RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions
© Ordinario et al; licensee BioMed Central Ltd. 2009
Received: 15 July 2009
Accepted: 28 October 2009
Published: 28 October 2009
Gene conversion depends upon the same factors that carry out more general process of homologous recombination, including homologous gene targeting and recombinational repair. Among these are the RAD51 paralogs, conserved factors related to the key recombination factor, RAD51. In chicken and other fowl, gene conversion (templated mutation) diversifies immunoglobulin variable region sequences. This allows gene conversion and recombinational repair to be studied using the chicken DT40 B cell line, which carries out constitutive gene conversion and provides a robust and physiological model for homology-directed repair in vertebrate cells.
We show that DT40 contains constitutive nuclear foci of the repair factors RAD51D and XRCC2, consistent with activated homologous recombination. Single-cell imaging of a DT40 derivative in which the rearranged and diversifying immunoglobulin λR light chain gene is tagged with polymerized lactose operator, DT40 PolyLacO-λR, showed that RAD51D and XRCC2 localize to the diversifying λR gene. Colocalizations correlate both functionally and physically with active immunoglobulin gene conversion. Ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of gene conversion, and conversion tracts were significantly longer in RAD51D than XRCC2 transfectants.
These results demonstrate direct functions of RAD51D and XRCC2 in immunoglobulin gene conversion, and also suggest that modulation of levels of repair factors may be a useful strategy to promote gene correction in other cell types.
Ig gene conversion is initiated by the B cell-specific enzyme, activation-induced deaminase (AID) [10–13]. AID deaminates C to U in transcribed Ig genes, producing a U·G mismatch [14–17]; uracil-DNA glycosylase (UNG) removes U to produce an abasic site [18–21]; and the MRE11/RAD50/NBS1 (MRN) complex promotes gene conversion  using its abasic lyase activity to cleave at abasic sites .
Gene conversion and gene targeting are both impaired by deficiencies in factors involved in homology-directed repair, including MRE11 ; NBS1 [25, 26]; the five RAD51 paralogs, RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 [27–30]; and BRCA1 and BRCA2 [30, 31]. At the Ig genes, deficiencies of these factors, or deletion of  or repressive chromatin modifications at  the ψVλ donors does not simply diminish the clonal rate of gene conversion, but alters the mutational spectrum so that nontemplated mutations appear, analogous to those produced in somatic hypermutation in activated mammalian B cells.
To better understand the gene conversion pathway and how it may relate to other processes of recombinational repair, we have defined the localization and functions of RAD51D and XRCC2 in DT40 B cells. We find that RAD51D and XRCC2 form constitutive foci in normally proliferating DT40 cells. Single-cell imaging of DT40 PolyLacO-λR cells, in which the rearranged and expressed λR light chain gene can be visualized directly, showed that RAD51D and XRCC2 localize to the rearranged λR allele. Colocalization reflects function in the diversification mechanism, as it is diminished upon expression of Ugi, which inhibits UNG activity; and correlates with enrichment at the rearranged λR allele. In addition, ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of Ig gene conversion, and gene conversion tracts were significantly longer in RAD51D than XRCC2 transfectants. These results support a model in which RAD51D and XRCC2 participate directly in Ig gene conversion. They also support the notion that modulation of levels of repair factors may be useful for gene therapy strategies based on targeted gene correction.
RAD51, RAD51D-GFP and XRCC2-GFP form nuclear foci in DT40 B cells
To determine whether RAD51D or XRCC2 form nuclear foci, we examined DT40 derivatives stably transfected with RAD51D-GFP or XRCC2-GFP, expressing C-terminal GFP-tagged proteins to ensure specificity. As the GFP-tagged proteins did not produce a sufficiently strong signal for direct imaging, we stained with anti-GFP antibodies to amplify the signals, and imaged cells by fluorescence microscopy. Staining with anti-GFP antibodies produced no background in untransfected DT40 cells (e.g. Figure 2, center left); but revealed constitutive punctate nuclear foci in normally proliferating DT40 RAD51D-GFP cells (e.g. Figure 2, center right; 36% of cells contained at least 3 foci; n > 200). A parallel analysis of DT40 XRCC2-GFP transfectants similarly revealed constitutive punctate nuclear foci in normally proliferating cells (e.g. Figure 2, right; 33% of cells contained at least 3 foci; n > 200).
RAD51D-GFP and XRCC2-GFP localize to the rearranged λR gene
We assayed λR/RAD51 colocalizations in asynchronous DT40 PolyLacO-λR RFP-LacI cells, and observed colocalizations in 6.5% of cells (n = 167; e.g. Figure 3B, arrows). λR/RAD51D-GFP colocalizations were observed in 15% of DT40 PolyLacO-λR RFP-LacI RAD51D-GFP cells (n = 153; e.g. Figure 3C, arrows). λR/XRCC2-GFP colocalizations were observed in 17.5% of DT40 PolyLacO-λR RFP-LacI XRCC2-GFP cells (n = 434; e.g. Figure 3D, arrows).
