Activation‐induced cytidine deaminase induces DNA break repair events more frequently in the Ig switch region than other sites in the mammalian genome

Lee, Shauna A.; Parsa, Jahan-Yar; Martín, Alberto; Baker, Mark D.

doi:10.1002/eji.200737654

Cited by 1 publication

(3 citation statements)

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“…We previously showed that exogenous plasmid DNA incorporates into sites of dsDNA breaks caused by AID in hybridomas. 30 Using this same assay, we found approximately 7-fold higher levels of incorporation of the neomycin resistance plasmid in AID-high Ramos 7 and 80 clones 33 compared with the AID-negative Ramos 1 clone (Figure 6C), suggesting that the AID-high clones have more dsDNA breaks in their genome. In (A) Mice were immunized with NP-CGG and 11 days later were harvested to assess caspase levels in the GC via flow cytometry.…”

Section: Aid Levels Correlate With Apoptosis and Dsdna-break Formatiomentioning

confidence: 81%

“…Cells were grown in Iscove modified Dulbecco medium (Invitrogen) and 10% bovine calf serum (HyClone). Western blots for AID expression were performed as previously described 30 with mouse anti-human AID (Cell Signaling Technology) and rabbit polyclonal anti-␤ actin (Abcam) as per the manufacturer's instructions. A naturally low-expressing AID clone of Ramos (R1) was electroporated with 10 g linearized pCEP4-AID in Iscove modified Dulbecco medium at 250 V, 960 F, plated into 96-well plates, and selected for transfectants using 0.8 mg/mL hygromycin B to express human AID in this cell line.…”

Section: In Vitro Cell Culturementioning

confidence: 99%

“…Double-stranded DNA (dsDNA) breaks were assayed using a plasmid integration assay as previously described 30 using electroporation conditions as previously described. 8 Transient transfection efficiencies were calculated by electroporation of Ramos cells with the pmaxGFP plasmid (Amaxa) and analyzed by flow cytometry 24 hours later.…”

Section: Dsdna Break Analysesmentioning

confidence: 99%

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AID constrains germinal center size by rendering B cells susceptible to apoptosis

Zaheen

Boulianne

Parsa

et al. 2009

Blood

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IntroductionA fundamental hallmark of humoral immunity is the ability to produce class-switched antibodies that have enhanced affinity for antigen. This process, termed affinity maturation, allows clonally selected B cells to refine their response to theoretically target any antigen with high specificity. After activation, B cells mutate the immunoglobulin (Ig) locus in a process known as somatic hypermutation (SHM). 1 Mutated clones that have acquired increased affinity for antigen preferentially expand over their low-affinity counterparts. The selection process occurs in the germinal center (GC), a transient microenvironment that forms in secondary lymphoid tissue shortly after antigen exposure. 2 The GC provides a competitive setting for B cells whereby ineffectual clones are actively cleared from the system via apoptosis, although the processes that govern this selection still remain vague.SHM and class switch recombination (CSR) are initiated by the enzyme activation-induced cytidine deaminase (AID), 3,4 which deaminates cytidines to uridines specifically at the Ig locus. 5-10 AID-generated uridines are then engaged by various DNA repair pathways that either lead to the generation of point mutations in the antibody variable region or recombinogenic events that lead to CSR. AID Ϫ/Ϫ mice lack mutations at their Ig locus and are incapable of producing class-switched antibodies. 4 Interestingly, these mice were previously shown to harbor an abnormally high proportion of splenic GC B cells. 11 This phenotype is also recapitulated in humans with AID deficiencies. 3 Although a link between reduced gut immunity (resulting from an inability to produce mucosal IgA) and peripheral GC formation was hypothesized to account for these abnormalities, this remains to be conclusively proven, and the possibility of a B cell-intrinsic effect that could explain the profound expansion of GC B cells has not been examined.GC B cells represent a unique lymphoid compartment of actively proliferating cells where numerous apoptotic factors must synergize to induce the elimination of nonproductive clones. Factors contributing to the intrinsic pathway of apoptosis, such as Bcl-2, Bcl-x L , and Bim, 12-17 and the extrinsic pathway, such as Fas, [18][19][20][21][22] have been implicated in GC selection. The unique physiology of these cells makes them highly susceptible to disease progression, often serving as etiologic sites for autoimmune and malignant B cells. 13,[23][24][25][26] Recent evidence has implicated AID as a fundamental contributor to the genetic aberrations that lead to these disease phenotypes. 24,[27][28][29] Given the importance of apoptosis as a parameter for both GC B-cell selection and lymphomagenesis, we investigated the relationship between AID-induced DNA mutation and cell death to understand how this enzyme may impinge on survival and death within the GC niche. Here we report that many of the potentially harmful AID-induced genetic alterations may indeed lead to the death of these cells within the GC. Methods Mice...

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Section: Aid Levels Correlate With Apoptosis and Dsdna-break Formatiomentioning

confidence: 81%

Section: In Vitro Cell Culturementioning

confidence: 99%