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 http://www.jimmunol.org/content/176/1/365 Downloaded from References This article cites 15 articles, 2 of which you can access for free at: http://www.jimmunol.org/content/176/1/365.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology 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 result- ing 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 tran- sitions 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 Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 366 Ig HYPERMUTATION IN UNG-DEFICIENT DT40 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.