λR/RAD51D-GFP colocalization depends upon AID-initiated DNA damage
We have previously established the approximate level of background colocalizations in DT40 PolyLacO-λR cells of two other factors essential for Ig gene diversification, Polη-GFP  and E2A . Polη-GFP colocalizes with λR in 11.4% of normally proliferating DT40 PolyLacO-λR RFP-LacI Polη-GFP cells, but in only 5.4% DT40 PolyLacO-λR RFP-LacI Polη-GFP Ugi transfectants . As E2A acts upstream of AID, background for colocalizations of E2A was determined somewhat differently, by comparing localizations to the rearranged and unrearranged λ gene . Colocalized λR/E2A foci are evident in 26% of DT40 PolyLacO-λR GFP-LacI cells, and in only 6.3% of DT40 PolyLacO-λU GFP-LacI cells (in which the unrearranged λU allele is tagged with PolyLacO). Thus, for the three factors analyzed thus far, RAD51D-GFP, Polη-GFP and E2A, background colocalization is in the range of 5.4 - 7.0%. By this criterion, the fraction of cells exhibiting λR/RAD51 colocalizations is not significantly different from background, while the fraction of cells exhibiting colocalizations of RAD51D-GFP or XRCCC2-GFP is highly significant (P = 0.0001, χ2 test).
RAD51D-GFP is enriched at the rearranged but not unrearranged λ allele
To further establish that the observed colocalizations at λR reflect events critical to the mechanism of Ig gene conversion, we compared physical association of RAD51D-GFP with the rearranged and diversifying or unrearranged and inactive λ alleles by chromatin immunoprecipitation (ChIP). Chromatin was immunoprecipitated from normally proliferating DT40 RAD51D-GFP cells with a polyclonal anti-GFP antibody or with nonspecific IgG control antibodies, and amplicons from either the rearranged or unrearranged Vλ region were amplified in duplex PCR reactions, along with a control amplicon, ovalbumin, as previously described [23, 36]. The rearranged VλR region was 2.7-fold enriched relative to the ovalbumin control (Figure 5B, left), but there was no significant enrichment of the unrearranged VλU region (1.2-fold; Figure 5B, right). The level of enrichment was reproducible in three independent experiments. It is comparable to levels previously documented for association of RAD51D at breaks generated by I-SceI cleavage in mammalian cells ; and also in the range for association of other repair factors at target loci in eukaryotic cells, where interactions may be transient or occur only in only a small fraction of cells [23, 36, 38].
RAD51D or XRCC2 expression accelerated Ig gene diversification in chicken B cells, but not human B cells
To confirm that RAD51D or XRCC2 expression affects homology-directed repair but not other processes that diversify V region sequence, we asked if stable expression of RAD51D or XRCC2 affected clonal diversification in derivatives of the constitutively hypermutating human B cell line Ramos. The fraction of sIgM loss variants was almost identical in stable Ramos transfectants expressing RAD51D-GFP or XRCC2-GFP as in GFP control transfectants (P = 0.73 and 0.78, respectively, Mann-Whitney U test; data not shown). Thus ectopic expression of RAD51D and XRCC2 promotes homology-directed repair but does not influence somatic hypermutation, which is homology-independent.
Ectopic expession of either RAD51D or XRCC2 promotes gene conversion
To ask whether ectopic RAD51D or XRCC2 expression promoted gene conversion or other mutagenic pathways, single sIgM- cells of transfectants were isolated by flow cytometry, and Vλ regions were PCR-amplified and sequenced. After elimination of germline and duplicate sequences, sequences carrying unique mutations or combinations of mutations were aligned with the sequences of the ψVλ regions. Potential donors for each mutation were identified, if present in the ψVλ array, and the minimum homology that existed between potential ψVλ donors and recipient genes was determined for each sequence. Following the established convention [29, 33]), changes from the germline sequence that shared a region of identity at least 9 bp in length with one or more ψVλ donors were identified as gene conversion events. Changes with no match, or which matched within a region < 9 bp in length, were identified as point mutations.
A similar analysis was carried out on 19 independently transfected lines expressing XRCC2, from which sequences of 162 Vλ regions were determined, and 27 unique sequences further analyzed. These harbored a total of 77 single base changes, 75 of which matched sequences of ψVλ donors, and thus appeared to result from gene conversion (Figure 7B; Additional File 2). Thus, in XRCC2 transfectants, 97% of sequence changes match sequences in ψVλ donors.
