DNA polymerase κ deficiency does not affect somatic hypermutation in mice

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  • 0014-2980/02/1111-3152$17.50+.50/0 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

    DNA polymerase k deficiency does not affectsomatic hypermutation in mice

    Dominik Schenten1,3, Valerie L. Gerlach2, Caixia Guo2, Susana Velasco-Miguel2,Christa L. Hladik2, Charles, L. White2, Errol C. Friedberg2, Klaus Rajewsky1,3 andGloria Esposito1

    1 Institute for Genetics, University of Cologne, Cologne, Germany2 Laboratory of Molecular Pathology, Department of Pathology, University of Texas

    Southwestern Medical Center, Dallas, USA3 Center for Blood Research, Boston, USA

    Somatic hypermutation (SH) in B cells undergoing T cell-dependent immune responses gen-erates high-affinity antibodies that provide protective immunity. Most current models of SHpostulate the introduction of a nick into the DNA and subsequent replication-independent,error-prone short-patch synthesis by one or more DNA polymerases. The PolK (DinB1) geneencodes a specialized mammalian DNA polymerase called DNA polymerase (pol ), amember of the recently discovered Y family of DNA polymerases. The mouse PolK gene isexpressed at high levels in the seminiferous tubules of the testis and in the adrenal cortex,and at lower levels in most other cells of the body including B lymphocytes. In vitro studiesshowed that pol can act as an error-prone polymerase, although they failed to ascribe aclear function to this enzyme. The ability of pol to generate mutations when extending prim-ers on undamaged DNA templates identifies this enzyme as a potential candidate for theintroduction of nucleotide changes in the immunoglobulin (Ig) genes during the process ofSH. Here we show that pol -deficient mice are viable, fertile and able to mount a normalimmune response to the antigen (4-hydroxy-3-nitrophenyl)acetyl-chicken + -globulin (NP-GC). They also mutate their Ig genes normally. However, pol -deficient embryonic fibro-blasts are abnormally sensitive to killing following exposure to ultraviolet (UV) radiation, sug-gesting a role of pol in translesion DNA synthesis.

    Key words: B cell / Somatic hypermutation / DNA polymerase

    Received 5/6/02Revised 14/8/02Accepted 3/9/02

    [I 23245]

    Abbreviations: ES cells: Embryonic stem cells GC: Ger-minal center MEF: Mouse embryonic fibroblast NP-CG: (4-Hydroxy-3-nitrophenyl) acetyl chicken + -globulin SH:Somatic hypermutation pol k : DNA polymerase

    1 Introduction

    In T cell-dependent immune responses, nave B cells aretriggered to proliferate in specific structures present insecondary lymphoid organs, the germinal centers (GC)[1]. Here, they modify their rearranged variable (V) genesby somatic hypermutation (SH) to generate high-affinityantibodies against the immunizing antigen. B cells carry-ing V genes with mutations leading to the generation ofhigh-affinity antibodies are preferentially selected anddifferentiate into memory and antibody-secreting plasmacells [2]. During SH Ig genes acquire point mutations at ahigh rate (around 103/bp/generation) [3, 4]. Transcription

    is essential for this process and mutations accumulate ina 2-kb window downstream of the promoter [58]. Dele-tions and duplications have been observed leading tothe conclusion that SH involves the generation of DNAstrand breaks [9, 10]. Recently, the presence of DNAdouble-strand breaks in Ig genes of cells undergoing SHhas been indeed demonstrated [11, 12]. However, themolecular origin of these lesions and their significancefor the SH process is still unclear.

    While cis-acting elements required for SH have beenidentified [6, 7], little is known about the molecular com-ponents necessary for this process. The GC B cell-specific activation-induced cytidine deaminase (AID) isthe first protein found to be essential for SH [13, 14],although its function remains elusive. Most current mod-els of SH postulate the introduction of nicks into the DNAand subsequent replication-independent, error-proneshort-patch synthesis by one or more DNA polymerases[1517]. Evidence for the role of novel error-prone poly-

    3152 D. Schenten et al. Eur. J. Immunol. 2002. 32: 31523160

  • Fig. 1. Generation of PolK/ mice. (A) Schematic representation of the gene targeting in the PolK locus by homologous recombi-nation. 129/Ola-derived ES cells were targeted with a vector containing the loxP-flanked exon 6 and a neomycyin resistance cas-sette for positive selection. A thymidine kinase gene was used to select against random integration of the vector. Only exons 5and 6 of the wild-type locus are shown. Rectangles represent coding DNA, filled triangles indicate loxP sites, and bold lines showregions of homology. E, exon; B, BamH I site; tk, thymidine kinase gene; neor, neomycin resistance gene. (B) Cre-mediated dele-tion of exon 6 and the neomycin resistance cassette. A Southern blot of BamH I-digested tail DNA from wild-type, heterozygousand homozygous mice, respectively, is shown. A probe containing exon 5 of the PolK locus was used. The wild-type fragmentmigrates at 9.9 kb and the fragment from the targeted locus migrates at 5.2 kb.

    merases in SH has recently been presented [1820], indi-cating the synergistic action of different polymerases inthe process.

