Induction of homologous recombination between sequence repeats by the activation induced cytidine deaminase (AID) protein.

Jean-Marie Buerstedde,David G Schatz,Noel Lowndes

doi:10.7554/elife.03110

Abstract

The activation induced cytidine deaminase (AID) protein is known to initiate somatic hypermutation, gene conversion or switch recombination by cytidine deamination within the immunoglobulin loci. Using chromosomally integrated fluorescence reporter transgenes, we demonstrate a new recombinogenic activity of AID leading to intra- and intergenic deletions via homologous recombination of sequence repeats. Repeat recombination occurs at high frequencies even when the homologous sequences are hundreds of bases away from the positions of AID-mediated cytidine deamination, suggesting DNA end resection before strand invasion. Analysis of recombinants between homeologous repeats yielded evidence for heteroduplex formation and preferential migration of the Holliday junctions to the boundaries of sequence homology. These findings broaden the target and off-target mutagenic potential of AID and establish a novel system to study induced homologous recombination in vertebrate cells.DOI: http://dx.doi.org/10.7554/eLife.03110.001.

Highlights

The activation induced cytidine deaminase (AID) is essential for all types of B cell-specific immunoglobulin (Ig) gene diversification—somatic hypermutation (SH), gene conversion (GC), and class switch recombination (CSR) (Muramatsu et al, 2000; Revy et al, 2000; Arakawa et al, 2002; Harris et al, 2002)
Red fluorescence loss was stimulated 10- to 30-fold in the presence of the human Ig lambda enhancer Diversification Activator (DIVAC) (Buerstedde et al, 2014). These results, combined with our previous extensive analyses of green fluorescence protein (GFP)-based reporter constructs (Blagodatski et al, 2009; Buerstedde et al, 2014), suggested that the loss of red fluorescence was due to inactivation of the red fluorescence protein (RFP) transgenes by AID dependent, DIVAC-stimulated hypermutation events
Ten sequences showed uniform deletions of one RSV repeat and the intervening sequence with no other sequence changes as expected for faithful recombination of repeats (RR) events. These results demonstrate that DT40 and CH12 cells carried out both RSV and switch region recombination, but the absolute and relative frequencies of switch recombination were about 5 and 30-fold higher, respectively, in induced CH12 cells

Summary

Introduction

The activation induced cytidine deaminase (AID) is essential for all types of B cell-specific immunoglobulin (Ig) gene diversification—somatic hypermutation (SH), gene conversion (GC), and class switch recombination (CSR) (Muramatsu et al, 2000; Revy et al, 2000; Arakawa et al, 2002; Harris et al, 2002). AID-dependent double-strand breaks—believed to be generated by deamination of nearby cytidines on both strands of sequence repeats within switch regions—have been detected during CSR (Wuerffel et al, 1997; Petersen et al, 2001; Rush et al, 2004; Schrader et al, 2005) These breaks are normally joined by non-homologous end joining leading to the deletion of the intervening DNA sequence, but erroneous repair may lead to chromosomal translocations (reviewed by Boboila et al, 2012). It is uncertain, whether double-strand breaks routinely accompany SH, since breaks within hypermutating V segments were subsequently found to be AID independent (Papavasiliou and Schatz, 2002; Bross and Jacobs, 2003). It remains unclear whether GC is initiated by a double strand-break or by a single-strand nick (Yabuki et al, 2005; Nakahara et al, 2009)

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