RAD51D expression increases gene conversion tract lengths
Gene conversion repair tract lengths can be estimated by determining the boundaries of homology between each tract recipient and its possible ψVλ donors. These homologies were determined for the DT40 RAD51D and DT40 XRCC2 transfectants. A mutation was scored as templated only if there was homology to a ψVλ donor within a window at least 9 nt in length, so 9 nt was the lower limit on tract length. Homologies ranged from 9-81 bp in Vλ regions of RAD51D transfectants (Figure 7A), and from 9-14 bp in Vλ regions of XRCC2 transfectants (Figure 7B). The average minimum repair tract length was thereby estimated to be 18.9 bp in RAD51D transfectants; and 9.2 bp in XRCC2 transfectants. This difference is highly significant (P < 0.001, Mann-Whitney U test). The average minimum tract length in control GFP transfectants was 9.9 bp (Additional File 3), significantly different from RAD51D transfectants (P = 0.012, Mann-Whitney U test) but comparable to XRCC2 transfectants (P = 0.11, Mann-Whitney U test). Thus, RAD51D expression resulted in longer tract lengths.
We also distinguished the fraction of templated events that contained two or more templated base changes, rather than a single base change. In the DT40 RAD51D transfectants, a minimum of 36 gene conversion events could account for all the templated changes in the sequenced Vλ regions, 33 (92%) of which caused changes at two or more nt; and three that caused a change at a single nt (Figure 7C). (This latter category could in principle result from a nontemplated mutation that coincidentally matched germline sequence.) In the DT40 XRCC2 transfectants, a minimum of 38 gene conversion events could account for all mutation tracts, 17 (45%) resulting in ≥ 2 changes and 21 (55%) resulting in a single nt change (Figure 7C). Thus, the fraction of gene conversion events producing ≥ 2 templated base differences was strikingly different between RAD51D and XRCC2 transfectants (P < 0.0001, Fisher's exact test).
The five RAD51 paralogs have previously been shown to be necessary for Ig gene conversion in experiments demonstrating that ablation of any of these genes causes AID-initiated mutagenesis to switch from a templated to a nontemplated repair pathway [27–30]. We have presented several kinds of evidence consistent with direct function of both RAD51D and XRCC2 in Ig gene conversion. Imaging colocalizations with λR provided one snapshot of events in Ig gene conversion. We documented λR/RAD51D-GFP colocalizations in 15% of cells, and λR/XRCC2-GFP colocalizations in 17.5% of cells, both significantly greater than background. Colocalizations were shown to correlate both functionally and physically with active Ig gene conversion. In addition, ectopic expression of either RAD51D or XRCC2 accelerated Ig gene conversion in chicken DT40 B cells, but did not affect the rate of diversification in human Ramos B cells, which depend upon low fidelity polymerases and not homologous recombination to repair DNA damage initiated by the activity of AID. Notably, expression of RAD51D but not XRCC2 increased the conversion tract length.
DT40 cells are unusual in that they contain constitutive foci of repair factors, including the key recombination factor, RAD51  as well as the RAD51D and XRCC2 foci that we have documented. These constitutive foci probably reflect the recombinationally active state of DT40 B cells, which support ongoing Ig gene conversion and very efficient homologous gene targeting. While it is not possible to confirm that gene conversion is ongoing at a specific λR allele imaged in a single cell, expression of Ugi, which inhibits gene conversion, caused a 50% reduction in the fraction of cells exhibiting λR/RAD51D-GFP colocalizations. This functional analysis suggests that about half the colocalizations observed are at sites of active gene conversion. Other colocalizations may reflect background inherent to confocal microscopy, a possibility consistent with evidence that expression of Ugi similarly reduced λR/Polη-GFP colocalizations by approximately 50% . Participation in the gene conversion mechanism was further supported by establishing that RAD51D-GFP is specifically enriched at the rearranged λR allele, which undergoes diversification; and not at the inactive, unrearranged λU allele.
The fraction of cells in which colocalizations are evident will be determined both by the fraction of cells in which colocalizations occur and by the duration of colocalization. λR/RAD51 colocalizations were evident in only 6.5% of cells, not significantly greater than background. The absence of a significant fraction of cells exhibiting λR/RAD51 colocalizations could mean that RAD51 associations with the diversifying Ig genes are relatively transient; alternatively, Ig gene conversion may represent a specialized pathway in which recombination does not depend upon RAD51.
Accelerated gene conversion and increased tract length in DT40 RAD51D transfectants
Expression of either RAD51D or XRCC2 caused comparable 3-fold acceleration in the rate of diversification, as measured by the sIgM loss assay. Distinct mechanisms could account for this increase. Expression of these factors may cause a greater fraction of cells to carry out productive diversification, or alter the reaction kinetics of diversification in individual cells.
Gene conversion tracts were significantly longer in DT40 RAD51D than DT40 XRCC2 transfectants. In the latter cell lines tract length was identical to that in control DT40 GFP transfectants. Gene conversion tract length is almost certainly closely regulated in vivo, as tract length has clear biological consequences. Short conversion tracts may be advantageous at diversifying Ig genes as they enable the recipient gene to accumulate a patchwork of mutations from multiple different donors, which contributes diversity to the repertoire. Short conversion tracts will also cause modulated rather than drastic changes in antibody specificity. In contrast, longer conversion tracts would tend to create greater diversity in the same number of rounds of diversification; but would overwrite not only germline sequence but also sequence from previous rounds of gene conversion, in effect erasing mutations.
