Immunoglobulin Class switch recombination in the absence of artemis
Blood, 2009Co-Authors: Pauline Soulassprauel, Gwenael Le Guyader, Paola Riveramunoz, Vincent Abramowski, Sylvia Bruneau, Alain FischerAbstract:Nonhomologous end-joining DNA repair factors, including Artemis, are all required for the repair of DNA double-strand breaks, which occur during the assembly of the variable antigen recognition domain of B-cell receptors and T-cell receptors through the V(D)J recombination. Mature B cells further shape their Immunoglobulin repertoire on antigen recognition notably through the Class switch recombination (CSR) process. To analyze the role of Artemis during CSR, we developed a mature B-cell-specific Artemis conditional knockout mouse to bypass the absence of B cells caused by its early deficit. Although CSR is not overwhelmingly affected in these mice, Class switching to certain isotypes is clearly reduced both in vitro on B-cell activation and in vivo after keyhole limpet hemocyanin immunization. The reduced CSR in Artemis-deficient B cells is accompanied by the increase in DNA microhomology usage at CSR junctions, the imprint of an alternative DNA end-joining pathway. Likewise, significant increase in DNA microhomology usage is the signature of CSR junctions obtained from human RS-SCID patients harboring hypomorphic Artemis mutations. Altogether, this indicates that Artemis participates in the repair of a subset of DNA breaks generated during CSR.
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Reduced Immunoglobulin Class switch recombination in the absence of Artemis.
Blood, 2009Co-Authors: Paola Rivera-munoz, Alain Fischer, Vincent Abramowski, Pauline Soulas-sprauel, Gwenael Le Guyader, Sylvia Bruneau, Frederic Paques, Jeanpierre De VillartayAbstract:Non homologous end joining (NHEJ) DNA repair factors, including Artemis, are all required for the repair of DNA double strand breaks (dsb), which occur during the assembly of the variable antigen recognition domain of B cell receptors (BCR) and T cell receptors (TCR) through the V(D)J recombination. Mature B cells further shape their Ig repertoire upon antigen recognition notably through the Class switch recombination process (CSR). To analyze the role of Artemis during CSR we developed a mature B cell specific Artemis conditional KO mouse to bypass the absence of B cells caused by its early deficit. Although CSR is not overwhelmingly affected in these mice, Class switching to certain isotypes is clearly reduced both in vitro upon B cell activation and in vivo after KLH immunization. The reduced CSR in Artemis deficient B cells is accompanied by the increase in DNA microhomology usage at CSR junctions, the imprint of an alternative DNA end-joining pathway. Likewise, significant increase in DNA microhomology usage is the signature of CSR junctions obtained from human RS-SCID patients harboring hypomorphic Artemis mutations. Altogether this indicates that Artemis participates in the repair of a subset of DNA breaks generated during CSR.
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human pms2 deficiency is associated with impaired Immunoglobulin Class switch recombination
Journal of Experimental Medicine, 2008Co-Authors: Sophie Peron, Alain Fischer, Ayse Metin, Pauline Gardes, Mariealexandra Alyanakian, Eamonn Sheridan, Christian P KratzAbstract:Immunoglobulin (Ig) Class switch recombination (CSR) deficiencies are rare primary immunodeficiencies characterized by the lack of switched isotype (IgG/IgA/IgE) production. In some cases, CSR deficiencies can be associated with abnormal somatic hypermutation. Analysis of CSR deficiencies has helped reveal the key functions of CSR-triggering molecules, i.e., CD40L, CD40, and effector molecules such as activation-induced cytidine deaminase and uracil N-glycosylase. We report a new form of B cell–intrinsic CSR deficiency found in three patients with deleterious, homozygous mutations in the gene encoding the PMS2 component of the mismatch repair machinery. CSR was found partially defective in vivo and markedly impaired in vitro. It is characterized by the defective occurrence of double-strand DNA breaks (DSBs) in switch regions and abnormal formation of switch junctions. This observation strongly suggests a role for PMS2 in CSR-induced DSB generation.
