X-ray repair cross-complementing protein 1 (XRCC1) deficiency enhances class switch recombination and is permissive for alternative end joining

Abstract

DNA double-strand breaks (DSBs) are essential intermediates in Ig gene rearrangements: V(D)J and class switch recombination (CSR). In contrast to V(D)J recombination, which is almost exclusively dependent on nonhomologous end joining (NHEJ), CSR can occur in NHEJ-deficient cells via a poorly understand backup pathway (or pathways) often termed alternative end joining (A-EJ). Recently, several components of the single-strand DNA break (SSB) repair machinery, including XRCC1, have been implicated in A-EJ. To determine its role in A-EJ and CSR, Xrcc1 was deleted by targeted mutation in the CSR proficient mouse B-cell line, CH12F3. Here we demonstrate that XRCC1 deficiency slightly increases the efficiency of CSR. More importantly, Lig4 and XRCC1 double-deficient cells switch as efficiently as Lig4-deficient cells, clearly indicating that XRCC1 is dispensable for A-EJ in CH12F3 cells during CSR.A DNA double-strand break (DSB) is one of the most severe forms of DNA damage and can result in chromosome loss or translocations. A variety of endogenous and exogenous sources can induce DSBs, including ionizing radiation, reactive oxygen species, and some chemicals. On the other hand, physiological processes during lymphocyte development such as V(D)J and Ig class switch recombination (CSR) rely on DSBs to rearrange genetic information in somatic cells.V(D)J recombination is a site-specific DNA recombination initiated by the RAG proteins, which are evolved from an ancient DNA transposase. The RAG complex recognizes specific DNA sequences called recombination signal sequences (RSS) and cuts the DNA on one side of the RSS. The ensuing repair of the four DNA ends that are produced from a pair of cleavage events results in joining of subexonic coding fragments to form an exon encoding the antigen-binding domain of a B-or T-cell receptor. In contrast, CSR in antigen-stimulated mature B cells is a regionally specific recombination between two repetitive regions [called switch (S) regions] that precede each of the constant regions (1). Looping out intervening sequences between two S regions allows the expression of a new constant region that was further downstream and results in a switch of Ig class (or isotype) from IgM to IgG, IgE, or IgA (2). CSR is initiated by activation-induced cytidine deaminase (AID) that converts DNA cytosines into uracils at S regions. Through mechanisms that are not yet fully understood, repair of AID-generated uracils in the S region ultimately results in DSBs (2), which serve as critical intermediates in an overall cut-and-paste chromosomal deletion (2).In vertebrate cells, DSB repair mechanisms generally fall into two major categories: homologous recombination (HR) and nonhomologous end joining (NHEJ) (3). HR relies on the presence of another copy of DNA sequences that are highly similar to the one harboring the DSB. Copying genetic information from the intact copy allows high-fidelity repair of the DSB. In complex genomes rich in repetitive DNA sequences, HR is restricted to S and G2 phase of the ce...

Repair of Double-Strand Breaks by End Joining

“…Studies in mammalian cells have implicated DNA ligase III (Lig3) as the major alt-NHEJ ligase (Audebert et al 2004;Wang et al 2005;Sallmyr et al 2008;Simsek et al 2011;Chiruvella et al 2012). As a cofactor of Lig3, XRCC1 is also suggested to be involved (Audebert et al 2004;Saribasak et al 2011), although more recent studies suggest that XRCC1 may be dispensable (Boboila et al 2012;Han et al 2012). A possible contribution to such discrepancies is that Lig1 (the only other mammalian ligase) has also been shown to support some level of alt-NHEJ (Liang et al 2008;Simsek et al 2011).…”

MBD4 Facilitates Immunoglobulin Class Switch Recombination

“…S4A in the supplemental material). The CH12 cell line can be induced to undergo IgA switching (25) and has been used for targeted gene deletion (26)(27)(28)(29)(30). The 5= and 3= Mbd4 homology arms, of 2.85 and 5.34 kb, respectively, were cloned into the targeting vector pLNTK.…”

Apurinic/Apyrimidinic Endonuclease 1 Is the Essential Nuclease during Immunoglobulin Class Switch Recombination

“…1A) to attempt disruption of the APE1 gene in a mouse B cell line (CH12F3) that is capable of robust cytokine-induced CSR. The gene-targeting strategy utilized has been described previously (21)(22)(23)(24). Gene targeting would delete exon 4 of the APE1 gene, which contains ϳ52% of the coding sequence (Fig.…”

“…Individual IgA-positive clones were isolated by limiting dilutions of cytokine-stimulated cultures in 96-well plates. Switch junctions were amplified with primers KY761 (5=-AACTCTCCA GCCACAGTAATGACC-3=) and KY743 (5=-GAGCTCGTGGGAGTGTC AGTG-3=) as previously described (22)(23)(24). PCR products were sequenced at the Genomic Core Facility at Michigan State University.…”