Possible implications for gene correction strategies
There is considerable interest in the possibility of correcting mutations associated with genetic disease by gene correction . In this approach, a DNA break is targeted at or near a defective gene, and ensuing DNA repair uses a homologous donor to correct the genetic defect, thereby restoring gene function. Strategies for elevating the efficiency of gene correction have focused on each of the steps in this pathway. Efforts to design nucleases that create specific breaks have met with encouraging recent success [40–43]. A critical limitation is the relatively low efficiency of homology-directed repair in most vertebrate cell types. In a few cases it has been shown that homology-directed repair, or the related process of homologous gene targeting, can be enhanced by increasing or diminishing levels of specific repair factors [44–49]. This has suggested that systematic analysis of the ability of repair factors to stimulate homology-directed repair might identify useful strategies to promote targeted gene correction. Our results identify RAD51D and XRCC2 as potential candidates for such approaches.
It is not possible to know whether a factor that promotes gene conversion at the Ig genes in chicken B lymphocytes would have a similar function in other cell types or other species. Nonetheless, by establishing that ectopic expression of repair factors can enhance homologous recombination in this context, our results provide proof in principle for the likely utility of extending this approach to other targets and other cell types. The ability of RAD51D expression to augment repair tract length is of particular potential utility for application to targeted gene correction. In targeted gene correction, repair tract length in effect determines how near a target mutation nuclease cleavage can occur and still promote useful repair. Thus, longer tracts are predicted to be advantageous in this context. The evidence that ectopic expression of RAD51D enhances both the clonal efficiency of gene conversion and repair tract length suggests the utility of considering both these parameters in future efforts to promote gene correction.
DT40 contains constitutive nuclear foci of the repair factors RAD51D and XRCC2, consistent with activated homologous recombination. Single-cell imaging of DT40 PolyLacO-λR cells showed that RAD51D and XRCC2 localize to the diversifying λR gene. Colocalization correlates with function in diversification, and with physical association with the rearranged λR allele. Ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of gene conversion, and conversion tracts were significantly longer in RAD51D than XRCC2 transfectants. These results demonstrate direct functions of RAD51D and XRCC2 in immunoglobulin gene conversion, and also suggest that modulation of levels of repair factors may be a useful strategy for gene correction in other cell types.
RAD51D and XRCC2 cDNAs were isolated from the human B cell line, Raji, as mammalian RAD51 paralogs are known to function in chicken cells . Following reverse transcription with the ProtoScript first strand cDNA synthesis kit (New England BioLabs, Ipswich, MA), RAD51D cDNA was amplified with the forward primer 5'-TAAGATCTACCATGGGCGTGCTCAGGGTC-3' and the reverse primers 5'-ATACCGGTGGTCATGTCTGATCACCCTG-3' or 5'-ATACCGGTGGTGTCTGATCACCCTGTAA-3', which carries an in-frame stop codon. PCR products were cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA), excised with BglII and AgeI, and subcloned into the pEGFP-N1 vector (Clontech, Mountain View, CA) to generate pRAD51D-GFP and pRAD51D, respectively. XRCC2 cDNA was similarly amplified with the forward primer 5'-CACCATGTGTAGTGCCTTCCATAGGGCTGAGTCT-3' and the reverse primer 5'-TCAACAAAATTCAACCCCACTTTCTCC-3' containing an in-frame stop codon and cloned into the pcDNA3.1/V5-His-D-TOPO vector (Invitrogen, Carlsbad, CA) to generate pXRCC2; or cDNA amplified with the primers 5'-AAAAAGGTACCGATGTGTAGTGCCTTCCATAGGGC-3' and 5'AAAAAACCGGTCCAC-AAAATTCAACCCCACTTTCTC-3', excised with KpnI and AgeI, and cloned into the pEGFP-N1 vector to generate pXRCC2-GFP. The Ugi expression vector  was provided by Dr. Michael Neuberger (Medical Research Council Laboratory of Molecular Biology, Cambridge, UK).
Cell culture, transfection and analysis
Cell culture, transfection and cell cycle analysis were carried out as described [22, 34]. Control experiments confirmed that cell proliferation and cell cycle distribution were unaltered in all stable transfectants prior to further analysis. Western blotting with antibodies against the tag was performed to verify comparable levels of expression of C-terminal tagged RAD51D or XRCC2 in stable transfectants (not shown). Uracil glycosylase activity was assayed  to verify Ugi expression.
