scholarly journals The Transcription Elongation Complex Directs Activation-Induced Cytidine Deaminase-Mediated DNA Deamination

2006 ◽  
Vol 26 (11) ◽  
pp. 4378-4385 ◽  
Author(s):  
Eva Besmer ◽  
Eleonora Market ◽  
F. Nina Papavasiliou
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Elongation Complex ◽  
Single Stranded Dna ◽  
Molecular Features ◽  
Activation Induced Cytidine Deaminase ◽  
Dna Deamination ◽  
Dna Substrate

ABSTRACT Activation-induced cytidine deaminase (AID) is a single-stranded DNA deaminase required for somatic hypermutation of immunoglobulin (Ig) genes, a key process in the development of adaptive immunity. Transcription provides a single-stranded DNA substrate for AID, both in vivo and in vitro. We present here an assay which can faithfully replicate all of the molecular features of the initiation of hypermutation of Ig genes in vivo. In this assay, which detects AID-mediated deamination in the context of transcription by Escherichia coli RNA polymerase, deamination targets either strand and declines in efficiency as the distance from the promoter increases. We show that AID binds DNA exposed by the transcribing polymerase, implicating the polymerase itself as the vehicle which distributes AID on DNA as it moves away from the promoter.

scholarly journals The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

2009 ◽  
Vol 206 (5) ◽  
pp. 1057-1071 ◽  
Author(s):  
Hong Ming Shen ◽  
Michael G. Poirier ◽  
Michael J. Allen ◽  
Justin North ◽  
Ratnesh Lal ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Naked Dna ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Dna Strands ◽  
Ampicillin Resistance Gene ◽  
Cytidine Deamination

The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes.

scholarly journals MSH2–MSH6 stimulates DNA polymerase η, suggesting a role for A:T mutations in antibody genes

2005 ◽  
Vol 201 (4) ◽  
pp. 637-645 ◽  
Author(s):  
Teresa M. Wilson ◽  
Alexandra Vaisman ◽  
Stella A. Martomo ◽  
Patsa Sullivan ◽  
Li Lan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Base Excision Repair ◽  
Somatic Hypermutation ◽  
Excision Repair ◽  
Cytidine Deaminase ◽  
Abasic Site ◽  
Cell Extracts ◽  
Activation Induced Cytidine Deaminase ◽  
Base Excision

Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2–MSH6 mismatch repair heterodimer and DNA polymerase (pol) η, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2–MSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol η in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2–MSH6 stimulates the catalytic activity of pol η in vitro. These observations suggest that the interaction between MSH2–MSH6 and DNA pol η stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs.

scholarly journals Single-Stranded DNA Structure and Positional Context of the Target Cytidine Determine the Enzymatic Efficiency of AID

2007 ◽  
Vol 27 (23) ◽  
pp. 8038-8048 ◽  
Author(s):  
Mani Larijani ◽  
Alberto Martin
Keyword(s):  
Target Genes ◽  
Cytidine Deaminase ◽  
Single Stranded Dna ◽  
Stem Loop ◽  
Double Stranded Dna ◽  
Antibody Diversification ◽  
Multiple Structures ◽  
Optimal Target

ABSTRACT Activation-induced cytidine deaminase (AID) initiates antibody diversification processes by deaminating immunoglobulin sequences. Since transcription of target genes is required for deamination in vivo and AID exclusively mutates single-stranded DNA (ssDNA) in vitro, AID has been postulated to mutate transcription bubbles. However, since ssDNA generated by transcription can assume multiple structures, it is unknown which of these are targeted in vivo. Here we examine the enzymatic and binding properties of AID for different DNA structures. We report that AID has minimal activity on stem-loop structures and preferentially deaminates five-nucleotide bubbles. We compared AID activity on cytidines placed at various distances from the single-stranded/double-stranded DNA junction of bubble substrates and found that the optimal target consists of a single-stranded NWRCN motif. We also show that high-affinity binding is required for but does not necessarily lead to efficient deamination. Using nucleotide analogues, we show that AID's WRC preference (W = A or T; R = A or G) involves the recognition of a purine in the R position and that the carbonyl or amino side chains of guanosine negatively influence specificity at the W position. Our results indicate that AID is likely to target short-tract regions of ssDNA produced by transcription elongation and that it requires a fully single-stranded WRC motif.

