Mutations Gene Conversion and Induces Transition Uracil DNA Glycosylase Disruption Blocks Ig

Uracil DNA Glycosylase Disruption Blocks Ig Gene Conversion and Induces Transition Mutations

This information is current as Huseyin Saribasak, Nesibe Nur Saribasak, Fatih M. Ipek, of September 29, 2021. Joachim W. Ellwart, Hiroshi Arakawa and Jean-Marie Buerstedde J Immunol 2006; 176:365-371; ; doi: 10.4049/jimmunol.176.1.365

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Uracil DNA Glycosylase Disruption Blocks Ig Gene Conversion and Induces Transition Mutations1

Huseyin Saribasak,* Nesibe Nur Saribasak,* Fatih M. Ipek,* Joachim W. Ellwart,† Hiroshi Arakawa,* and Jean-Marie Buerstedde2*‡

Ig gene conversion is most likely initiated by activation-induced cytidine deaminase-mediated cytosine deamination. If the resulting uracils need to be further processed by uracil DNA glycosylase (UNG), UNG inactivation should block gene conversion and induce transition mutations. In this study, we report that this is indeed the phenotype in the B cell line DT40. Ig gene conversion is almost completely extinguished in the UNG-deficient mutant and large numbers of transition mutations at C/G bases accumulate within the rearranged Ig L chain gene (IgL). The mutation rate of UNG-deficient cells is about seven times higher than that of pseudo V gene-deleted (␺V؊) cells in which mutations arise presumably after uracil excision. In addition, UNG-deficient cells show relatively more mutations upstream and downstream of the VJ segment. This suggests that hypermutating B cells process acti- Downloaded from vation-induced cytidine deaminase-induced uracils with approximately one-seventh of uracils giving rise to mutations depending on their position. The Journal of Immunology, 2006, 176: 365–371.

he activation-induced cytidine deaminase (AID)3 gene V(D)J segments, it is an interesting question whether gene con- tightly regulates all three B cell- specific processes of Ig version, in analogy to hypermutation and switch recombination, T repertoire formation: hypermutation, gene conversion, requires the processing of uracils by UNG. It was reported (11) http://www.jimmunol.org/ and switch recombination (1–4). Based on the findings that uracil that expression of an Ugi transgene in DT40 reduced gene con- DNA glycosylase (UNG)-deficient mice and human patients show version to 30% of the wild-type level, suggesting that UNG activ- reduced switch recombination and a shift of mutations at C/G ity enhances gene conversion. However, this phenotype remains bases toward transitions (5, 6), it was postulated that AID exerts its difficult to interpret, because, contrary to expectation, no evidence action within the Ig loci by the deamination of cytosines to uracils for an increased mutation rate was found, and it was unclear how which are subsequently further processed by UNG. efficiently and specifically Ugi transgene expression inhibited One of the best cellular systems to study the mechanism of UNG activity. AID-induced gene conversion and hypermutation is the chicken B We, therefore, decided to disrupt both copies of the UNG gene cell line DT40 (7). DT40 diversifies its rearranged Ig L chain gene in DT40 and analyze the mutant phenotype with respect to Ig gene by guest on September 29, 2021 (IgL) almost exclusively by ␺V-templated gene conversions in an conversion and hypermutation. AID-dependent fashion (3). The gene conversion events typical of wild-type DT40 are, however, replaced by frequent single nucle- Materials and Methods otide substitutions when homologous recombination factors like Isolation of chicken UNG cDNA the RAD51 paralogues XRCC2, XRCC3, and Rad51B are inacti- ␺ The EST dkfz426_17p6r1 of the bursal EST database (12) shows signifi- vated (8), or the nearby V locus is removed (9). The hypermu- cant homology to the murine UNG cDNA. The corresponding clone was tation activity elicited by the block of gene conversion shares sequenced by bidirectional primer walk and found to contain a 1.1-kb many features with Ig hypermutation in mice and human B cells, cDNA insert including the full-length UNG open reading frame of 299 aa. but is limited to C/G bases. Expression of the dominant-negative The amino acid sequence of the chicken UNG is 79 and 75% identical to the human and murine UNG, respectively. inhibitor of UNGs, Ugi, shifted the mutation spectrum toward transitions in hypermutating XRCC2-deficient DT40 clones (10), pro- Construction of gene targeting and cDNA expression constructs viding first evidence that uracil is a likely intermediate in the hy- Primers derived from the chicken UNG cDNA were used to amplify the permutation process. genomic locus by long-range PCR using DNA from DT40 as template. The Since the studies cited above indicate that Ig gene conversion is exon-intron structure deduced from the PCR fragments was verified when initiated by AID-induced cytosine deamination in the rearranged the chicken genome sequence, including the UNG locus on chromosome 15, was released (13). The targeting constructs were made by cloning ap- propriate genomic fragments upstream and downstream of floxed marker *Institute of Molecular Radiobiology, GSF, Neuherberg, Germany; and †Institute of genes (14). The constructs were linearized by NotI before transfection. The Molecular Immunology, GSF, Mu¨nchen-Grosshardern, Germany UNG expression cassette was made by cloning the chicken UNG cDNA ␤ Received for publication July 27, 2005. Accepted for publication September 29, 2005. downstream of the -actin promoter and upstream of an IRES-GPT sequence. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Cell culture with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the European Framework VI Grant “Geninteg” and the Cell culture, transfection, selection of stable transfectants, and marker re- DFG Sonderforschungsbereich “Networks in Genome Expression and Maintenance.” cycle by transient Cre induction were performed as previously described (3). 2 Address correspondence and reprint requests to Dr. Jean-Marie Buerstedde, Institute of Molecular Radiobiology, GSF, Ingolsta¨dter Landstrasse 1, D-85764 Neuherberg, UNG activity assay Germany. E-mail address: [email protected] 3 Abbreviations used in this paper: AID, activation-induced cytidine deaminase; Exponentially growing cells were washed two times with PBS and resus- UNG, uracil DNA glycosylase; sIgM, surface IgM. pended at a concentration of ϳ103 cells/␮l in a buffer containing 25 mM