Cells (~3 × 105) were deposited onto glass slides using Shandon Cytospin3 (800 rpm, 4 min; Thermo Fisher Scientific, Waltham, MA), fixed with 2% paraformaldehyde for 20 min, permeabilized with 0.5% NP-40 for 15 min, and stained as previously described [22, 34, 50]. Primary antibodies for staining were monoclonal anti-GFP (3E6, 1:100; Molecular Probes, Eugene, OR) and polyclonal anti-RAD51 (1:500; provided by Dr. Charles Radding, Yale University, New Haven, CT). Secondary antibodies (Molecular Probes, Eugene, OR) were: for anti-GFP, Alexa Fluor 488-conjugated anti-mouse IgG (1:1500); and for anti-RAD51, Alexa Fluor 488- and 594-conjugated anti-rabbit IgG (1:1500). Specific recognition of chicken RAD51 was verified by immunoblotting (data not shown). Stained cells were visualized using Leica SP1 confocal (Leica Microsystems, Bannockburn, IL) and DeltaVision deconvolution (Applied Precision, Issaquah, WA) microscopes with 60× and 100× objectives, and images were processed using Leica LCS (Leica Microsystems, Bannockburn, IL) and softWoRx (Applied Precision, Issaquah, WA) software.
To image PolyLacO, DT40 PolyLacO-λR cells were stably transfected with an RFP-LacI expression construct (a derivative of p3'SS-GFP-LacI [51, 52] in which GFP was replaced with DsRed-monomer (Clontech, Mountain View, CA), and visualized as above. Cells that contained RAD51, RAD51D-GFP or XRCC2-GFP foci superimposed with RFP-LacI foci were considered positive for colocalization. Cross sectional images measuring 0.2 μm apart were analyzed with a line profile tool of the image software to confirm colocalization occurred within the same focal plane. Fluorescent signals were sometimes partially rather than completely overlapping, which may reflect the considerable distance (~17 kb) between the PolyLacO-tag and the Vλ region; both configurations were scored as localization. Significance of colocalization was analyzed with the Pearson's χ2 test.
Chromatin preparation and immunoprecipitation (IP) were performed as described previously [23, 36]. Anti-GFP antibody was purchased from Abcam (Cambridge, MA). Amplifications were performed using Fast-Start Taq polymerase (Roche, Indianapolis, IN) and the following oligonucleotide primers: for the DT40 rearranged Vλ region, 5'-GCCGTCACTGATTGCCGTTTTCTCCCCTC-3' and 5'-CGAGACGAGGTCAGCGACTCACCTAGGAC-3'; for the unrearranged Vλ, 5'-CAGGAATGGAGGTGGGACT-3' and 5'-GCCGTCACTGATTGCCGTTTTCTCCCCTC-3' (one of the rearranged Vλ region oligos); for ovalbumin, 5'-ATTGCGCATTGTTATCCACA-3' and 5'-TAAGCCCTGCCAGTTCTCAT-3'. PCR products were quantified using ImageQuant software (Amersham, Piscataway, NJ). Enrichment was calculated as the ratio of the Vλ amplicon to the ovalbumin amplicon, normalized to the ratio of products from IP with polyspecific IgG antibodies: Enrichment Vλ = [anti-GFP (Vλ/ovalbumin)]/[IgG (Vλ/ovalbumin)]. Enrichment was compared at three template concentrations, to confirm that assays were within the linear range of PCR. Results shown are representative of three amplifications from two independent chromatin preparations.
Ig diversification rates and VλR sequence analysis
The sIgM loss assay was used to quantitate Ig gene diversification [22, 29, 30, 33]. Briefly, independent transfectants (typically 30-65 clones) were cultured for 4-6 wk posttransfection, stained with monoclonal anti-chicken IgM conjugated to RPE (1:500; SouthernBiotech, Birmingham, AL), and analyzed by flow cytometry. The percentage of sIgM- cells was calculated as the ratio of the number of cells with 8-fold or greater decrease in RPE intensity to the RPE of the sIgM+ population [29, 30]. Statistical analyses used the R software package http://www.r-project.org, and sIgM loss levels in transfectants were compared to vector controls using the Mann-Whitney U test.
Rearranged Vλ regions were amplified from flow-sorted single sIgM- cells and sequenced as described [22, 33]. Sequence alignment was done with the Sequencher program (Gene Codes Corporation, Ann Arbor, MI). A mutation in Vλ was designated as templated if one or more base substitutions within a 9 bp tract exactly matched to a ψVλ region; and otherwise as nontemplated . Gene conversion tract lengths and mutational spectra of RAD51D and XRCC2 transfectants were compared using the Mann-Whitney U test and Fisher's exact test, respectively.
We thank Drs. Yansong Gu, Brian Kennedy, David Morris, Dennis Willerford, and all members of the Maizels laboratory for valuable discussions, and David Bednarski for assistance with DNA sequencing. This research was supported by NIH R01 GM041712 and RL1 GM084434 to N.M. E.O. is grateful to NIH training programs T32 GM07223 and T32 AG00057 for support.