High Expression of Activation-Induced Cytidine Deaminase and in Vivo Class Switch Recombination in Mantle Cell Lymphoma: Further Support for Antigen Involvement in Lymphomagenesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1538-1538
Author(s):  
Aliki Xochelli ◽  
Fotini Marantidou ◽  
Evangelia Stalika ◽  
Lesley-Ann Sutton ◽  
Alba Navarro ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Splice Variants ◽  
Cytidine Deaminase ◽  
Gene Repertoire ◽  
Transcript Levels ◽  
Class Switch ◽  
Ighv Gene ◽  
Activation Induced Cytidine Deaminase ◽  
Mantle Cell

Abstract Abstract 1538 According to the WHO 2008 Classification, the cellular origin of mantle cell lymphoma (MCL) is traced to a peripheral B cell of the inner mantle zone, mostly of naïve pre-germinal center type. This notion, however, is seriously challenged by both the remarkable restrictions of the immunoglobulin gene repertoire in MCL and, furthermore, by the fact that the great majority of cases exhibit imprints of somatic hypermutation (SHM) in rearranged IGHV genes, ranging from (mostly) minimal to pronounced. These findings support an antigen-driven origin for MCL, at least for a substantial fraction of the entire cohort. Activation-induced cytidine deaminase (AID) is induced in B cells following contact with antigen and is critically implicated in both somatic hypermutation (SHM) and class switch recombination (CSR). Although the available information about AID expression and in vivo CSR in MCL is limited and contradictory, at least some MCL cases have been reported to express AID and undergo ongoing CSR. With this in mind, here we investigated AID-mRNA isoform expression and isotype switch events in a large series of MCL cases and explored possible associations with IGHV gene repertoire and SHM status. Overall, 107 cases were included in the study and tumor-involved diagnostic tissue samples of different types were evaluated, including: fresh-frozen lymph nodes (LN, n=53), peripheral blood (PB, n=42), spleen (n=5), bone marrow biopsies (n=3) and other (n=4). The neoplastic lymphocytic infiltration ranged from 52–98% (median 80%). Thirty-five of 107 cases (32.7%) carried IGHV genes with 100% identity to the germline (GI) whereas the remaining 72 cases bore some imprint of SHM: in particular, 48/107 cases (44.9%) carried IGHV genes with 97–99.9% GI and, finally, 24/107 cases (22.4%) carried IGHV genes with <97% GI. In keeping with the literature, the IGHV gene repertoire of the present cohort was remarkably biased, with the IGHV3–21, IGHV4–34, IGHV3–23 and IGHV1–8 genes accounting for 55.1% of cases. Profiling of AID mRNA expression was performed by RQ-PCR for the full-length AID (AID-FL) as well as the most frequent splice variants, namely AID-ΔE4a (lacking the first 30 nucleotides from exon 4), and AID-ΔE4 (loss of the entire exon 4). AID transcript levels were calculated as the percentage of AID copy number divided by the copy number of the reference transcript (c-ABL). AID-FL transcripts were detected in 104/107 (97%) cases whereas the AID-ΔE4a and AID-ΔE4 splice variants were detected in 72/107 (67.3%) and 107/107 cases (100%), respectively. The median values for AID-FL, AID-ΔE4a and AID-ΔE4 transcripts were 4.45%, 0.133% and 0.918%, respectively. AID transcript levels varied between different cases by up to 5-log for AID-FL transcripts and 4-log for splice variants. Not unexpectedly, the median transcript levels in LN samples were higher (up to 1-log) compared to PB samples. A highly significant (p<0.001) association was noted between medium-to-high AID-FL transcript levels (AID-FL/ABL○1%) and IGHV GI 100%. Given the difference in tissue origin of our samples, we also performed a separate analysis for LN samples only and found that cases with 100% IGHV GI expressed high AID-FL transcript levels (AID-FL/ABL○10%) significantly (p=0.04) more frequently than cases carrying mutated IGHV genes. Isotype switch events were investigated in 41 cases: overall, 4 cases (9.7%), all with GI<100%, carried alternative tumor-derived Cγ (n=1) or Cα (n=3) transcripts. In conclusion, the present analysis documents AID expression in the vast majority of MCL, thus corroborating our previous hypothesis for antigen involvement in MCL ontogeny. Ongoing CSR events appear to be a feature of MCL, further supporting an activated status, at least for subset of cases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID

