Single-Stranded DNA Breaks Adjacent to Cytosines Occur during Ig Gene Class Switch Recombination

Arudchandran, Arulvathani; Bernstein, Ralph M.; Max, Edward E.

doi:10.4049/jimmunol.173.5.3223

Cited by 22 publications

(21 citation statements)

References 37 publications

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“…LM-PCR has been previously used to detect endogenous singleand double-strand breaks in Ig genes from B cells (33)(34)(35). In this study, the breaks were exogenously created at sites of dU by UNG/ APE1, and then analyzed by LM-PCR.…”

Section: Discussionmentioning

confidence: 99%

Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

Martomo

Yang

et al. 2005

The Journal of Immunology

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Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and class switch recombination of Ig genes in B cells. Although AID has been shown to deaminate deoxycytidine to deoxyuridine in DNA in vitro, there is no physical evidence for increased uracils in DNA from cells expressing AID in vivo. We used several techniques to detect uracil bases in a gene that was actively transcribed in Escherichia coli cells expressing AID. Plasmid DNA containing the gene was digested with uracil-DNA glycosylase to remove uracil, and apurinic/apryimidinic endonuclease to nick the abasic site. The nicked DNA was first analyzed using alkaline gel electrophoresis, in which there was a 2-fold increase in the linear form of the plasmid after AID induction compared with plasmid from noninduced bacteria. Second, using a quantitative denaturing Southern blot technique, the gene was predominantly nicked in the nontranscribed strand compared with the transcribed strand. Third, using ligation-mediated PCR, the nicks were mapped on the nontranscribed strand and were located primarily at cytosine bases. These data present direct evidence for the presence of uracils in DNA from cells that are induced to express AID, and they are preferentially generated at cytosines in the nontranscribed strand during transcription.

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Section: Discussionmentioning

confidence: 99%

Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

Martomo

Yang

et al. 2005

The Journal of Immunology

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“…Blunt-ended broken DNA can be ligated to the blunt end of the adaptor directly, while staggered broken DNA ends can be converted to blunt ends by T4 DNA polymerase before ligation. The staggered end of the adaptor and single-stranded DNA breaks are not expected to be ligated in this assay [53]. Following induction of TbTIF2 RNAi for 24 h, we Figure 6, the Ethidium bromide (EtBr)-stained LMPCR products are shown at the top, the Southern blot result is shown in the middle, and the PCR products using primers specific to TbRAP1 are shown at the bottom as a loading control.…”

Section: Depletion Of Tbtif2 Increased Dsbs In Subtelomeric Bessmentioning

confidence: 99%

Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity

Jehi

2014

Cell Res

122

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Subtelomeres consist of sequences adjacent to telomeres and contain genes involved in important cellular functions, as subtelomere instability is associated with several human diseases. Balancing between subtelomere stability and plasticity is particularly important for Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. T. brucei regularly switches its major variant surface antigen, variant surface glycoprotein (VSG), to evade the host immune response, and VSGs are expressed exclusively from subtelomeres in a strictly monoallelic fashion. Telomere proteins are important for protecting chromosome ends from illegitimate DNA processes. However, whether they contribute to subtelomere integrity and stability has not been well studied. We have identified a novel T. brucei telomere protein, T. brucei TRF-Interacting Factor 2 (TbTIF2), as a functional homolog of mammalian TIN2. A transient depletion of TbTIF2 led to an elevated VSG switching frequency and an increased amount of DNA double-strand breaks (DSBs) in both active and silent subtelomeric bloodstream form expression sites (BESs). Therefore, TbTIF2 plays an important role in VSG switching regulation and is important for subtelomere integrity and stability. TbTIF2 depletion increased the association of TbRAD51 with the telomeric and subtelomeric chromatin, and TbRAD51 deletion further increased subtelomeric DSBs in TbTIF2-depleted cells, suggesting that TbRAD51-mediated DSB repair is the underlying mechanism of subsequent VSG switching. Surprisingly, significantly more TbRAD51 associated with the active BES than with the silent BESs upon TbTIF2 depletion, and TbRAD51 deletion induced much more DSBs in the active BES than in the silent BESs in TbTIF2-depleted cells, suggesting that TbRAD51 preferentially repairs DSBs in the active BES.

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“…The requirement for at least part of the non-homologous endjoining double-strand break DNA repair machinery in CSR suggests that these abasic sites lead to breaks that trigger the switch reaction [15][16][17]. Indeed, AID-dependent DNA breaks have been extensively documented for the switch regions [17][18][19][20][21][22]. In SHM, the resolution of the AID-generated uracil in the DNA is less clear.…”

Section: Introductionmentioning

confidence: 99%

Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA

et al. 2008

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Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate nonhomologous recombination in the case of class-switch recombination. In vitro studies have demonstrated that AID deaminates single-stranded but not double-stranded substrates unless AID is in a complex with RPA and the substrate is actively undergoing transcription. However, it is not clear whether AID deaminates its substrates primarily as a monomer or as a higher order oligomer. To examine the oligomerization state of AID alone and in the presence of single stranded DNA substrates of various structures, including loops embedded in double-stranded DNA, we used atomic force microscopy (AFM) to visualize AID protein alone or in complex with DNA. Surprisingly, AFM results indicate that most AID molecules exist as a monomer and that it binds single-stranded DNA substrates as a monomer at concentrations where efficient deamination of single-stranded DNA substrates occur. The rate of deamination, under conditions of excess and limiting protein, also imply that AID can deaminate single-stranded substrates as a monomer. These results imply that nonphosphorylated AID is catalytically active as a monomer on single stranded DNA in vitro, including single-stranded DNA found in loops similar to those transiently formed in the immunoglobulin switch regions during transcription.

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Single-Stranded DNA Breaks Adjacent to Cytosines Occur during Ig Gene Class Switch Recombination

Cited by 22 publications

References 37 publications

Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

Trypanosoma brucei TIF2 suppresses VSG switching by maintaining subtelomere integrity

Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA

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