Mutagen-induced recombination in mammalian cells in vitro

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<ul><li><p>Mutation Research, 284 (1992) 37-51 37 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00 </p><p>MUT 0375 </p><p>Mutagen-induced recombination in mammalian cells in vitro </p><p>Dennis Hellgren Department of Clinical Genetics, Karolinska Hospital, Karolinska Institute, S-104 O1 Stockholm, Sweden </p><p>(Accepted 30 March 1992) </p><p>Keywords: Recombination; Mutagen; Mammalian cells in vitro </p><p>Summary </p><p>It is now clear from in vitro studies that mutagens induce recombination in the ceil, both homologous and nonhomologous exchanges. The recombination events induced are extrachromosomal events, ex- changes between extrachromosomal DNA and chromosomes, and inter- as well as intrachromosomal exchanges. However, not all types of DNA damage can induce recombination. The mechanisms involved in the induction process are not known but may involve activation of DNA repair systems. In addition, stimulation of mRNA transcription by mutagens, different recombination pathways and how the assay system is constructed may affect the frequency and characteristics of the observed recombination events. </p><p>Correspondence: (present address) Dennis Hellgren, Environ- mental Medicine Unit, CNT/NOVUM, Hiilsoviigen 7, S-141 57 Huddinge, Sweden. </p><p>Abbreviations: AAF, 2-acetylaminofluorene; N-Aco-AAF, N- acetoxy-2-acetylaminofluorene; aprt, adeninephosphoribosyl- transferase; m-AMSA, 4'-(acridinylamino)methanesulfon-m- anisidine; AT, ataxia telangiectasia; BPDE, (+)-anti 7,8-dihy- droxy-9,10-epoxy,7,8,9,10-tetrahydrobenzo[a]pyrene; BrdUrd, bromodeoxyuridine; CHO, Chinese hamster ovary cells; EBV, Epstein-Barr virus; ENU, ethylnitrosourea; ES, embryonic stem cells; HMT, 4'-hydroxymethyl-4,5',8-trimethylpsoralen; HN2, nitrogen mustard; HIV-1, human immunodeficiency virus type 1; lAP, intracisternal A particle; LTR, long termi- nal repeat; MMC, mitocycin C; MMS, methyl methanesul- fonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; Mo- Musv, Moloney murine sarcoma virus; 1-NOP, 1-nitro- sopyrene; 4-NQO, 4-nitroquinoline-l-oxide; SCID, severe combined immunodeficiency; spr2, small, proline rich 2; SV40, simian virus 40; TK, thymidine kinase; TPA, 10-O-tetrade- eanoylphorbol-13-acetate; XP, xeroderma pigmentosum; ts, temperature sensitive. </p><p>Introduction </p><p>During recent years homologous recombina- tion has been shown to be involved in the activa- tion of protooncogenes by erroneous rejoining of different chromosomal regions. Similarly, muta- tion may arise by unequal exchanges between sister chromatids causing deletion in one and insertion in the other. In some cases, the DNA sequences at the recombination break points have shown homology to the signal sequences used by recombinases when rearranging immunoglobulin and T-cell receptor genes [1]. Repetitive DNA such as Alu sequences have also been shown to participate in genetic recombination [2,3]. Cir- cumstantial evidence indicates that the recombi- nation process is actively mediated and thus may be induced under certain circumstances, such as treatment of cells with mutagenic agents. This is also borne out by recent research. </p></li><li><p>38 </p><p>A number of studies have clearly shown that mammalian cells are able to recombine 'artificial' substrates in vivo. Bacteriophages, eukaryotic viruses and plasmids have been used as model substrates. In many cases the recombination events occur extrachromosomally, prior to inte- gration into the genome. This applies in general for plasmids. </p><p>Recombinagenic activity has also been demon- strated in extracts from cells [4,5]. Studies using plasmids have shown that the ability of cells to recombine substrates extrachromosomally peaks during the S-phase of the cell cycle [6]. This has also been found for chromosomal integration of </p><p>retrovirus vectors [7] and