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 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 Activation-induced Deaminase Functions in a Postcleavage Step of the Somatic Hypermutation Process

2002 ◽  
Vol 195 (9) ◽  
pp. 1193-1198 ◽  
Author(s):  
F. Nina Papavasiliou ◽  
David G. Schatz
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Dna Lesions ◽  
Dominant Negative ◽  
Constant Region ◽  
Region Gene ◽  
Variable Regions ◽  
Class Switch ◽  
Activation Induced Deaminase

Activation of B cells by antigen fuels two distinct molecular modifications of immunoglobulin (Ig) genes. Class-switch recombination (CSR) replaces the Igμ heavy chain constant region with a downstream constant region gene, thereby altering the effector function of the resulting antibodies. Somatic hypermutation (SHM) introduces point mutations into the variable regions of Ig genes, thereby changing the affinity of antibody for antigen. Mechanistic overlap between the two reactions has been suggested by the finding that both require the activation-induced cytidine deaminase (AID). It has been proposed that AID initiates both CSR and SHM by activating a common nuclease. Here we provide evidence that cells lacking AID, or expressing a dominant negative form of the protein, are still able to incur DNA lesions in SHM target sequences. The results indicate that an intact cytidine deaminase motif is required for AID function, and that AID acts downstream of the initial DNA lesions in SHM.

scholarly journals Genetic loss of the ubiquitin ligase RNF126, an AID interacting partner and modifier, affects strand targeting during somatic hypermutation of antibody genes

2018 ◽  
Author(s):  
Nicholas Economos ◽  
Rebecca K Delker ◽  
Pete Stavropoulos ◽  
F. Nina Papavasiliou
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Point Mutations ◽  
Variable Region ◽  
Dna Breaks ◽  
Switch Region ◽  
Post Translational Modifications ◽  
Class Switch ◽  
Double Stranded Dna ◽  
Activation Induced Cytidine Deaminase

AbstractActivation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) in B lymphocytes by catalyzing the introduction of deoxyuracil: deoxyguanine mismatches into the DNA of the transcribed Ig locus. Repair pathways then process these mismatches to produce point mutations in the Ig variable region or double-stranded DNA breaks in the switch region followed by deletional recombination. It has been suggested that post-translational modifications on AID mediate a number of these different decisions, ranging from global targeting (Ig vs the genome), local targeting (variable vs switch region; transcribed vs non-transcribed strand) as well as process-appropriate DNA repair. Here we demonstrate that absence of RNF126, an E3 ligase shown to mono-ubiquitylate AID, results in a specific strand targeting defect in SHM, producing substantial G>C bias; strickingly, loss of RNF126 was also associated with tandem indels within the variable region (JH4 intron) but only a slight increase in the types of chromosomal translocations that are characteristic of deregulated AID. Conversely, these findings suggest that mono-ubiquitination of AID, likely in situ, is necessary for the proper removal of the protein from the non-transcribed strand, thus producing both optimal patterns of SHM and also limiting the number of indels within the target locus.

scholarly journals MBD4 Facilitates Immunoglobulin Class Switch Recombination

2016 ◽  
Vol 37 (2) ◽  
Author(s):  
Fernando Grigera ◽  
Robert Wuerffel ◽  
Amy L. Kenter
Keyword(s):  
Excision Repair ◽  
Cytidine Deaminase ◽  
Ectopic Expression ◽  
Class Switch Recombination ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Base Excision

ABSTRACT Immunoglobulin heavy chain class switch recombination (CSR) requires targeted formation of DNA double-strand breaks (DSBs) in repetitive switch region elements followed by ligation between distal breaks. The introduction of DSBs is initiated by activation-induced cytidine deaminase (AID) and requires base excision repair (BER) and mismatch repair (MMR). The BER enzyme methyl-CpG binding domain protein 4 (MBD4) has been linked to the MMR pathway through its interaction with MutL homologue 1 (MLH1). We find that when Mbd4 exons 6 to 8 are deleted in a switching B cell line, DSB formation is severely reduced and CSR frequency is impaired. Impaired CSR can be rescued by ectopic expression of Mbd4. Mbd4 deficiency yields a deficit in DNA end processing similar to that found in MutS homologue 2 (Msh2)- and Mlh1-deficient B cells. We demonstrate that microhomology-rich S-S junctions are enriched in cells in which Mbd4 is deleted. Our studies suggest that Mbd4 is a component of MMR-directed DNA end processing.

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.

Molecular analysis of a large cohort of patients with the hyper immunoglobulin M (IgM) syndrome

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 1881-1890 ◽  
Author(s):  
Wen-I Lee ◽  
Troy R. Torgerson ◽  
Michael J. Schumacher ◽  
Leman Yel ◽  
Qili Zhu ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Cd40 Ligand ◽  
Immunoglobulin M ◽  
Uracil Dna Glycosylase ◽  
Molecular Defects ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Cd40 Activation ◽  
Common Variable

AbstractThe hyper immunoglobulin M (IgM) syndrome (HIGM), characterized by recurrent infections, low serum IgG and IgA, normal or elevated IgM, and defective class switch recombination and somatic hypermutation, is a heterogenous disorder with at least 5 distinct molecular defects, including mutations of the genes coding for the CD40 ligand (CD40L) and IKK-gamma (NEMO) genes, both X-linked; and mutations of CD40, activation-induced cytidine deaminase (AICDA), and uracil-DNA glycosylase (UNG), associated with autosomal recessive HIGM syndromes. To investigate the molecular basis of HIGM, we determined the prevalence of mutations affecting these 5 genes in a cohort of 140 patients (130 males and 10 females). Those patients without a molecular diagnosis were subsequently evaluated for mutations of the following genes: inducible CO-stimulator molecule (ICOS), ICOS ligand (ICOSL), and if male, Bruton tyrosine kinase (Btk) and SLAM-associated protein (SAP/SH2D1A). We found mutations of CD40L in 98 males; AICDA in 4 patients (3 males, 1 female); UNG in one adult male; and Btk in 3 boys. Of the remaining 25 males, one infant with hypohidrotic ectodermal dysplasia had a mutation of NEMO. None of the remaining 33 patients (24 males/9 females) had mutations affecting CD40, ICOS, ICOSL, or SH2D1, and are best classified as common variable immune deficiency (CVID), although other genes, including some not yet identified, may be responsible.

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