HEPES, 5 mM EDTA, 1 mM DTT, 10% glycerol, and a mixture of pro- Results tease inhibitors (10). The cells were then freeze-fractured by liquid nitro- UNG inactivation does not affect cell viability in DT40 gen and centrifuged at 6500 rpm for 10 min at 4°C. The supernatant was transferred to a new tube and then centrifuged again at 5000 rpm for 5 min The 5Ј and 3Ј arms of the UNG knockout constructs were designed at 4°C. The supernatant of this second centrifugation was stored as cell to insert an early in-frame stop codon at codon 66 and delete the extract at Ϫ20°C. UNG activity was measured by incubating 5 ␮l of cell extract with 10 pmol of a 5Ј FITC-labeled oligonucleotide containing a region encoding the highly conserved codons 67–120. Two ver- single uracil at position 23 (5Ј-TCACCTGCTCCGGGGGTGGCAGUTA sions of the constructs, named pUngBsr and pUngGpt, included a CGGCTGGAAGTTACTATTATGGCTG-3Ј) in a final volume of 10 ␮l floxed blasticidine S resistance gene and a floxed guanine phos- for2hat37°C. Control reactions were incubated with an oligonucleotide phoribosyl transferase gene (14), respectively (Fig. 1A). Targeted in which the uracil was replaced by cytidine. Because AP endonuclease activity in the extracts was found to be insufficient for complete cleavage integration was detected by PCR using a primer located upstream of the abasic site after uracil excision, 5 ␮l of 0.5 M NaOH was added to of the 5Ј targeting arm of the construct (ung1, Fig. 1A) along with the reaction and incubation was continued for1hat37°C. The DNA was a primer from the resistance marker. ␮ then precipitated by ethanol and resuspended in 10 l of formamide-load- pUngBsr Ϫ ing buffer before being analyzed on a 15% Tris-borate-EDTA-urea PAGE Transfection of into a sIgM ( ) variant of the DT40 clone R gel. After electrophoresis, gel images were visualized using a Fuji Film AID (9) yielded a targeting frequency of ϳ1 in 10. One of the (FLA-3000) phosphor imager. heterozygous UNGϩ/Ϫ clones was then transfected by pUngGpt. This construct integrated frequently into the already targeted UNG allele, Ig reversion assays and sequence analysis but only one clone with targeted integration into the other allele was Ͼ Quantification of surface IgM (sIgM) expression by FACS and sequence found upon screening of 300 transfectants. The identified UNG- analysis of L chain VJ regions have been described previously (3, 9). deficient clone showed normal cell viability and cloning efficiency, Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. UNG gene disruption. A, Aligned maps of the chicken UNG locus, the targeting constructs, and the disrupted locus after targeting integration of the constructs and marker excision. The locations of the primers used to con- firm the UNG gene disruption are indicated. B, Analysis of the UNG locus by PCR. The AIDR precursor clone is compared with the AID- RUNGϪ/Ϫ mutant. C, UNG assay using cell extracts. The structure of the oligonucleotide sub- strate is shown below the gel image. The Journal of Immunology 367 suggesting that the low recovery of homozygous clones was not Ig frameshift repair by gene conversion is blocked in the related to a growth defect of UNG-deficient cells. Excision of the