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pathophysiology of b cell intrinsic Immunoglobulin Class switch recombination deficiencies
Advances in Immunology, 2007Co-Authors: Anne Durandy, Sophie Peron, Nadine Taubenheim, Alain FischerAbstract:B-cell intrinsic Immunoglobulin Class switch recombination (Ig-CSR) deficiencies, previously termed hyper-IgM syndromes, are genetically determined conditions characterized by normal or elevated serum IgM levels and an absence or very low levels of IgG, IgA, and IgE. As a function of the molecular mechanism, the defective CSR is variably associated to a defect in the generation of somatic hypermutations (SHMs) in the Ig variable region. The study of Ig-CSR deficiencies contributed to a better delineation of the mechanisms underlying CSR and SHM, the major events of antigen-triggered antibody maturation. Four Ig-CSR deficiency phenotypes have been so far reported: the description of the activation-induced cytidine deaminase (AID) deficiency (Ig-CSR deficiency 1), caused by recessive mutations of AICDA gene, characterized by a defect in CSR and SHM, clearly established the role of AID in the induction of the Ig gene rearrangements underlying CSR and SHM. A CSR-specific function of AID has, however, been detected by the observation of a selective CSR defect caused by mutations affecting the C-terminus of AID. Ig-CSR deficiency 2 is the consequence of uracil-N-glycosylase (UNG) deficiency. Because UNG, a molecule of the base excision repair machinery, removes uracils from DNA and AID deaminates cytosines into uracils, that observation indicates that the AID-UNG pathway directly targets DNA of switch regions from the Ig heavy-chain locus to induce the CSR process. Ig-CSR deficiencies 3 and 4 are characterized by a selective CSR defect resulting from blocks at distinct steps of CSR. A further understanding of the CSR machinery is expected from their molecular definition.
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human uracil dna glycosylase deficiency associated with profoundly impaired Immunoglobulin Class switch recombination
Nature Immunology, 2003Co-Authors: Kohsuke Imai, Nadia Catalan, Monique Forveille, Bodil Kavli, Patrick Revy, Shigeaki Nonoyama, Geir Slupphaug, Hans E Krokan, Hans D. Ochs, Alain FischerAbstract:Activation-induced cytidine deaminase (AID) is a 'master molecule' in Immunoglobulin (Ig) Class-switch recombination (CSR) and somatic hypermutation (SHM) generation, AID deficiencies are associated with hyper-IgM phenotypes in humans and mice. We show here that recessive mutations of the gene encoding uracil–DNA glycosylase (UNG) are associated with profound impairment in CSR at a DNA precleavage step and with a partial disturbance of the SHM pattern in three patients with hyper-IgM syndrome. Together with the finding that nuclear UNG expression was induced in activated B cells, these data support a model of CSR and SHM in which AID deaminates cytosine into uracil in targeted DNA (Immunoglobulin switch or variable regions), followed by uracil removal by UNG.
Frederick W Alt - One of the best experts on this subject based on the ideXlab platform.
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kinase dependent structural role of dna pkcs during Immunoglobulin Class switch recombination
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Jennifer L Crowe, Frederick W Alt, Zhengping Shao, Xiaobin S Wang, Peichi Wei, Wenxia Jiang, Brian J Lee, Verna M EstesAbstract:The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a Classical nonhomologous end-joining (cNHEJ) factor. Loss of DNA-PKcs diminished mature B cell Class switch recombination (CSR) to other isotypes, but not IgG1. Here, we show that expression of the kinase-dead DNA-PKcs (DNA-PKcsKD/KD) severely compromises CSR to IgG1. High-throughput sequencing analyses of CSR junctions reveal frequent accumulation of nonproductive interchromosomal translocations, inversions, and extensive end resection in DNA-PKcsKD/KD, but not DNA-PKcs−/−, B cells. Meanwhile, the residual joints from DNA-PKcsKD/KD cells and the efficient Sµ-Sγ1 junctions from DNA-PKcs−/− B cells both display similar preferences for small (2–6 nt) microhomologies (MH). In DNA-PKcs−/− cells, Sµ-Sγ1 joints are more resistant to inversions and extensive resection than Sµ-Se and Sµ-Sµ joints, providing a mechanism for the isotype-specific CSR defects. Together, our findings identify a kinase-dependent role of DNA-PKcs in suppressing MH-mediated end joining and a structural role of DNA-PKcs protein in the orientation of CSR.
