scholarly journals APE1- and APE2-dependent DNA breaks in immunoglobulin class switch recombination

2007 ◽  
Vol 204 (12) ◽  
pp. 3017-3026 ◽  
Author(s):  
Jeroen E.J. Guikema ◽  
Erin K. Linehan ◽  
Daisuke Tsuchimoto ◽  
Yusaku Nakabeppu ◽  
Phyllis R. Strauss ◽  
...  
Keyword(s):  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Uracil Dna Glycosylase ◽  
Cytosine Deamination ◽  
Abasic Sites ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Antibody Class

Antibody class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded breaks (DSBs) in switch-region DNA. The initial steps in DSB formation have been elucidated, involving cytosine deamination by activation-induced cytidine deaminase and generation of abasic sites by uracil DNA glycosylase. However, it is not known how abasic sites are converted into single-stranded breaks and, subsequently, DSBs. Apurinic/apyrimidinic endonuclease (APE) efficiently nicks DNA at abasic sites, but it is unknown whether APE participates in CSR. We address the roles of the two major mammalian APEs, APE1 and APE2, in CSR. APE1 deficiency causes embryonic lethality in mice; we therefore examined CSR and DSBs in mice deficient in APE2 and haploinsufficient for APE1. We show that both APE1 and APE2 function in CSR, resulting in the DSBs necessary for CSR and thereby describing a novel in vivo function for APE2.

scholarly journals The roles of APE1, APE2, DNA polymerase β and mismatch repair in creating S region DNA breaks during antibody class switch

2008 ◽  
Vol 364 (1517) ◽  
pp. 645-652 ◽  
Author(s):  
Carol E Schrader ◽  
Jeroen E.J Guikema ◽  
Xiaoming Wu ◽  
Janet Stavnezer
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Cytidine Deaminase ◽  
Dna Breaks ◽  
Dna Polymerase Β ◽  
Cytosine Deamination ◽  
Abasic Sites ◽  
Class Switch ◽  
Single Strand Breaks ◽  
Antibody Class

Immunoglobulin class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded DNA breaks (DSBs) in immunoglobulin switch region DNA. The initial steps of DSB formation have been elucidated: cytosine deamination by activation-induced cytidine deaminase (AID) and the generation of abasic sites by uracil-DNA glycosylase (UNG). We show that abasic sites are converted into single-strand breaks (SSBs) by apurinic/apyrimidinic endonucleases (APE1 and APE2). If SSBs are near to each other on opposite strands, they will generate DSBs; but if distal from each other, mismatch repair appears to be required to generate DSBs. The resulting S region DSBs occur at dC residues that are preferentially targeted by AID. We also investigate whether DNA polymerase β, which correctly repairs SSBs resulting from APE activity, attempts to repair the breaks during CSR. We find that although polymerase β does attempt to repair S region DNA breaks in switching B cells, the frequency of AID-instigated breaks appears to outnumber the SSBs repaired correctly by polymerase β, and thus some DSBs and mutations are generated. We also show that the S region DSBs are introduced and resolved during the G1 phase of the cell cycle.

scholarly journals Regulation of class switch recombination and somatic mutation by AID phosphorylation

2008 ◽  
Vol 205 (11) ◽  
pp. 2585-2594 ◽  
Author(s):  
Kevin M. McBride ◽  
Anna Gazumyan ◽  
Eileen M. Woo ◽  
Tanja A. Schwickert ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Somatic Mutation ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Class Switch Recombination ◽  
Variable Region ◽  
Dna Breaks ◽  
Class Switching ◽  
Class Switch

Activation-induced cytidine deaminase (AID) is a mutator enzyme that initiates somatic mutation and class switch recombination in B lymphocytes by introducing uracil:guanine mismatches into DNA. Repair pathways process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region DNA. However, AID can also produce off-target DNA damage, including mutations in oncogenes. Therefore, stringent regulation of AID is required for maintaining genomic stability during maturation of the antibody response. It has been proposed that AID phosphorylation at serine 38 (S38) regulates its activity, but this has not been tested in vivo. Using a combination of mass spectrometry and immunochemical approaches, we found that in addition to S38, AID is also phosphorylated at position threonine 140 (T140). Mutation of either S38 or T140 to alanine does not impact catalytic activity, but interferes with class switching and somatic hypermutation in vivo. This effect is particularly pronounced in haploinsufficient mice where AID levels are limited. Although S38 is equally important for both processes, T140 phosphorylation preferentially affects somatic mutation, suggesting that posttranslational modification might contribute to the choice between hypermutation and class switching.

