X-ray repair cross-complementing protein 1 (XRCC1) deficiency enhances class switch recombination and is permissive for alternative end joining Li Han1, Weifeng Mao1,2, and Kefei Yu3 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 Edited* by Frederick W. Alt, Howard Hughes Medical Institute, Harvard Medical School, Children’s Hospital, Immune Disease Institute, Boston, MA, and approved February 10, 2012 (received for review December 15, 2011) DNA double-strand breaks (DSBs) are essential intermediates in Ig the DNA end, the DNA-dependent protein kinase (DNA-PKcs) gene rearrangements: V(D)J and class switch recombination (CSR). that regulates end joining by phosphorylating other proteins (in- In contrast to V(D)J recombination, which is almost exclusively de- cluding itself), and the ligase complex containing XLF, XRCC4, pendent on nonhomologous end joining (NHEJ), CSR can occur in and DNA ligase 4. Also involved is a growing list of auxiliary NHEJ-deficient cells via a poorly understand backup pathway (or factors, including end processing nucleases (e.g., Artemis) and pathways) often termed alternative end joining (A-EJ). Recently, polymerases (μ and λ), polynucleotide kinases, 53BP1, and many several components of the single-strand DNA break (SSB) repair DNA damage response proteins (ATM, H2AX, Chk1, etc.). machinery, including XRCC1, have been implicated in A-EJ. To de- Although both V(D)J and class switch recombination rely on termine its role in A-EJ and CSR, Xrcc1 was deleted by targeted the generation and repair of DSBs, the dependence on NHEJ mutation in the CSR proficient mouse B-cell line, CH12F3. Here we is distinctively different between these two reactions. Whereas demonstrate that XRCC1 deficiency slightly increases the efficiency RAG-generated DSBs are almost exclusively joined by NHEJ, S of CSR. More importantly, Lig4 and XRCC1 double-deficient cells region breaks can be joined in NHEJ-deficient cells at a reduced switch as efficiently as Lig4-deficient cells, clearly indicating that but still considerable rate (4–6). DSB repair in the absence of an XRCC1 is dispensable for A-EJ in CH12F3 cells during CSR. intact NHEJ system has been collectively termed alternative end joining (A-EJ) (3, 4). A-EJ could be a component-substitution DNA double-strand break (DSB) is one of the most severe form of NHEJ or a distinct pathway (or pathways) (7–9). So far, Aforms of DNA damage and can result in chromosome loss or components of A-EJ have not been conclusively defined. A-EJ translocations. A variety of endogenous and exogenous sources has attracted much research attention recently because of its can induce DSBs, including ionizing radiation, reactive oxygen implication in chromosomal translocations that could lead to species, and some chemicals. On the other hand, physiological oncogenic transformations (10). Many translocation junctions processes during lymphocyte development such as V(D)J and have microhomology (DNA sequences that can be assigned to Ig class switch recombination (CSR) rely on DSBs to rearrange either of the two ends), which is characteristic of A-EJ. A-EJ is genetic information in somatic cells. sometimes called microhomology-mediated end joining (9). V(D)J recombination is a site-specific DNA recombination However, the presence of microhomology at the junction is not initiated by the RAG proteins, which are evolved from an an- a criterion to distinguish A-EJ from NHEJ, as NHEJ also prefers cient DNA transposase. The RAG complex recognizes specific short homology between the two ends (9, 11, 12). DNA sequences called recombination signal sequences (RSS) The final stage of DSB repair depends on DNA ligases. Ver- and cuts the DNA on one side of the RSS. The ensuing repair of tebrates have three ATP-dependent ligases (I, III, and IV) (13). the four DNA ends that are produced from a pair of cleavage Lig4 appears to be dedicated to NHEJ as no other function has events results in joining of subexonic coding fragments to form been described for Lig4 outside the NHEJ realm. XRCC4 an exon encoding the antigen-binding domain of a B- or T-cell complexes with and stabilizes Lig4. Deficiency of either Lig4 or receptor. In contrast, CSR in antigen-stimulated mature B cells XRCC4 essentially abolishes NHEJ. Conceivably, joining of S is a regionally specific recombination between two repetitive re- region breaks in Lig4-deficient cells must depend on Lig1 and/or gions [called switch (S) regions] that precede each of the con- Lig3. Normally, Lig1 complexes with proliferating cell nuclear stant regions (1). Looping out intervening sequences between antigen and is recruited to the replication fork to join Okazaki two S regions allows the expression of a new constant