bsr absence of UNG and gpt marker cassettes from the double-targeted clone yielded a The three clones, AIDR, AIDRUNGϪ/Ϫ, and AIDRUNGR, carry a clone named AIDRUNGϪ/Ϫ that was used for further analysis. R Ϫ/Ϫ frameshift mutation in their rearranged L chain V segment, which can Genomic PCR of AID UNG confirmed the homozygous deletion be corrected by gene conversion giving rise to sIgMϩ revertants. To of part of the UNG loci (Fig. 1B). R Ϫ/Ϫ determine how inactivation of the UNG gene affects frameshift repair AID UNG was transfected by an UNG cDNA expression by gene conversion, sIgM(ϩ) subpopulations were measured for 24 vector in which the UNG gene is expressed as a bicistronic mes- subclones of each of the three clones and the stable sIgM(Ϫ) control sage along with the gpt transgene. One of the mycophenolic acid Ϫ R R clone DT40 IgL . Representative FACS profiles and the calculation resistant transfectants was named AID UNG and retained for of the average percentages of gated events reveal a striking reduction further analysis. of sIgM(ϩ) cells for AIDRUNGϪ/Ϫ (Fig. 2A). It remains uncertain Ϫ/Ϫ UNG activity is strongly reduced in UNG-deficient cells whether sIgM reversion still occurs at a low level in UNG cells or whether it is entirely blocked, since the average of sIgM(ϩ) events in R R Ϫ/Ϫ R R Cell extracts of the AID , AID UNG , and AID UNG clones AIDRUNGϪ/Ϫ subclones (0.35%) is only slightly above the back- were incubated with a uracil-containing oligonucleotide. Excision ground noise seen in DT40 IgLϪ (0.24%). The lack of sIgM reversion of the uracil by UNG leads to an abasic site, which can be detected is partially complemented in the AIDRUNGR clone and sequence by the appearance of a shorter oligonucleotide after NaOH-medi- analysis of sIgM(ϩ) subpopulations confirmed that the L chain ated strand cleavage (Fig. 1C). In the absence of in vitro UNG frameshift was indeed repaired by gene conversions (data not shown). treatment, the amount of cleavage product was strongly reduced in These results demonstrate that inactivation of UNG severely compro- Downloaded from R Ϫ/Ϫ extracts of AID UNG cells compared with extracts of wild- mises Ig gene conversion. type and reconstituted AIDRUNGR cells. This confirms that the UNG activity is severely compromised by the UNG gene disrup- tions and that most of the activity in wild-type cell extracts is UNG-deficient cells rapidly lose sIgM linked to UNG. Nevertheless, a low level of cleavage was still The Ig mutation rate is most conveniently quantified in sIgM(ϩ) R Ϫ/Ϫ detectable after incubation with AID UNG extract (Fig. 1C, clones by measuring the appearance of sIgM(Ϫ) cells due to delete- http://www.jimmunol.org/ lane 2), but not in the “No extract” control (Fig. 1C, lane 5) upon rious mutations (9). To study whether UNG inactivation induces Ig prolonged incubation time. Because the UNG gene disruption most hypermutation, sIgM(ϩ) cells of an AIDRUNGR subclone were likely behaves like a null mutation, the residual activity may reflect sorted, exposed to different levels of tamoxifen, and subcloned. This the presence of one or more backup UNGs. procedure allowed the isolation of the following sIgM(ϩ) clones: 1) by guest on September 29, 2021