    PolK (DinB1) is expressed at high levels in mouse testis,but also at lower levels in a wide variety of other tissuesincluding spleen [21, 22]. The physiological function(s) ofthe pol protein is unknown. It is a highly conservedpolymerase belonging to the UmuC/DinB/Rad30/Rev1family, recently renamed the Y family of polymerases[23], and shares extensive amino acid homology with theSOS-induced error-prone DNA polymerase Pol IV, theproduct of the E. coli dinB gene [21]. Pol lacks detect-able 3-5 proofreading exonuclease activity and copiesundamaged DNA with a single-base substitution errorrate of 6103 in vitro [24, 25]. Overexpression ofmurine pol in a mouse cell line results in about a tenfoldincrease of spontaneous mutagenesis [26]. Moreover, acomparison between the mutational patterns of SH andpol in vitro suggested a possible contribution of pol toSH [27]. These features mark pol as a potential candi-date for a specialized DNA polymerase required for SHor the replicative bypass (translesion synthesis) of someform of spontaneous base damage. We have generatedmice deficient for pol to investigate its contribution tothe accumulation of mutations in the Ig genes and haveexamined the role of the polymerase in protection

    against the effects of UV radiation, which is known toresult in oxidative damage to DNA.

    2 Results and discussion

    2.1 Generation of PolK-deficient mice

    To inactivate pol function, we modified the mouse PolK(DinB1) locus by gene targeting. We flanked exon 6 withloxP sites rendering it susceptible to Cre recombinase-mediated deletion. Additionally, we introduced a loxP-flanked neomycin resistance gene as selection marker.Exon 6 was chosen for two reasons. First, it contains twoessential catalytic residues: aspartate 197 and glutamate198. Replacement of these two amino acids by alanineresidues results in a complete loss of the DNA polymer-ase function in vitro [25, 26]. Second, mRNA splicingfrom exon 5 to exon 7 leads to a frame-shift mutation.The wild-type PolK locus, the modified locus afterhomologous recombination with the targeting vector andthe locus after Cre-mediated recombination are depictedin Fig. 1A. Two independent ES clones were used to gen-erate chimeric mice; both transmitted the targeted alleleinto the germ line. We achieved deletion of the neomycinresistance gene and exon 6 in vivo by crossing the chi-meras to a deleter mouse (Fig. 1B). Mice homozygousfor the deletion of exon 6 are viable, present at the

    Eur. J. Immunol. 2002. 32: 31523160 Pol is not a major contributor to somatic hypermutation 3153

  • Fig. 2. PolK/ mice are unable to express pol . (A) RT-PCRof PolK+/+, PolK+/ and PolK/ mice with primers annealing inexon 5 and downstream of exon 6, respectively. (B) Northernhybridization of equal amounts of RNA from PolK+/+ andPolK/ mice with a PolK-specific probe spanning nucleo-tides 4831493 of the cDNA sequence. (C) Immunohistologyof sections from testis with an mAb against human pol .Shown are sections from a wild-type mouse (left panel) andfrom a pol -deficient mouse (right panel).

    expected Mendelian ratio and do not exhibit obviousabnormalities. In contrast to pol [2830] and pol g [31,32], lack of pol protein does not interfere with embry-onic development.

    To confirm the inactivation of the PolK gene, we usedreverse transcription (RT)-PCR to amplify PolK tran-scripts from testis using primers spanning exon 6. Asshown in Fig. 2A, cDNA from PolK+/+ mice gave rise totwo alternative splice products. In contrast, cDNA fromPolK/ mice gave rise to one PCR product only, which isshorter than the larger wild-type product and consistentwith the lack 153 base pairs corresponding to exon 6 inthe mRNA (Fig. 2A), as confirmed by sequencing (notshown). Additionally, Northern blot analysis using equalamounts of mRNA from wild-type and mutant micerevealed that the intensity of the band from the latter wasfive times less than the band from the wild-type sample(Fig. 2B). The presence of a frame-shift mutation leadingto premature stop codons presumably renders mRNAlacking exon 6 less stable than wild-type mRNA.

    We analyzed histological sections of mouse testis byimmunohistochemistry to confirm the absence of pol

    protein in the mutant mice. Frozen sections of testis fromeither wild-type or PolK-deficient mice were incubatedwith a monoclonal antibody against human pol protein(S. Velasco-Miguel et al., manuscript in preparation).Pol protein was mainly localized in the nuclei of sper-matocytes and round spermatids of the seminiferoustubules in wild-type animals (Fig. 2C). A more completedescription of the distribution of pol during mouse sper-matogenesis will be published elsewhere (S. Velasco-Miguel, et al., manuscript in preparation). In contrast, nosignal could be detected in mice homozygous for thedeletion of exon 6 (Fig. 2C).