- Reynaud CA, Anquez V, Grimal H, Weill JC: A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell. 1987, 48 (3): 379-388. 10.1016/0092-8674(87)90189-9View ArticlePubMedGoogle Scholar
- Thompson CB, Neiman PE: Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment. Cell. 1987, 48 (3): 369-378. 10.1016/0092-8674(87)90188-7View ArticlePubMedGoogle Scholar
- McCormack WT, Thompson CB: Chicken IgL variable region gene conversions display pseudogene donor preference and 5' to 3' polarity. Genes Dev. 1990, 4 (4): 548-558. 10.1101/gad.4.4.548View ArticlePubMedGoogle Scholar
- Sayegh CE, Demaries SL, Pike KA, Friedman JE, Ratcliffe MJ: The chicken B-cell receptor complex and its role in avian B-cell development. Immunol Rev. 2000, 175: 187-200. 10.1111/j.1600-065X.2000.imr017507.xView ArticlePubMedGoogle Scholar
- Arakawa H, Buerstedde JM: Immunoglobulin gene conversion: insights from bursal B cells and the DT40 cell line. Dev Dyn. 2004, 229 (3): 458-464. 10.1002/dvdy.10495View ArticlePubMedGoogle Scholar
- Sale JE: Immunoglobulin diversification in DT40: a model for vertebrate DNA damage tolerance. DNA Repair (Amst). 2004, 3 (7): 693-702. 10.1016/j.dnarep.2004.03.042View ArticleGoogle Scholar
- Yamazoe M, Sonoda E, Hochegger H, Takeda S: Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40. DNA Repair (Amst). 2004, 3 (8-9): 1175-1185. 10.1016/j.dnarep.2004.03.039View ArticleGoogle Scholar
- Maizels N: Immunoglobulin gene diversification. Annu Rev Genet. 2005, 39: 23-46. 10.1146/annurev.genet.39.073003.110544View ArticlePubMedGoogle Scholar
- Buerstedde JM, Reynaud CA, Humphries EH, Olson W, Ewert DL, Weill JC: Light chain gene conversion continues at high rate in an ALV-induced cell line. EMBO J. 1990, 9 (3): 921-927.PubMed CentralPubMedGoogle Scholar
- Muramatsu M, Kinoshita K, Fagarasan S, Yamada S, Shinkai Y, Honjo T: Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell. 2000, 102 (5): 553-563. 10.1016/S0092-8674(00)00078-7View ArticlePubMedGoogle Scholar
- Revy P, Muto T, Levy Y, Geissmann F, Plebani A, Sanal O, Catalan N, Forveille M, Dufourcq-Labelouse R, Gennery A, et al: Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell. 2000, 102 (5): 565-575. 10.1016/S0092-8674(00)00079-9View ArticlePubMedGoogle Scholar
- Arakawa H, Hauschild J, Buerstedde JM: Requirement of the Activation-Induced Deaminase (AID) gene for immunoglobulin gene conversion. Science. 2002, 295 (5558): 1301-1306. 10.1126/science.1067308View ArticlePubMedGoogle Scholar
- Harris RS, Sale JE, Petersen-Mahrt SK, Neuberger MS: AID is essential for immunoglobulin V gene conversion in a cultured B cell line. Curr Biol. 2002, 12 (5): 435-438. 10.1016/S0960-9822(02)00717-0View ArticlePubMedGoogle Scholar
- Petersen-Mahrt SK, Harris RS, Neuberger MS: AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature. 2002, 418 (6893): 99-103. 10.1038/nature00862View ArticlePubMedGoogle Scholar
- Bransteitter R, Pham P, Scharff MD, Goodman MF: Activation-induced cytidine deaminase deaminates deoxycytidine on single-stranded DNA but requires the action of RNase. Proc Natl Acad Sci USA. 2003, 100 (7): 4102-4107. 10.1073/pnas.0730835100PubMed CentralView ArticlePubMedGoogle Scholar
- Chaudhuri J, Tian M, Khuong C, Chua K, Pinaud E, Alt FW: Transcription-targeted DNA deamination by the AID antibody diversification enzyme. Nature. 2003, 422 (6933): 726-730. 10.1038/nature01574View ArticlePubMedGoogle Scholar
- Ramiro AR, Stavropoulos P, Jankovic M, Nussenzweig MC: Transcription enhances AID-mediated cytidine deamination by exposing single-stranded DNA on the nontemplate strand. Nat Immunol. 2003, 4 (5): 452-456. 10.1038/ni920View ArticlePubMedGoogle Scholar
- Di Noia J, Neuberger MS: Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature. 2002, 419: 43-48. 10.1038/nature00981View ArticlePubMedGoogle Scholar
- Imai K, Slupphaug G, Lee WI, Revy P, Nonoyama S, Catalan N, Yel L, Forveille M, Kavli B, Krokan HE, et al: Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch recombination. Nat Immunol. 2003, 4 (10): 1023-1028. 10.1038/ni974View ArticlePubMedGoogle Scholar