FIGURE 2. sIgM expression analysis of UNG-deficient and control clones. A, FACS anti- IgM staining profiles of representative subclones derived initially from sIgM(Ϫ) clones. Average percentages of events falling into sIgM(ϩ) gates based on the measurement of 24 subclones are shown below the profiles. B, FACS anti-IgM staining profiles of representative subclones derived initially from sIgM(ϩ) clones. Average percentages of events falling into sIgM(Ϫ) gates based on the measurement of 48 subclones of the AIDRUNGϪ/Ϫ clone and 24 subclones of the other clones are shown below the profiles. 368 Ig HYPERMUTATION IN UNG-DEFICIENT DT40 Downloaded from http://www.jimmunol.org/

FIGURE 3. Mapping of mutations to the consensus sequence of the rearranged Ig L gene. Nucleotide substitutions identified in the region from the leader sequence to the J-C intron were mapped onto the likely sequence of the precursor cell for the subclone. All point mutations identified in the AIDRUNGϪ/Ϫ and AIDR␺VϪ subclones after 6 wk of culture are shown above and below the precursor sequence, respectively. Hot spot motifs (RGYW and its complement WRCY) are highlighted in bold letters. In the case that mutations at the same position are Ͼ8, they are shown with their numbers. by guest on September 29, 2021 AIDRUNGR retaining both floxed AID and UNG expression cassettes, UNG inactivation induces a high rate of Ig transition mutations Ϫ/Ϫ Ϫ/Ϫ 2) AID R Ϫ/Ϫ UNG lacking the AID and the UNG cassettes, and 3) To determine the cause of the rapid sIgM loss in AID UNG AIDRUNGϪ/Ϫ clone1–3 retaining the AID, but lacking the UNG cas- cells, the rearranged Ig L chain genes were sequenced for two sette. The appearance of sIgM(Ϫ) subpopulations was measured in Ϫ Ϫ Ϫ AIDRUNG / and two AIDR␺V subclones 6 wk after subclon- parallel in subclones of these clones, as well as in subclones of control ϩ R R␺ Ϫ ing. As expected from the results of the sIgM analysis, sequences sIgM( ) AID and AID V (9) clones (Fig. 2B). This analysis R Ϫ/Ϫ revealed dramatically increased percentages of sIgM(Ϫ) cells for all of AID UNG cells showed an extraordinarily high number of three AIDRUNGϪ/Ϫ clones (49.00, 53.93, and 34.57%, respectively) single nucleotide substitutions at G/C bases and only few gene compared with the AIDR precursor clone (1.50%). AIDRUNGϪ/Ϫ conversion tracts (Fig. 3). The average number of mutations per ϳ R Ϫ/Ϫ subclones displayed on average twice as many sIgM(Ϫ) cells than sequence was 7-fold higher in sequences of AID UNG than R Ϫ AIDR␺VϪ clones (23.84%), which accumulate L chain hypermuta- that of AID ␺V cells (Table I). Almost all mutations of R Ϫ/Ϫ tions due to the loss of ␺V gene conversion donors. The rise of AID UNG cells (98%), but only about one-third of the muta- Ϫ sIgM(Ϫ) was significantly reduced in the AIDRUNGR clone (7.18%) tions of AIDR␺V cells (40%) were transitions (Fig. 4A). Muta- upon reintroduction of an UNG transgene. The AIDϪ/ϪUNGϪ/Ϫ tions in both cell populations occurred preferentially at the known clone remained stably sIgM(ϩ), showing that UNG disruption only hypermutation hot spots (ϳ50%, data not shown), almost exclusively causes sIgM(Ϫ) loss in the presence of AID expression. at G/C bases and in about equal numbers at G and C bases. The