    2.2 PolK-deficient mice are fertile

    As shown in Fig. 2C, the overall histological structure ofthe testis from pol -deficient mice is indistinguishablefrom that of wild-type controls. We also failed to detectabnormalities in the shape and mobility of sperm cellsfrom mice lacking pol . Furthermore, both male andfemale PolK mutants are fertile and the litter size doesnot differ from wild-type mice (data not shown). At thepresent time the functional significance of the high levelsof tissue-specific expression of PolK in the mouse testisis not clear. Several other error-prone DNA polymerasesare also highly expressed in the testis. In particular, pol Qand pol u share a similar expression pattern with pol [33,34]. It is therefore conceivable that pol , pol Q and pol userve similar functions in the testis and in PolK/ micethe other two polymerases may compensate for the lossof pol activity. Hopefully, the generation of mice defi-cient for different combinations or all three DNA polymer-ases will clarify this issue.

    2.3 PolK-deficient embryonic fibroblasts aresensitive to killing by UV radiation

    Pol is one of multiple specialized DNA polymerases thatmay be used in cells to bypass sites of base damage thatstall or arrest normal DNA replication. Purified pol is notable to bypass thymine dimers, [64] photoproducts(quantitatively major photoproducts produced by UVradiation) or cisplatin lesions in vitro [24]. However, thepurified enzyme is able to bypass thymine glycol lesionsin vitro (P. Fischhaber, V. L. Gerlach, S. S. Wallace, J. W.Feaver and E. C. Friedberg, unpublished observations).Thymine glycols are a form of oxidized thymine that canbe produced by various treatments of cells, includingexposure to UV radiation. We therefore compared thesensitivity of embryonic fibroblasts (MEF) from homozy-gous mutant and wild-type mice. As shown in Fig. 3, thepol -deficient cells are as sensitive to UV radiation asMEF from homozygous mutant Xpc mice defective

    3154 D. Schenten et al. Eur. J. Immunol. 2002. 32: 31523160

  • Fig. 3. PolK-deficient cells are abnormally sensitive to killingfollowing exposure to UV radiation. Survival was measuredas described in the text. Closed circles PolK+/+ MEF; closedtriangles, PolK/MEF; open squares Xpc/ MEF.

    Fig. 4. Antibody response of PolK-deficient and control miceto NP-GC. Mice were immunized with NP-CG and the serumconcentrations of NP-specific IgG1 were measured 14 dayspost immunization in an ELISA assay. Each dot representsthe serum titer of an individual mouse.

    tive in genome-wide DNA repair but proficient intranscription-coupled nucleotide excision repair [35].These results indicate that in the absence of pol someform of UV radiation damage (possibly thymine glycol)results in cell killing.

    2.4 PolK-deficient mice show normal B and Tcell compartments

    Cell proliferation and viability may be impaired by theabsence of a DNA polymerase. We therefore investi-gated whether pol -deficiency affects B and T cell popu-lations. PolK-deficient mice exhibited normal numbers ofsplenocytes when compared to heterozygous controlanimals. We also did not observe differences in the pro-portion of bone marrow B cells and mature B cells fromblood and spleen in PolK+/ and PolK/ mice (data notshown). Highly proliferating GC B cells do not appear tobe affected by the absence of functional pol since wedid not observe any significant variations betweenmutant and control animals with respect to B220+PNA+ Bcells (data not shown). The analysis of splenic T cell pop-ulations also did not reveal abnormalities (data notshown). Thus, pol deficiency does not result in anymajor developmental defect in either B or T cell compart-ments.

    2.5 PolK-deficient mice respond to the T cell-dependent antigen NP-CG

    We next investigated whether PolK/ B cells are able tomount a normal T cell-dependent immune response anddifferentiate into plasma cells secreting antigen-specific

    Ig. Groups of four age-matched mice were immunizedwith NP-CG and the levels of NP-specific IgG1 weredetermined 14 days later. The serum levels of IgG1 weresimilar in PolK/, PolK+/, PolK+/+ and PolKfl/fl (conditionalPolK mutants; D.S., K.R., and G.E., unpublished) mice(Fig. 4). Hence, pol -deficient B cells are able to partici-pate in T cell-dependent immune responses comparablyto control B cells.

    The mechanisms of SH and class switch recombination(CSR) share common elements [13, 36]. We thereforemeasured the levels of the different Ig isotypes in theblood of homozygous mutants and both heterozygousand wild-type controls. No differences were noticedbetween the different groups of animals (data notshown).

    2.6 PolK-deficient GC B cells efficiently mutatetheir Ig genes

    To investigate whether pol is involved in SH of Ig genes,groups of 79-week-old (2 PolK/, 2 PolK+/ and 1 PolKfl/fl

    animals) and 8-month-old (4 PolK/ mice, designatedPolK/*) mice were immunized with NP-CG. At 14 dayspost immunization GC B cells were isolated and ana-lyzed for the level and pattern o...