- Di Noia JM, Neuberger MS: Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID-mediated deoxycytidine deamination. Eur J Immunol. 2004, 34 (2): 504-508. 10.1002/eji.200324631View ArticlePubMedGoogle Scholar
- Saribasak H, Saribasak NN, Ipek FM, Ellwart JW, Arakawa H, Buerstedde JM: Uracil DNA glycosylase disruption blocks Ig gene conversion and induces transition mutations. J Immunol. 2006, 176 (1): 365-371.View ArticlePubMedGoogle Scholar
- Yabuki M, Fujii MM, Maizels N: The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions. Nat Immunol. 2005, 6 (7): 730-736. 10.1038/ni1215View ArticlePubMedGoogle Scholar
- Larson ED, Cummings WJ, Bednarski DW, Maizels N: MRE11/RAD50 cleaves DNA in the AID/UNG-dependent pathway of immunoglobulin gene diversification. Mol Cell. 2005, 20 (3): 367-375. 10.1016/j.molcel.2005.09.018View ArticlePubMedGoogle Scholar
- Yamaguchi-Iwai Y, Sonoda E, Sasaki MS, Morrison C, Haraguchi T, Hiraoka Y, Yamashita YM, Yagi T, Takata M, Price C, et al: Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells. EMBO J. 1999, 18 (23): 6619-6629. 10.1093/emboj/18.23.6619PubMed CentralView ArticlePubMedGoogle Scholar
- Tauchi H, Kobayashi J, Morishima K, Van Gent DC, Shiraishi T, Verkaik NS, VanHeems D, Ito E, Nakamura A, Sonoda E, et al: Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells. Nature. 2002, 420 (6911): 93-98. 10.1038/nature01125View ArticlePubMedGoogle Scholar
- Nakahara M, Sonoda E, Nojima K, Sale JE, Takenaka K, Kikuchi K, Taniguchi Y, Nakamura K, Sumitomo Y, Bree RT, et al: Genetic evidence for single-strand lesions initiating Nbs1-dependent homologous recombination in diversification of Ig V in chicken B lymphocytes. PLoS Genet. 2009, 5 (1): e1000356- 10.1371/journal.pgen.1000356PubMed CentralView ArticlePubMedGoogle Scholar
- Takata M, Sasaki MS, Sonoda E, Fukushima T, Morrison C, Albala JS, Swagemakers SM, Kanaar R, Thompson LH, Takeda S: The Rad51 paralog Rad51B promotes homologous recombinational repair. Mol Cell Biol. 2000, 20 (17): 6476-6482. 10.1128/MCB.20.17.6476-6482.2000PubMed CentralView ArticlePubMedGoogle Scholar
- Takata M, Sasaki MS, Tachiiri S, Fukushima T, Sonoda E, Schild D, Thompson LH, Takeda S: Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol Cell Biol. 2001, 21 (8): 2858-2866. 10.1128/MCB.21.8.2858-2866.2001PubMed CentralView ArticlePubMedGoogle Scholar
- Sale JE, Calandrini DM, Takata M, Takeda S, Neuberger MS: Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation. Nature. 2001, 412 (6850): 921-926. 10.1038/35091100View ArticlePubMedGoogle Scholar
- Hatanaka A, Yamazoe M, Sale JE, Takata M, Yamamoto K, Kitao H, Sonoda E, Kikuchi K, Yonetani Y, Takeda S: Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for Rad51 paralogs in homologous recombination. Mol Cell Biol. 2005, 25 (3): 1124-1134. 10.1128/MCB.25.3.1124-1134.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Longerich S, Orelli BJ, Martin RW, Bishop DK, Storb U: Brca1 in immunoglobulin gene conversion and somatic hypermutation. DNA Repair (Amst). 2008, 7 (2): 253-266. 10.1016/j.dnarep.2007.10.002View ArticleGoogle Scholar
- Arakawa H, Saribasak H, Buerstedde JM: Activation-induced cytidine deaminase initiates immunoglobulin gene conversion and hypermutation by a common intermediate. PLoS Biol. 2004, 2 (7): E179- 10.1371/journal.pbio.0020179PubMed CentralView ArticlePubMedGoogle Scholar
- Cummings WJ, Yabuki M, Ordinario EC, Bednarski DW, Quay S, Maizels N: Chromatin structure regulates gene conversion. PLoS Biol. 2007, 5 (10): e246- 10.1371/journal.pbio.0050246PubMed CentralView ArticlePubMedGoogle Scholar
- Yabuki M, Ordinario EC, Cummings WJ, Fujii MM, Maizels N: E2A acts in cis in G1 phase of cell cycle to promote Ig gene diversification. J Immunol. 2009, 182 (1): 408-415.PubMed CentralView ArticlePubMedGoogle Scholar
- Ordinario EC, Yabuki M, Larson RP, Maizels N: Temporal regulation of Ig gene diversification revealed by single-cell imaging. J Immunol. 2009, 183 (7): 4545-4553. 10.4049/jimmunol.0900673PubMed CentralView ArticlePubMedGoogle Scholar