for adenovirus [8]. It is likely that DNA replication with its concomitant unfolding of nucleosomes makes DNA more ac- cessible to enzymes that have recombinagenic activity. Induction of RNA transcription also seems to increase the probability of recombina- tion, both in yeast and in mammalian cells [9-14]. </p><p>These few examples indicate that many cellu- lar processes can influence the frequency of ho- mologous recombination which further compli- cates the attempts to study its mechanisms and its role in mutagenesis. Since this review will only cover mutagen-induced recombination in mammalian cells the reader is referred to other </p><p>TABLE 1 </p><p>FREQUENCY OF BACKGROUND RECOMBINATION IN MITOTIC CELLS </p><p>Cells Frequency (F) Type Type Reference or rate (R) * of recombination of vector construct </p><p>CHO 7 x 10 -5 (F) interchromosomal endogenous markers 105 Rat 3 5 x 10-6 (R) a interchromosomal TK gene lacking promoter ~ 106 </p><p>4 x 10-3 (R) b intrachromosomal functional TK gene b 106 FM3A (mouse) 1.4 x 10-6 (R) intrachromosomal? endogenous repetitive 64 </p><p>sequence CHO 6.8 x 10 6 (R) intrachromosomal/ tandem neo gene construct 107 </p><p>interchromosomal AB1 ES cells 4.3 x 10 6 (R) c intrachromosomal duplicated hprt exon 3 108 </p><p>construct 3.8x 10 3 (R) d intrachromosomal duplicated Hox-2.6 108 </p><p>construct E-14TG2a 8.7 x 10-7 (F) e intrachromosomal duplicated hprt exon 3 109 </p><p>ES cells construct DBA mice 0.9 X 10-7 (F) f intrachromosomal/ recombination between 110 </p><p>interchromosomal retroviral LTR sequences LM205 5 10 s (R) intrachromosomal/ promoterless neo gene g 63 Human cells interchromosomal </p><p>3T6 0.13-1 X 10 -6 (R) interchromosomal tandem neo gene construct 111 3T6 5-30 x 10- 6 (F) intrachromosomal/ tandem neo gene construct 112 </p><p>interchromosomal </p><p>* Figures given are the sum of recombination events such as gene conversion, deletions, etc., since most assays do not discriminate between the various events. F = frequency, recombinants per number of cells; R = rate, events per cell per generation. </p><p>a The TK promoter was inserted downstream of the TK coding sequence. An unequal exchange between chromatids inserts the promoter in the correct position. </p><p>h The loss of a functional TK gene was scored, in 20% of the cases the TK gene was deleted. c The duplication of hprt exon 3 was created by gene targeting. d The duplication of Hox-2.6 was created during gene targeting. c The duplication of hprt exon 3 was created by gene targeting. f A natural mutation due to integration of an ecotropic murine leukemia virus provirus into a gene. This causes a lightening of </p><p>coat color. Reversion due to homologous recombination between the two LTRs, removing the virus except a single LTR, can easily be scored since the mouse then becomes intensely colored in an otherwise light coat color strain. </p><p>g The SV40 promoter/enhancer was inserted downstream of the neo gene. The gene becomes active when the promoter has been relocated to the 5' side of the neo gene through an event involving gene duplication. </p></li><li><p>reviews for further aspects on recombination [15- 20]. </p><p>Definitions In this review, the term homologous recombi- </p><p>nation will be used for exchanges between DNA sequences with extensive homology, for example between two mutated selectable markers. The term nonhomologous recombination is reserved for exchanges between sequences with a low de- gree of homology or none at all. The borderline between homologous and nonhomologous recom- bination is a difficult one to draw and will not be further discussed here. </p><p>39 </p><p>Extrachromosomal recombination refers to ex- changes between or within DNA molecules that are located outside the chromosomes, such as plasmids or viruses. </p><p>DNA integration means insertion of foreign DNA, such as plasmids, into a chromosome, gen- erally through nonhomologous