Table I. Mutation comparisonsa

Mutation Analysis Type of Mutation

Culture Total Total Mut/ Cell Line Time (wk) Region Mutations Sequences bp(10Ϫ3) PM GC Del Dup I

AIDRUNGϪ/Ϫ 1 LVJ-intron (1360 bp) 610 374 1.15 583 24c 2—1 6 LVJ-intron (1360 bp) 963 95 7.38 954 5 4 — — 6 EFI␣ (500 bp) 2 78 0.05 2 — — — — AIDϪ/ϪUNGϪ/Ϫ 6 VJ-intron (480 bp) 1 95 0.02 1 — — — — AIDR ␺VϪ 6 LVJ-intron (1372 bp) 603 376 1.14 592 — 7 4 —

a Mut, mutation; PM, point mutation; GC, gene conversion; Del, deletion; Dup, duplication; I, insertion. b Only point mutations were counted. c Conversion events seem to have occurred early during clone expansion from one subclone. The Journal of Immunology 369 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Comparison of the mutations from AIDRUNGϪ/Ϫ and AIDR␺VϪ cells. A, Analysis of the mutation pattern of Ig L chain sequences 6 wk after subcloning. Only single nucleotide substitutions are included. The ratios of transition (trs) to transversion (trv) are shown in numbers and percentages. B, Distribution of mutations along the rearranged L chain gene. The number of mutations occurring within adjacent 10-bp intervals is plotted along the rearranged L chain gene sequence.

Ϫ Ϫ Ϫ Ϫ Ϫ / ␣ R / number of mutations at A and T is so low that it is difficult to rule out UNG cells and sequences of the EF1 gene from AID UNG artifacts due to PCR amplification. Likewise, very few mutations were cells (Table I), confirming that the mutation activity induced by UNG detected in sequences of rearranged VJ regions from AIDϪ/ disruption is AID dependent and Ig locus specific. 370 Ig HYPERMUTATION IN UNG-DEFICIENT DT40

Table II. Mutation distributions

Point Mutations

Upstream V Downstream J Total Cell Line Sequences (560 bp) VJ (313 bp) (487 bp) (1360 bp)

AIDRUNGϪ/Ϫ (1 wk) 374 141a (24%)b 381 (65%) 61 (11%) 583 AIDRUNGϪ/Ϫ (6 wk) 95 140 (15%) 609 (64%) 205 (21%) 954 AIDR␺VϪ (6 wk) 376 22 (4%) 540 (91%) 30 (5%) 592

a The number of mutations b The percentage of mutations out of total.

Mutations from AIDRUNGϪ/Ϫ and AIDR␺VϪ cells are tions, their respective numbers within windows of 10 bases were differently distributed plotted against the rearranged Ig L chain gene sequence (Fig. 4B). Ϫ Ϫ If uracils are not removed in the absence of UNG until the next This display shows that AIDRUNG / mutations are more scat- Ϫ replication cycle, transition mutations of UNG-negative cells will tered and less focused on the VJ regions than AIDR␺V muta- mark the positions of all AID-induced uracils. In contrast, hyper- tions. To rule out that the difference is an artifact due to the di- Ϫ Ϫ mutations of UNG-positive cells indicate the positions of only vergent mutation load, AIDRUNG / sequences were determined those uracils that escaped error-free repair after UNG processing. 1 wk after subcloning. The average number of mutations per se- Downloaded from To compare the location of AIDRUNGϪ/Ϫ and AIDR␺VϪ muta- quence in this culture is similar to the average in the 6-wk http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. A model explaining the regulation of Ig gene conversion and Ig hypermutation by AID and UNG. The Journal of Immunology 371