- Larson ED, Duquette ML, Cummings WJ, Streiff RJ, Maizels N: MutSα binds to and promotes synapsis of transcriptionally activated immunoglobulin switch regions. Curr Biol. 2005, 15 (5): 470-474. 10.1016/j.cub.2004.12.077View ArticlePubMedGoogle Scholar
- Rodrigue A, Lafrance M, Gauthier MC, McDonald D, Hendzel M, West SC, Jasin M, Masson JY: Interplay between human DNA repair proteins at a unique double-strand break in vivo. EMBO J. 2006, 25 (1): 222-231. 10.1038/sj.emboj.7600914PubMed CentralView ArticlePubMedGoogle Scholar
- Wolner B, van Komen S, Sung P, Peterson CL: Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast. Mol Cell. 2003, 12 (1): 221-232. 10.1016/S1097-2765(03)00242-9View ArticlePubMedGoogle Scholar
- Porteus MH, Connelly JP, Pruett SM: A look to future directions in gene therapy research for monogenic diseases. PLoS Genet. 2006, 2 (9): e133- 10.1371/journal.pgen.0020133PubMed CentralView ArticlePubMedGoogle Scholar
- Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PD, Holmes MC: Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005, 435 (7042): 646-651. 10.1038/nature03556View ArticlePubMedGoogle Scholar
- Ashworth J, Havranek JJ, Duarte CM, Sussman D, Monnat RJ, Stoddard BL, Baker D: Computational redesign of endonuclease DNA binding and cleavage specificity. Nature. 2006, 441 (7093): 656-659. 10.1038/nature04818PubMed CentralView ArticlePubMedGoogle Scholar
- Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, et al: An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol. 2007, 25 (7): 778-785. 10.1038/nbt1319View ArticlePubMedGoogle Scholar
- Szczepek M, Brondani V, Buchel J, Serrano L, Segal DJ, Cathomen T: Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol. 2007, 25 (7): 786-793. 10.1038/nbt1317View ArticlePubMedGoogle Scholar
- Liu Y, Maizels N: Coordinated response of mammalian Rad51 and Rad52 to DNA damage. EMBO Reports. 2000, 1: 85-90. 10.1093/embo-reports/kvd002PubMed CentralView ArticlePubMedGoogle Scholar
- Yanez RJ, Porter AC: Differential effects of Rad52p overexpression on gene targeting and extrachromosomal homologous recombination in a human cell line. Nucleic Acids Res. 2002, 30 (3): 740-748. 10.1093/nar/30.3.740PubMed CentralView ArticlePubMedGoogle Scholar
- Di Primio C, Galli A, Cervelli T, Zoppe M, Rainaldi G: Potentiation of gene targeting in human cells by expression of Saccharomyces cerevisiae Rad52. Nucleic Acids Res. 2005, 33 (14): 4639-4648. 10.1093/nar/gki778PubMed CentralView ArticlePubMedGoogle Scholar
- Vasileva A, Linden RM, Jessberger R: Homologous recombination is required for AAV-mediated gene targeting. Nucleic Acids Res. 2006, 34 (11): 3345-3360. 10.1093/nar/gkl455PubMed CentralView ArticlePubMedGoogle Scholar
- So S, Nomura Y, Adachi N, Kobayashi Y, Hori T, Kurihara Y, Koyama H: Enhanced gene targeting efficiency by siRNA that silences the expression of the Bloom syndrome gene in human cells. Genes Cells. 2006, 11 (4): 363-371. 10.1111/j.1365-2443.2006.00944.xView ArticlePubMedGoogle Scholar
- Iiizumi S, Kurosawa A, So S, Ishii Y, Chikaraishi Y, Ishii A, Koyama H, Adachi N: Impact of non-homologous end-joining deficiency on random and targeted DNA integration: implications for gene targeting. Nucleic Acids Res. 2008, 36 (19): 6333-6342. 10.1093/nar/gkn649PubMed CentralView ArticlePubMedGoogle Scholar
- Liu Y, Li M-J, Lee EY-HP, Maizels N: Localization and dynamic relocalization of mammalian Rad52 during the cell cycle and in response to DNA damage. Curr Biol. 1999, 9: 975-978. 10.1016/S0960-9822(99)80427-8View ArticlePubMedGoogle Scholar
- Robinett CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont AS: In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition. J Cell Biol. 1996, 135 (6 Pt 2): 1685-1700. 10.1083/jcb.135.6.1685View ArticlePubMedGoogle Scholar
- Belmont AS, Straight AF: In vivo visualization of chromosomes using lac operator-repressor binding. Trends Cell Biol. 1998, 8 (3): 121-124. 10.1016/S0962-8924(97)01211-7View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.