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epigenetic tethering of aid to the donor switch region during Immunoglobulin Class switch recombination
Journal of Experimental Medicine, 2011Co-Authors: Beena Jeevanraj, Isabelle Robert, Vincent Heyer, Adeline Page, Jing Wang, Florence Cammas, Frederick W Alt, Regine LossonAbstract:Immunoglobulin Class switch recombination (CSR) is initiated by double-stranded DNA breaks (DSBs) in switch regions triggered by activation-induced cytidine deaminase (AID). Although CSR correlates with epigenetic modifications at the IgH locus, the relationship between these modifications and AID remains unknown. In this study, we show that during CSR, AID forms a complex with KAP1 (KRAB domain–associated protein 1) and HP1 (heterochromatin protein 1) that is tethered to the donor switch region (Sμ) bearing H3K9me3 (trimethylated histone H3 at lysine 9) in vivo. Furthermore, in vivo disruption of this complex results in impaired AID recruitment to Sμ, inefficient DSB formation, and a concomitant defect in CSR but not in somatic hypermutation. We propose that KAP1 and HP1 tether AID to H3K9me3 residues at the donor switch region, thus providing a mechanism linking AID to epigenetic modifications during CSR.
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influence of switch region length on Immunoglobulin Class switch recombination
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Ali A Zarrin, Jing Wang, Ming Tian, Tiffany Borjeson, Frederick W AltAbstract:The Class and effector functions of antibodies are modulated through the process of Ig heavy chain Class switch recombination (CSR). CSR occurs between switch (S) regions that lie upstream of the various Ig heavy chain constant region exons. Molecular analyses of S-region functions have been hampered by their large size and repetitive nature. To test potential relationships between S-region size and efficiency of CSR, we generated normal B lymphocytes in which the 12-kb S region flanking the Cγ1 exons (Sγ1) was replaced with synthetic or endogenous S regions of various lengths. Replacement of Sγ1 with 1- and 2-kb synthetic sequences representing the Sγ1 core repeats or a 4-kb portion of the core endogenous Sγ1 region supported CSR frequencies that directly correlated with S-region length. These findings indicate that S-region size is an important factor in determining endogenous CSR efficiency. Moreover, these results also will allow the development of a systematic system to test the function of various S-region motifs by replacing endogenous S regions with synthetic S regions controlled for size effects.
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an evolutionarily conserved target motif for Immunoglobulin Class switch recombination
Nature Immunology, 2004Co-Authors: Ali A Zarrin, Frederick W Alt, Jayanta Chaudhuri, Nicole Stokes, Dhruv Kaushal, Louis Du Pasquier, Ming TianAbstract:Immunoglobulin H Class-switch recombination (CSR) occurs between switch regions and requires transcription and activation-induced cytidine deaminase (AID). Transcription through mammalian switch regions, because of their GC-rich composition, generates stable R-loops, which provide single-stranded DNA substrates for AID. However, we show here that the Xenopus laevis switch region S(mu), which is rich in AT and not prone to form R-loops, can functionally replace a mouse switch region to mediate CSR in vivo. X. laevis S(mu)-mediated CSR occurred mostly in a region of AGCT repeats targeted by the AID-replication protein A complex when transcribed in vitro. We propose that AGCT is a primordial CSR motif that targets AID through a non-R-loop mechanism involving an AID-replication protein A complex.
Bernardo Reinasanmartin - One of the best experts on this subject based on the ideXlab platform.
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the cohesin complex regulates Immunoglobulin Class switch recombination
Journal of Experimental Medicine, 2013Co-Authors: Annesophie Thomasclaudepierre, Isabelle Robert, Vincent Heyer, Adeline Page, Ebe Schiavo, Marjorie Fournier, Bernardo ReinasanmartinAbstract:Immunoglobulin (Ig) Class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to switch regions and by the subsequent generation of double-stranded DNA breaks (DSBs). These DNA breaks are ultimately resolved through the nonhomologous end joining (NHEJ) pathway. We show that during CSR, AID associates with subunits of cohesin, a complex previously implicated in sister chromatid cohesion, DNA repair, and the formation of DNA loops between enhancers and promoters. Furthermore, we implicate the cohesin complex in the mechanism of CSR by showing that cohesin is dynamically recruited to the Sμ-Cμ region of the IgH locus during CSR and that knockdown of cohesin or its regulatory subunits results in impaired CSR and increased usage of microhomology-based end joining.
