Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences

Roy, Deepankar; Yu, Kefei; Lieber, Michael R.

doi:10.1128/mcb.01251-07

Cited by 134 publications

(142 citation statements)

References 48 publications

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“…A likely candidate for this structure is an extended R loop, in which the nascent RNA rehybridizes with the template DNA strand behind the elongating RNAP. Indeed, evidence for the formation of R loops was obtained for transcripts containing G stretches, i.e., when extrastable RNA/DNA duplex was formed (11). R-loop formation could exacerbate transcriptional blockage by preventing the rewinding of a DNA duplex upstream of the RNA polymerase, which contributes to transcription elongation (29,30).…”

Section: Mechanism Of Transcription Blockage By G N ∕C N Sequences Anmentioning

confidence: 99%

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Mechanisms and implications of transcription blockage by guanine-rich DNA sequences

Belotserkovskii

Liu

Tornaletti

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

135

143

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Various DNA sequences that interfere with transcription due to their unusual structural properties have been implicated in the regulation of gene expression and with genomic instability. An important example is sequences containing G-rich homopurinehomopyrimidine stretches, for which unusual transcriptional behavior is implicated in regulation of immunogenesis and in other processes such as genomic translocations and telomere function. To elucidate the mechanism of the effect of these sequences on transcription we have studied T7 RNA polymerase transcription of G-rich sequences in vitro. We have shown that these sequences produce significant transcription blockage in an orientation-, lengthand supercoiling-dependent manner. Based upon the effects of various sequence modifications, solution conditions, and ribonucleotide substitutions, we conclude that transcription blockage is due to formation of unusually stable RNA/DNA hybrids, which could be further exacerbated by triplex formation. These structures are likely responsible for transcription-dependent replication blockage by G-rich sequences in vivo.R-loops | DNA supercoiling | Hoogsteen base pairing | inosine | 7-deazaquanosine S equence-specific modulation of transcription, including transcription blockage or impediment, plays an important role in DNA transactions, for example, transcription-related mutagenesis and recombination (reviewed in refs. 1 and 2) and could also be responsible for several severe genetic diseases (reviewed in refs. 3-5).Among the DNA sequences that could affect transcription are GC-rich homopurine-homopyrimidine (hPu/hPy) stretches. These sequences could form unusual DNA structures, including triplexes and G quadruplexes (reviewed in refs. 3-5), which have been implicated in several transcription-dependent phenomena (for example, see refs. 6-9).Another important property of these sequences is a dramatic asymmetry in the stabilities of RNA/DNA duplexes: The rPu/dPy duplex is significantly more stable, whereas the rPy/dPu duplex is less stable than a DNA/DNA duplex of the same sequence (10). The increased stability of rPu/dPy duplexes is likely responsible for stable R-loop formation by these sequences (11), although alternative DNA structures might also be involved (8,12,13).The simplest example of GC-rich hPu/hPy sequences, the G n ∕C n repeats, is abundant in various genomes, including transcribed domains (14, 15).The G 32 ∕C 32 stretch was previously shown to stall DNA replication in Escherichia coli plasmids in vivo (16). Remarkably, this effect was observed only when the sequence was transcribed, which led to a model stipulating that this sequence stalled an elongating RNA polymerase, and the stalled transcription complex, in turn, blocked the replication machinery (16).To elucidate the mechanism of transcription blockage by this sequence, we have studied its effect on T7 RNA polymerase (T7 RNAP) transcription in vitro, using various sequence modifications and solution conditions that allowed us to discriminate between possible D...

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Section: Mechanism Of Transcription Blockage By G N ∕C N Sequences Anmentioning

confidence: 99%

“…The increased stability of rPu/dPy duplexes is likely responsible for stable R-loop formation by these sequences (11), although alternative DNA structures might also be involved (8,12,13).…”

mentioning

confidence: 99%

Mechanisms and implications of transcription blockage by guanine-rich DNA sequences

Belotserkovskii

Liu

Tornaletti

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

135

143

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“…It has been hypothesized that switch regions have evolved to contain frequent AGCT sequences, which contains WRC hotspots on both strands, to promote a high level of deamination (22). These potential hotspots are interspersed in otherwise G-rich clusters in the template strands that have been demonstrated to promote R-loop formation, exposing a single-stranded DNA substrate (44,45).…”

Section: Loop Graft Variants Effectively Restrict Hiv-bhagwat and Co-mentioning

confidence: 99%

Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification

Kohli

Maul

Guminski

et al. 2010

Journal of Biological Chemistry

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Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W ‫؍‬ A/T, R ‫؍‬ A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

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“…To explore the mechanism by which AID-mediated Top1 reduction augments S region cleavage and CSR, we speculated that aberrant supercoiling by Top1 protein reduction may induce non-B DNA structure in the S region so that Top1 cannot religate, resulting in irreversible cleavage by Top1. DNA structural alterations can be estimated by the DNA's sensitivity to bisulfite modification from dC to dU, which occurs in the single-stranded regions (6,7). The presence of consecutive C bases is suitable for determining the single-strandedness by the bisulfite assay (7).…”

Section: Top1mentioning

confidence: 99%

“…The S region transcripts seem to remain on the DNA template for a short period, thus facilitating the formation of R-loop. Lieber and colleagues reported that the bisulfite treatment of nuclear DNA from switch-induced spleen cells converted dC to dU in the S region, indicating the presence of single stranded DNA in the S region in activated B cells (6,7). R-loop may not survive long but lead to the formation of non-B structures including stem-loop, cruciform, and triplex in the S region because of abundant inverted repeats (3,(8)(9)(10).…”

mentioning

confidence: 99%

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

Kobayashi

Aida

Nagaoka

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5 to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the S region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.camptothecin ͉ non-B DNA ͉ translation suppression

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Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences

Cited by 134 publications

References 48 publications

Mechanisms and implications of transcription blockage by guanine-rich DNA sequences

Mechanisms and implications of transcription blockage by guanine-rich DNA sequences

Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

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