2010 ◽  
Vol 207 (1) ◽  
pp. 141-153 ◽  
Author(s):  
Meng Wang ◽  
Cristina Rada ◽  
Michael S. Neuberger
Keyword(s):  
Active Site ◽  
Somatic Hypermutation ◽  
Affinity Maturation ◽  
Mutation Spectrum ◽  
Nucleotide Substitutions ◽  
Local Sequence ◽  
Mutational Hotspots ◽  
Dna Deamination

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5′-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID’s active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine→pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID’s active site and DNA local sequence environment in determining in vivo hotspot dominance.

scholarly journals Strand-biased defect in C/G transversions in hypermutating immunoglobulin genes in Rev1-deficient mice

2006 ◽  
Vol 203 (2) ◽  
pp. 319-323 ◽  
Author(s):  
Jacob G. Jansen ◽  
Petra Langerak ◽  
Anastasia Tsaalbi-Shtylik ◽  
Paul van den Berk ◽  
Heinz Jacobs ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Translesion Synthesis ◽  
Uracil Dna Glycosylase ◽  
Abasic Sites ◽  
Activation Induced Cytidine Deaminase ◽  
Deficient Mice

Somatic hypermutation of Ig genes enables B cells of the germinal center to generate high-affinity immunoglobulin variants. Key intermediates in somatic hypermutation are deoxyuridine lesions, introduced by activation-induced cytidine deaminase. These lesions can be processed further to abasic sites by uracil DNA glycosylase. Mutagenic replication of deoxyuridine, or of its abasic derivative, by translesion synthesis polymerases is hypothesized to underlie somatic hypermutation. Rev1 is a translesion synthesis polymerase that in vitro incorporates uniquely deoxycytidine opposite deoxyuridine and abasic residues. To investigate a role of Rev1 in mammalian somatic hypermutation we have generated mice deficient for Rev1. Although Rev1−/− mice display transient growth retardation, proliferation of Rev1−/− LPS-stimulated B cells is indistinguishable from wild-type cells. In mutated Ig genes from Rev1−/− mice, C to G transversions were virtually absent in the nontranscribed (coding) strand and reduced in the transcribed strand. This defect is associated with an increase of A to T, C to A, and T to C substitutions. These results indicate that Rev1 incorporates deoxycytidine residues, most likely opposite abasic nucleotides, during somatic hypermutation. In addition, loss of Rev1 causes compensatory increase in mutagenesis by other translesion synthesis polymerases.

Activation-induced cytidine deaminase in chronic lymphocytic leukemia B cells: expression as multiple forms in a dynamic, variably sized fraction of the clone

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3333-3339 ◽  
Author(s):  
Emilia Albesiano ◽  
Bradley T. Messmer ◽  
Rajendra N. Damle ◽  
Steven L. Allen ◽  
Kanti R. Rai ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Somatic Hypermutation ◽  
Splice Variants ◽  
Cytidine Deaminase ◽  
Prognostic Indicator ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Activation Induced Cytidine Deaminase

AbstractThe degree of somatic mutation of immunoglobulin variable (Ig V) region genes is an important prognostic indicator of clinical course and outcome in B-cell chronic lymphocytic leukemia (B-CLL), although the reason for this association remains unclear. Furthermore, some B-CLL cells continue to acquire Ig V gene mutations after the transforming event. Because activation-induced cytidine deaminase (AID) is an essential component of the canonical somatic hypermutation process in healthy B cells, its expression in B-CLL is potentially relevant to the disease. We detected full-length AID transcripts and 3 splice variants by conventional reverse transcription polymerase chain reaction (RT-PCR) in approximately 40% of the cases examined. More sensitive real-time quantitative PCR detected AID transcripts in virtually all B-CLL samples tested, although the range of transcript levels was very large between different cases and varied within individual cases over time. Limiting dilution assays revealed that AID expression was restricted to a small fraction of the leukemic cells in the blood. However, this small fraction is not unique in its ability to express AID, because in vitro stimulation of B-CLL cells with appropriate stimuli significantly increased the fraction of AID-expressing cells. These data suggest that AID-mediated DNA alterations may occur in a variably sized, minor subset of B-CLL cells at any given time.