region that fragments during DNA replication (13). Lig3 complexes with was further downstream and results in a switch of Ig class (or XRCC1 and was generally considered the ligase involved in isotype) from IgM to IgG, IgE, or IgA (2). CSR is initiated by single-strand break (SSB) repair pathways. It has been shown activation-induced cytidine deaminase (AID) that converts DNA that cellular Lig3 activity is dependent on XRCC1 (14), which is cytosines into uracils at S regions. Through mechanisms that are a scaffold protein that interacts with many other DNA repair not yet fully understood, repair of AID-generated uracils in the S factors (e.g., Parp1, Pol β, APE1, PNKP, aprataxin, etc.) (13). region ultimately results in DSBs (2), which serve as critical inter- Although the traditional view of Lig3 in nuclear DNA repair has mediates 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 Author contributions: K.Y. designed research; L.H. and W.M. performed research; L.H., nonhomologous end joining (NHEJ) (3). HR relies on the pres- W.M., and K.Y. analyzed data; and K.Y. wrote the paper. ence of another copy of DNA sequences that are highly similar to The authors declare no conflict of interest. the one harboring the DSB. Copying genetic information from the *This Direct Submission article had a prearranged editor. intact copy allows high-fidelity repair of the DSB. In complex 1L.H. and W.M. contributed equally to this work. genomes rich in repetitive DNA sequences, HR is restricted to S 2Present address: Department of Biotechnology, Dalian Medical University, Dalian and G2 phase of the cell cycle when sister chromatids are available 116044, China. as a source of homology. In contrast, NHEJ is the major DSB 3To whom correspondence should be addressed. E-mail: [email protected]. repair pathway that operates throughout the cell cycle. The core This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NHEJ components include the Ku70/86 heterodimer that binds to 1073/pnas.1120743109/-/DCSupplemental. 4604–4608 | PNAS | March 20, 2012 | vol. 109 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1120743109 Downloaded by guest on September 26, 2021 been recently challenged (15, 16), the importance of XRCC1 in from XRCC1+/+ cells, consistent with the lack of effect hap- SSB repair has been well established. lodeficiency has on XRCC1 expression levels in these cells. Because A-EJ relies on microhomology and because both Lig1 and Lig3 are SSB ligases, we considered the possibility that A-EJ Slightly Increased CSR in XRCC1-Deficient Cells. To determine results from a pair of XRCC1-dependent SSB ligations at DNA whether XRCC1 is involved in CSR, cell growth and CSR effi- ciency were compared between XRCC1 proficient (+/Δ) and ends containing long nucleotide overlaps. In addition, XRCC1 Δ Δ deficient (Δ/Δ) cells. XRCC1 / cells grow more slowly than wild- has recently been implicated in A-EJ (17, 18), along with other +/Δ SSB repair factors (19). To assess the role of XRCC1 in CSR and type or XRCC1 cells, as assessed by live cell counting, re- fi gardless of the presence or absence of cytokine stimulation (Fig. A-EJ, we deleted Xrcc1 in wild-type and Lig4-de cient CH12F3 Δ/Δ cells by targeted mutation. Here we demonstrate that XRCC1 2A). We considered that the apparent slow growth of XRCC1 fi fi cells might be attributable to increased cell death because of the de ciency alone slightly enhances the ef ciency of CSR. More Δ/Δ importantly, deletion of both Lig4 and Xrcc1 genesdoesnot inability of XRCC1 cells to repair oxidation-associated DNA fi damage. However, shifting cells from 20% oxygen to 3% only abolish CSR. In fact, cells de cient in both XRCC1 and Lig4 Δ/Δ switch as efficiently, or slightly better than, Lig4-deficient cells. partially improve the growth of XRCC1 cells. Although the mechanism underlying the slow growth These data demonstrate unequivocally that XRCC1 is dis- Δ Δ of XRCC1 / cells has not been determined, it is unlikely that pensable for A-EJ during CSR. Δ Δ XRCC1 / cells have an intrinsic proliferation defect because Δ Δ XRCC1 / cells undergo robust CSR (Fig. 2B), which is known to Results and Discussion Δ Δ be cell-proliferation dependent. In fact, XRCC1 / cells switch Gene Targeting of XRCC1. XRCC1 is a DNA repair factor that has Δ more efficiently than XRCC1+/ cells (Figs. 2B and 3D). The in- recently been implicated as a component of A-EJ (17, 18). Be- crease in CSR efficiency in the absence of XRCC1 is small cause XRCC1 is essential for mouse embryonic development (∼19%), but consistent (P = 0.014). The mechanism by which (20), there is a lack of direct genetic evidence for its involvement XRCC1 deficiency promotes CSR efficiency is unknown. One fi Δ Δ in CSR and A-EJ.