scholarly journals Inducible DNA breaks in Ig S regions are dependent on AID and UNG

2005 ◽  
Vol 202 (4) ◽  
pp. 561-568 ◽  
Author(s):  
Carol E. Schrader ◽  
Erin K. Linehan ◽  
Sofia N. Mochegova ◽  
Robert T. Woodland ◽  
Janet Stavnezer
Keyword(s):  
Cytidine Deaminase ◽  
Constant Region ◽  
Dna Breaks ◽  
Region Gene ◽  
Uracil Dna Glycosylase ◽  
Strand Breaks ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Constant Region Gene ◽  
Dna Strands

Class switch recombination (CSR) occurs by an intrachromosomal deletion whereby the IgM constant region gene (Cμ) is replaced by a downstream constant region gene. This unique recombination event involves formation of double-strand breaks (DSBs) in immunoglobulin switch (S) regions, and requires activation-induced cytidine deaminase (AID), which converts cytosines to uracils. Repair of the uracils is proposed to lead to DNA breaks required for recombination. Uracil DNA glycosylase (UNG) is required for most CSR activity although its role is disputed. Here we use ligation-mediated PCR to detect DSBs in S regions in splenic B cells undergoing CSR. We find that the kinetics of DSB induction corresponds with AID expression, and that DSBs are AID- and UNG-dependent and occur preferentially at G:C basepairs in WRC/GYW AID hotspots. Our results indicate that AID attacks cytosines on both DNA strands, and staggered breaks are processed to blunt DSBs at the initiating ss break sites. We propose a model to explain the types of end-processing events observed.

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.

scholarly journals Disparate roles of ATR and ATM in immunoglobulin class switch recombination and somatic hypermutation

2006 ◽  
Vol 203 (1) ◽  
pp. 99-110 ◽  
Author(s):  
Qiang Pan-Hammarström ◽  
Aleksi Lähdesmäki ◽  
Yaofeng Zhao ◽  
Likun Du ◽  
Zhihui Zhao ◽  
...  
Keyword(s):  
Ataxia Telangiectasia ◽  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
End Joining ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Variable Heavy

Class switch recombination (CSR) and somatic hypermutation (SHM) are mechanistically related processes initiated by activation-induced cytidine deaminase. Here, we have studied the role of ataxia telangiectasia and Rad3-related protein (ATR) in CSR by analyzing the recombinational junctions, resulting from in vivo switching, in cells from patients with mutations in the ATR gene. The proportion of cells that have switched to immunoglobulin (Ig)A and IgG in the peripheral blood seems to be normal in ATR-deficient (ATRD) patients and the recombined S regions show a normal “blunt end-joining,” but impaired end joining with partially complementary (1–3 bp) DNA ends. There was also an increased usage of microhomology at the μ-α switch junctions, but only up to 9 bp, suggesting that the end-joining pathway requiring longer microhomologies (≥10 bp) may be ATR dependent. The SHM pattern in the Ig variable heavy chain genes is altered, with fewer mutations occurring at A and more mutations at T residues and thus a loss of strand bias in targeting A/T pairs within certain hotspots. These data suggest that the role of ATR is partially overlapping with that of ataxia telangiectasia–mutated protein, but that the former is also endowed with unique functional properties in the repair processes during CSR and SHM.

scholarly journals Epigenetic tethering of AID to the donor switch region during immunoglobulin class switch recombination

2011 ◽  
Vol 208 (8) ◽  
pp. 1649-1660 ◽  
Author(s):  
Beena Patricia Jeevan-Raj ◽  
Isabelle Robert ◽  
Vincent Heyer ◽  
Adeline Page ◽  
Jing H. Wang ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Epigenetic Modifications ◽  
Dna Breaks ◽  
Switch Region ◽  
Heterochromatin Protein 1 ◽  
Immunoglobulin Class ◽  
Class Switch

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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