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parp1 facilitates alternative nhej whereas parp2 suppresses igh c myc translocations during Immunoglobulin Class switch recombination
Journal of Experimental Medicine, 2009Co-Authors: Isabelle Robert, Françoise Dantzer, Bernardo ReinasanmartinAbstract:Immunoglobulin Class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the Immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR.
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enhanced intra switch region recombination during Immunoglobulin Class switch recombination in 53bp1 b cells
European Journal of Immunology, 2007Co-Authors: Junjie Chen, André Nussenzweig, Bernardo Reinasanmartin, Michel C NussenzweigAbstract:Immunoglobulin Class switch recombination (CSR) is initiated by activation-induced cytidine deaminase (AID), an enzyme that deaminates cytidine residues in single-stranded DNA. U:G mismatches created by AID are processed to produce lesions that recruit and activate DNA damage response proteins including Ataxia-telangiectasia mutated (ATM), histone H2AX, Nijmegen breakage syndrome 1 (Nbs1), and p53 binding protein 1 (53BP1). Among these proteins, absence of 53BP1 produces the most severe impairment of Class switching. Here, we demonstrate that AID is targeted normally to switch region DNA and that intra-switch region recombination is enhanced in 53BP1–/– B cells. In addition, Sµ-Sγ1 switch region junctions cloned from 53BP1–/– B cells show unusual insertions suggestive of failed Class switching. Our data are consistent with a role for 53BP1 in stabilizing the synapsis of switch regions during CSR.
Tasuku Honjo - One of the best experts on this subject based on the ideXlab platform.
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Response to Comment on "Uracil DNA Glycosylase Activity Is Dispensable for Immunoglobulin Class Switch"
Science, 2004Co-Authors: Nasim A Begum, Tasuku HonjoAbstract:The major argument presented by Stivers in ([ 1 ][1]) is that the residual uracil (U) removal activities of single mutants, but not double mutants, of uracil DNA glycosylase (UNG) are sufficient to introduce double-strand breaks (DSBs) and induce Class switch recombination (CSR). First, there are no
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uracil dna glycosylase activity is dispensable for Immunoglobulin Class switch
Science, 2004Co-Authors: Nasim A Begum, Naoki Kakazu, Hitoshi Nagaoka, Laurie A Boyer, Detlev Biniszkiewicz, Rudolf Jaenisch, Kazuo Kinoshita, Masamichi Muramatsu, Reiko Shinkura, Tasuku HonjoAbstract:Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step in Immunoglobulin Class switch recombination (CSR). AID is proposed to deaminate cytosine to generate uracil (U) in either mRNA or DNA. In the second instance, DNA cleavage depends on uracil DNA glycosylase (UNG) for removal of U. Using phosphorylated histone γ-H2AX focus formation as a marker of DNA cleavage, we found that the UNG inhibitor Ugi did not inhibit DNA cleavage in Immunoglobulin heavy chain (IgH) locus during CSR, even though Ugi blocked UNG binding to DNA and strongly inhibited CSR. Strikingly, UNG mutants that had lost the capability of removing U rescued CSR in UNG–/– B cells. These results indicate that UNG is involved in the repair step of CSRyet by an unknown mechanism. The dispensability of U removal in the DNA cleavage step of CSR requires a reconsideration of the model of DNA deamination by AID.
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isolation of differentially expressed genes upon Immunoglobulin Class switching by a subtractive hybridization method using uracil dna glycosylase
Nucleic Acids Research, 1998Co-Authors: Manabu Sugai, Shigeru Kondo, Akira Shimizu, Tasuku HonjoAbstract:Immunoglobulin Class switch recombination enables B lymphocytes to sequentially express antibodies that have identical specificities but that differ in Class and effector function. Although several cis elements required for Class switch recombination have been identified, few trans -acting factors which are directly related to Class switching have been found. Previously we have developed an efficient in vitro Class switching system using a cell line, CH12F3-2. To clarify the molecular mechanism of Class switching, we intended to isolate genes induced with interleukin (IL)-4, transforming growth factor (TGF)-beta and CD40L using the in vitro Class switching system. For that purpose, an improved method for making subtracted cDNA libraries, using uracil DNA glycosylase, has been developed. This method can overcome a general problem of subtraction, that rare cDNAs are easily lost. This new subtraction method was applied to the CH12F3-2 switching system to isolate genes induced by stimulations with IL-4, TGF-beta and CD40L, and cDNAs encoding deiodinase 1 and SS32, an alternatively spliced form of the muscle LIM protein, were obtained. Their expression patterns in response to various combinations of stimuli and the time courses of the induction supported the usefulness of this method.