AIDR␺VϪ culture (Table I). Nevertheless, AIDRUNGϪ/Ϫ mutations late in ␺V cells after the UNG-mediated processing of uracils pro- from the two cultures are similarly spread along the L chain gene (Fig. vides further insight into how the hypermutation process is 4B), indicating that the distribution is not influenced by the mutation initiated. UNG-deficient cells accumulate L chain mutation seven average. The different distribution of AIDRUNGϪ/Ϫ and AIDR␺VϪ times faster than ␺V cells, indicating that about one of seven AID- mutations is also evident from the relative number of mutations induced uracils is processed into a mutation. This relative frequent located upstream of the V segment’s start, within the VJ coding region conversion of uracils into mutations suggests the presence of an and downstream of the J segment’s end (Table II). Whereas the VJ unusual error-prone repair pathway that specifically recognizes coding region encompasses 91% of the AIDR␺VϪ mutations, this AID-induced uracils. Interestingly, the distribution of mutations region contains only 65 and 64% of the AIDRUNGϪ/Ϫ mutations within the rearranged L chain gene varies between UNG-deficient from the 1- and 6-wk cultures, respectively (Table II). and ␺V-deleted cells. Although the majority of mutations arise within the VJ coding region in both cell types, relatively more Discussion mutations in UNG-deficient cells are located upstream of the V Inactivation of the UNG gene in AID expressing DT40 almost and downstream of the J coding regions. The most plausible ex- completely abolishes Ig gene conversions and activates a very high planation for this phenomenon is that UNG-mediated processing rate of transition mutations at C/G bases in the rearranged L chain of uracils is more error prone within the VJ segment than in the VJ region. This demonstrates conclusively that Ig gene conversion, flanking regions. Thus, the comparison of UNG and ␺V mutations like previously shown from hypermutation and switch recombina- provides the first evidence that the characteristic distribution of Ig tion (5, 6), requires UNG downstream of AID. Nevertheless, a hypermutation over the V(D)J regions reflects not only AID-me- minor alternative pathway seems to be responsible for the few diated cytosine deamination, but also UNG-mediated permutation Downloaded from gene conversion tracts still seen in UNG-deficient cells. of the resulting uracils in a position-dependent fashion. The results strongly support a model in which AID first deami- nates cytosine within the rearranged V(D)J segments, and the re- Acknowledgments sulting uracils are then processed by UNG to give rise to either We thank Randy Caldwell for help with the UNG assay and transition and transversion mutations or to gene conversions (Fig. Claire Brellinger and Andrea Steiner-Mezzadri for excellent technical

5). Inactivation of the UNG gene blocks the processing of uracils assistance. http://www.jimmunol.org/ and leads exclusively to transition mutations when uracil pairs with adenine in the next replication cycle. So far, this model had Disclosures been based on results showing that Ugi expression shifts Ig hy- The authors have no financial conflict of interest. permutations toward transitions in a XRCC2-deficient mutant (10) and inhibits Ig gene conversion in wild-type DT40 (11). However, References 1. Muramatsu, M., K. Kinoshita, S. Fagarasan, S. Yamada, Y. Shinkai, and the phenotype of the Ugi gene transfectants differ from the phe- T. Honjo. 2000. Class switch recombination and hypermutation require activa- notype of the UNG-deficient mutant in some features at odds with tion-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell the model. For example, Ugi expression increased transition mu- 102: 553–563.