Primary cutaneous follicle center lymphoma and primary cutaneous large B-cell lymphoma, leg type, are both targeted by aberrant somatic hypermutation but demonstrate differential expression of AID

Blood ◽  
2006 ◽  
Vol 107 (12) ◽  
pp. 4926-4929 ◽  
Author(s):  
Remco Dijkman ◽  
Cornelis P. Tensen ◽  
Maike Buettner ◽  
Gerald Niedobitek ◽  
Rein Willemze ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
B Cell Lymphoma ◽  
Molecular Features ◽  
Large B Cell Lymphoma ◽  
Activation Induced Cytidine Deaminase ◽  
Single Base Pair ◽  
Large B Cell

AbstractWe assessed primary cutaneous large B-cell lymphoma, leg type (PCLBCL, leg type; n = 13), and primary cutaneous follicle center lymphoma (PCFCL; n = 19) for somatic hypermutation (SHM) of BCL6, and aberrant SHM of MYC, RhoH/TTF, and PAX5. We demonstrate SHM of BCL6 in 8 PCLBCLs (62%), leg type, and 7 PCFCL patients (37%), and aberrant SHM in PAX5, RhoH/TTF, and/or MYC in 7 PCLBCLs (54%), leg type, and 10 PCFCL patients (53%). The majority of mutations consisted of single base-pair substitutions (n = 54) with rare deletions/insertions (n = 4), and displayed molecular features typical of the SHM process. Quantitative real-time PCR and immunohistochemical stainings for activation-induced cytidine deaminase, which is indispensable for SHM, demonstrated significantly higher expression in PCLBCL, leg type. Our results suggest that (aberrant) SHM may contribute to the pathogenesis of PCLBCL, leg type, and PCFCL and is not restricted to diffuse large B-cell lymphomas with an aggressive clinical behavior.

scholarly journals Recombinase-Mediated Cassette Exchange as a Novel Method To Study Somatic Hypermutation in Ramos Cells

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Linda B. Baughn ◽  
Susan L. Kalis ◽  
Thomas MacCarthy ◽  
Lirong Wei ◽  
Manxia Fan ◽  
...  
Keyword(s):  
Heavy Chain ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Antibody Diversity ◽  
Human Antibodies ◽  
Cassette Exchange ◽  
Activation Induced Cytidine Deaminase ◽  
Highly Expressed Genes ◽  
V Region

ABSTRACT Activation-induced cytidine deaminase (AID) mediates the somatic hypermutation (SHM) of immunoglobulin (Ig) variable (V) regions that is required for the generation of antibody diversity and for the affinity maturation of the antibody response against infectious agents and toxic substances. AID preferentially targets WRC (W = A/T, R = A/G) hot spot motifs, particularly WGCW motifs that create overlapping hot spots on both strands. In order to gain a better understanding of the generation of antibody diversity and to create a platform for the in vitro generation of affinity-matured antibodies, we have established a system involving recombinase-mediated cassette exchange (RMCE) to replace the V region and its flanking sequences. This makes it possible to easily manipulate the sequence of the Ig gene within the endogenous heavy chain of the Ramos human Burkitt’s lymphoma cell line. Here we show that the newly integrated wild-type (WT) VH regions introduced by RMCE undergo SHM similarly to non-RMCE-modified Ramos cells. Most importantly, we have shown that introducing a cluster of WGCW motifs into the complementary determining region 2 (CDR2) of the human heavy chain V region significantly raised the mutation frequency and number of mutations per sequence compared to WT controls. Thus, we have demonstrated a novel platform in Ramos cells whereby we can easily and quickly manipulate the endogenous human VH region to further explore the regulation and targeting of SHM. This platform will be useful for generating human antibodies with changes in affinity and specificity in vitro. IMPORTANCE An effective immune response requires a highly diverse repertoire of affinity-matured antibodies. Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Although a great deal has been learned about the regulation of AID, it remains unclear how it is preferentially targeted to particular motifs, to certain locations within the Ig gene and not to other highly expressed genes in the germinal center B cell. This is an important question because AID is highly mutagenic and is sometimes mistargeted to other highly expressed genes, including proto-oncogenes, leading to B cell lymphomas. Here we describe how we utilize recombinase-mediated cassette exchange (RMCE) to modify the sequence of the endogenous heavy chain locus in the Ramos Burkitt’s lymphoma cell line. This platform can be used to explore the regulation and targeting of SHM and to generate human antibodies with changes in affinity and specificity in vitro.

scholarly journals Human PMS2 deficiency is associated with impaired immunoglobulin class switch recombination

2008 ◽  
Vol 205 (11) ◽  
pp. 2465-2472 ◽  
Author(s):  
Sophie Péron ◽  
Ayse Metin ◽  
Pauline Gardès ◽  
Marie-Alexandra Alyanakian ◽  
Eamonn Sheridan ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Class Switch ◽  
Double Strand Dna Breaks ◽  
Switch Regions

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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