Michael R Lieber - One of the best experts on this subject based on the ideXlab platform.
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current insights into the mechanism of mammalian Immunoglobulin Class switch recombination
Critical Reviews in Biochemistry and Molecular Biology, 2019Co-Authors: Michael R LieberAbstract:Immunoglobulin (Ig) Class switch recombination (CSR) is the gene rearrangement process by which B lymphocytes change the Ig heavy chain constant region to permit a switch of Ig isotype from IgM to ...
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the role of g density in switch region repeats for Immunoglobulin Class switch recombination
Nucleic Acids Research, 2014Co-Authors: Zheng Z Zhang, Chihlin Hsieh, Nicholas R Pannunzio, Michael R LieberAbstract:The boundaries of R-loops are well-documented at Immunoglobulin heavy chain loci in mammalian B cells. Within primary B cells or B cell lines, the upstream boundaries of R-loops typically begin early in the repetitive portion of the switch regions. Most R-loops terminate within the switch repetitive zone, but the remainder can extend a few hundred base pairs further, where G-density on the non-template DNA strand gradually drops to the genome average. Whether the G-density determines how far the R-loops extend is an important question. We previously studied the role of G-clusters in initiating R-loop formation, but we did not examine the role of G-density in permitting the elongation of the R-loop, after it had initiated. Here, we vary the G-density of different portions of the switch region in a murine B cell line. We find that both Class switch recombination (CSR) and R-loop formation decrease significantly when the overall G-density is reduced from 46% to 29%. Short 50 bp insertions with low G-density within switch regions do not appear to affect either CSR or R-loop elongation, whereas a longer (150 bp) insertion impairs both. These results demonstrate that G-density is an important determinant of the length over which mammalian genomic R-loops extend.
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mechanism of r loop formation at Immunoglobulin Class switch sequences
The FASEB Journal, 2008Co-Authors: Deepankar Roy, Michael R LieberAbstract:Received 12 July 2007/Returned for modification 21 August 2007/Accepted 9 October 2007 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 T1 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). In addition, R-loop formation occurs in vivo at some G-rich transcript locations that are distinctly high for mitotic recombination in Saccharomyces cerevisiae, and this high recombination rate is reduced upon overexpression of S. cerevisiae RNase H1 (14). When prokaryotes lack topoisomerase activity, R-loops can form at a wider variety of sequences, and the lethality associated with this can be remedied by overexpression of Escherichia coli RNase H1 (7). In an avian lymphoid cell line, lack of the ASF2/SF2 RNA-binding protein favors R-loop formation at G-rich transcript locations in the genome, and expression of human RNase H1 can abolish the R-loop (19).
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mechanism of r loop formation at Immunoglobulin Class switch sequences
Molecular and Cellular Biology, 2008Co-Authors: Deepankar Roy, Michael R LieberAbstract: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 T1 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.
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r loops at Immunoglobulin Class switch regions in the chromosomes of stimulated b cells
Nature Immunology, 2003Co-Authors: Frederic Chedin, Chihlin Hsieh, Thomas E Wilson, Michael R LieberAbstract:The mechanism responsible for Immunoglobulin Class switch recombination is unknown. Previous work has shown that Class switch sequences have the unusual property of forming RNA-DNA hybrids when transcribed in vitro. Here we show that the RNA-DNA hybrid structure that forms in vitro is an R-loop with a displaced guanine (G)-rich strand that is single-stranded. This R-loop structure exists in vivo in B cells that have been stimulated to transcribe the γ3 or the γ2b switch region. The length of the R-loops can exceed 1 kilobase. We propose that this distinctive DNA structure is important in the Class switch recombination mechanism