2. Revy, P., T. Muto, Y. Levy, F. Geissmann, A. Plebani, O. Sanal, N. Catalan, by guest on September 29, 2021 tations only to 86% in the XRCC2-deficient mutant (10), whereas M. Forveille, R. Dufourcq-Labelouse, A. Gennery, et al. 2000. Activation-in- this number was 98% from the UNG-deficient mutant. Similarly, duced cytidine deaminase (AID) deficiency causes the autosomal recessive form Ugi expression reduced Ig gene conversion to 30% of wild-type of the hyper-IgM syndrome (HIGM2). Cell 102: 565–575. 3. Arakawa, H., J. Hauschild, and J. M. Buerstedde. 2002. Requirement of the levels (11), whereas the UNG gene disruption decreased gene con- activation-induced deaminase (AID) gene for immunoglobulin gene conversion. version almost 100-fold based on the L chain frameshift repair Science 295: 1301–1306. 4. Harris, R. S., J. E. Sale, S. K. Petersen-Mahrt, and M. S. Neuberger. 2002. AID assay. The most striking discrepancy to the model is the lack of is essential for immunoglobulin V gene conversion in a cultured B cell line. Curr. detectable hypermutation after Ugi expression in wild-type DT40. Biol. 12: 435–438. The difference between the results obtained after Ugi transfection 5. Rada, C., G. T. Williams, H. Nilsen, D. E. Barnes, T. Lindahl, and M. S. Neuberger. 2002. Immunoglobulin isotype switching is inhibited and somatic hypermutation and UNG disruption could be due to the fact that UNG-deficient perturbed in UNG-deficient mice. Curr. Biol. 12: 1748–1755. cells express a transfected AID, which may differ both in abun- 6. Imai, K., G. Slupphaug, W. I. Lee, P. Revy, S. Nonoyama, N. Catalan, L. Yel, dance and cell cycle regulation from endogenous AID. However, M. Forveille, B. Kavli, H. E. Krokan, et al. 2003. Human uracil-DNA glycosylase deficiency associated with profoundly impaired immunoglobulin class-switch re- the most straightforward interpretation seems to be that Ugi ex- combination. Nat. Immunol. 4: 1023–1028. pression in DT40 only partially inhibited UNG activity. 7. Arakawa, H., and J. M. Buerstedde. 2004. Immunoglobulin gene conversion: Insights from bursal B cells and the DT40 cell line. Dev. Dyn. 229: 458–464. Murine and human B cells do not use gene conversion for Ig 8. Sale, J. E., D. M. Calandrini, M. Takata, S. Takeda, and M. S. Neuberger. 2001. repertoire development and hypermutating chicken DT40 mutants Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into so- differ from primary B cells, as they do not mutate A/T bases. matic hypermutation. Nature 412: 921–926. 9. Arakawa, H., H. Saribasak, and J. M. Buerstedde. 2004. Activation-induced cy- Nevertheless, the initial steps leading to Ig gene conversion and tidine deaminase initiates immunoglobulin gene conversion and hypermutation hypermutations at C/G bases seem to be identical in all three cell by a common intermediate. PLoS Biol. 2: 967–974. systems. Thus, hypermutations at C/G bases are shifted toward 10. Di Noia, J. M., and M. S. Neuberger. 2002. Altering the pathway of immunoglobulin hypermutation by inhibiting uracil-DNA glycosylase. Nature 419: 43–48. transitions in UNG-deficient mice and human patients (5, 6), and 11. Di Noia, J. M., and M. S. Neuberger. 2004. Immunoglobulin gene conversion in B cells of the murine UNG/MSH2 double knockout which lack the chicken DT40 cells largely proceeds through an abasic site intermediate gener- ated by excision of the uracil produced by AID-mediated deoxycytidine deami- A/T mutator due to the MSH2 defect show a mutation spectrum nation. Eur. J. Immunol. 34: 504–508. very similar to the DT40 UNG-deficient mutant (15). 12. Abdrakhmanov, I., D. Lodygin, P. Geroth, H. Arakawa, A. Law, J. Plachy, If AID-induced uracils are not repaired in the absence of UNG, B. Korn, and J. M. Buerstedde. 2000. A large database of chicken bursal ESTs as a resource for the analysis of vertebrate gene function. Genome Res. 10: hypermutations in UNG-deficient DT40 cells will offer a unique 2062–2069. glimpse at the in vivo cytosine deamination activity of AID with- 13. International Chicken Genome Sequencing Consortium. 2004. Sequence and out subsequent selection or repair bias. The similar numbers of comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution. Nature 432: 695–716. mutations at C and G bases from UNG-deficient cells imply, for 14. Arakawa, H., D. Lodygin, and J. M. Buerstedde. 2001. Mutant loxP vectors for example, that the transcribed and the nontranscribed DNA strand selectable marker recycle and conditional knock-outs. BMC Biotechnol. 1: 7. 15. Rada, C., J. M. Di Noia, and M. S. Neuberger. 2004. Mismatch recognition and are equally accessible to AID. A comparison of the transition mu- uracil excision provide complementary paths to both Ig switching and the A/T- tations of UNG-deficient cells with hypermutations that accumu- focused phase of somatic mutation. Mol. Cell 16: 163–171.