Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4^−/− cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.DNA double-strand breaks (DSBs) are one of the most severe forms of DNA damage that can result from pathological conditions such as replication stress, exposure to ionizing radiation (IR), free radicals, or other DNA-damaging drugs or because of failed single-strand break repair (SSBR) (1, 2). In developing lymphocytes, programmed DSBs are essential intermediates for antigen receptor gene rearrangements, including V(D)J recombination and Ig heavy chain class switch recombination (CSR) (1, 2). Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are the two major pathways for DSB repair. Whereas HR is restricted to the S/G₂ phase of the cell cycle, NHEJ is active throughout the cell cycle and is generally considered the major pathway for DSB repair in mammals (1, 2).The NHEJ pathway has been extensively studied. The core components include the Ku70/Ku86 heterodimer, DNA-dependent protein kinase, X-ray cross complementation factor 4 (XRCC4), and DNA ligase IV (Lig4) (1, 2). Additional NHEJ factors include the Artemis nuclease, XRCC4-like factor (XLF) (or Cernunnos), Paralog of XRCC4 and XLF, and Polymerases µ and λ. Missing any of these factors results in various degrees of DSB repair deficits that are highly context-dependent. In general, cells lacking core components of NHEJ are hypersensitive to IR and abolished for V(D)J recombination but are only partially defective for CSR and competent for circulation of transfected linearized plasmids, suggesting that there exists an “alternative” way to join at least some types of DSBs. This alternative end-joining (A-EJ) pathway has recently become a focal area of research because of its implications in oncogenic chromosomal translocations (3), which are rare in NHEJ-proficient cells but much more frequent when NHEJ is compromised. Little is known about A-EJ other than it is kinetically slow and uses an increased level of microhomology (nucleotide overlaps that can be assigned to either of the two DNA ends) during joining (2, 4). A number of DNA repair factors, many of which are involved in SSBR, have been implicated in A-EJ (5), but the overall composition of A-EJ remains elusive. It is still unclear whether A-EJ is a distinct pathway, consists of multiple subpathways, or is merely an aberrant form of NHEJ with missing components substituted by compatible but less efficient factors. It is also unclear whether A-EJ contributes to DSB repair in NHEJ-proficient cells at all or is only active when NHEJ is compromised.Much of our understanding of mechanistic details of DSB repair has derived from studies of V(D)J recombination and CSR; both involving DSB intermediates (1). V(D)J recombination is initiated by the recombination-activating genes (RAGs) that bind and cleave at specific DNA sequences flanking the V, D, and J segments to assemble an exon encoding the variable (antigen binding) domain of the B- and T-cell receptors. CSR is initiated by activation-induced cytidine deaminase (AID) in antigen-stimulated B cells that changes the IgH constant (C) region to a different isotype. AID catalyzes DNA cytosine deamination (converting cytosines to uracils) at switch regions preceding each C region (6, 7). Processing of AID-generated uracils, through a mechanism still not fully characterized, leads to DSB formation. Although both processes use NHEJ to join DSBs, in cells missing any of the core components of NHEJ, CSR is only partially defective, whereas V(D)J recombination is completely abolished. It has been reported that the RAG complex holds the four broken ends in a postcleavage complex and directs VDJ-associated DSBs into the NHEJ pathway (8, 9). In contrast, significant levels of CSR can occur in the absence of any core NHEJ factors (10, 11–14), suggesting that switch region breaks are more accessible to alternative DSB repair pathways.Regardless of how broken DNA ends are processed, at least one DNA ligase is required to ligate the two ends. Vertebrates have three ATP-dependent DNA ligases (Lig1, Lig3, and Lig4) (15). Lig1 and Lig4 are conserved in all eukaryotes, whereas Lig3 is only present in vertebrates (15). Lig4 is a core component of the NHEJ pathway and functions exclusively in NHEJ. Cells deficient for Lig4, or its cofactor XRCC4, display the most severe phenotypes of NHEJ deficiency. In the absence of Lig4, A-EJ must rely on Lig1 or Lig3 (or both). It is generally accepted that the major role of Lig1 is to join Okazaki fragments during DNA replication, and this function is mediated by an interaction with the proliferating cell nuclear antigen (PCNA) (16). Lig3 is produced in somatic cells in two forms (mitochondrial and nuclear) via alternative translation initiation (17). It was recently shown that mitochondrial, but not nuclear, Lig3 is essential for cell viability (18, 19). Nuclear Lig3 stably interacts with the X-ray complementation factor 1 (XRCC1), a scaffold protein that is essential for base excision repair. For this reason, Lig3 is regarded as the primary DNA repair ligase for SSBR, although Lig1 has also been implicated in DNA repair (15).We have previously reported the disruption Lig4 and Lig1 in a mouse B-cell line (CH12F3) capable of robust cytokine-induced CSR (10). Lig4^−/− CH12F3 cells undergo kinetically slow but significant levels of CSR [∼50% of wild-type (WT) level at day 3] (10). Switch junctions isolated from Lig4^−/− CH12F3 cells show increased microhomology and no direct joins (10). In the present study, we focused on determining which of two remaining ligases (Lig1 and Lig3) is responsible for A-EJ during CSR in Lig4^−/− cells. To that end, we disrupted Lig1 and Lig3 (nuclear) individually in Lig4^−/− CH12F3 cells. We found that Lig1 and Lig3 have redundant functions in DNA repair in response to a variety of DNA-damaging agents and during repair by A-EJ during CSR.