recombinaton. This integration can be regarded as a random event. </p><p>Chromosomal recombination can be divided into intra- and interchromosomal recombination events. Intrachromosomal recombination involves a single chromosome and can result in gene con- version or loss of DNA through deletion. Gene </p><p>TABLE 2 </p><p>INDUCTION OF EXTRACHROMOSOMAL HOMOLOGOUS RECOMBINAT ION </p><p>(A) Treatment of DNA </p><p>Substrate DNA Cells Mutagenic Induction Comments Reference treatment </p><p>Adeno 5 mutants fibroblasts UV + 23 </p><p>ts SV40 viruses CV-1 UV + 24 monkey kidney cells UV - Both DNA and cells 25 </p><p>MMC - were treated UV + 26 </p><p>Herpes simplex Vero UV + 26 viruses LMTK + 26 </p><p>AT + 26 XP-A + 26 XP variant + 26 fibroblasts, normal + 26 </p><p>Herpes simplex fibroblasts, normal UV + 27 viruses fibroblasts, XPA UV + 27 </p><p>fibroblasts, variant UV + 27 </p><p>Herpes TK gene 2E5tk (CHO, normal) UV + 43 deletions UVL1 tk- (CHO mutant) UV + 43 </p><p>SupF mutants CV-1 (African green monkey) UV + 28 </p><p>(B) Treatment of cells </p><p>Cells Substrate DNA Treatment Induction Comments </p><p>VH10 mutants Adeno 5 deletion UV - XP-A XP-C XP var iant CV-I ts SV40 Monkey kidney cells ts SV40 - Both cells and viral </p><p>DNA treated </p><p>Reference </p><p>23 23 23 23 24 25 </p></li><li><p>40 </p><p>conversion occurs between homologous se- quences, whereby one sequence donates part of its sequence to the accepting sequence without undergoing any change. The acceptor sequence will become identical with the donor for a part of its length. </p><p>Interchromosomal recombination can occur between identical homologous chromosomes or nonidentical chromosomes. </p><p>The background frequency of recombination 'Background' recombination rates have been </p><p>estimated using various endogenous markers and artificial substrates. The rates for extrachromoso- mal recombination and integration of recombi- nant markers into the genome are difficult to estimate since the copy numbers of the extrachro- mosomal elements in each cell that undergoes recombination are usually difficult to estimate. </p><p>Examples of measured recombination rates are given in Table 1. These seem to indicate that there are no large differences between species. However, the constructs used are different, and it is therefore not possible to do a dose comparison of variations in the recombination rate between species. </p><p>Results from human gene mapping studies have shown that there are substantial differences in the meiotic recombination frequency between different regions within a chromosome and also between males and females [21]. This has also been found for mice [21]. It is not known if this is also the case for mitotic recombination. The rate of gene amplification in somatic cells varies de- pending on location in the genome [22]. This might be a reflection of regional differences in the recombination frequency in mitotic chromo- somes. </p><p>Induction of recombination by mutagenic agents </p><p>Extrachromosomal homologous recombination The induction of extrachromosomal recombi- </p><p>nation after mutagenic treatment has been stud- ied using viruses [23-27] and plasmids [28] as substrates. Both deletion mutants and tempera- ture sensitive variants of viruses have been used. The recombination is homologous since the par- ticipating substrates have common sequences in </p><p>which a recombination has to occur in order to create a functional product. Mutagenic agents studied include UV light and MMC (Table 2). </p><p>Induction of extrachromosomal recombination apparently depends on whether the cells or the substrate DNA has been treated. It has not been found in cells that have been pretreated before transfection, while pretreatment of substrate DNA with DNA damaging agents gives rise to an observable induction (Table 2). This is in contrast to recombination between vectors and chromoso- mal DNA, as described below. </p><p>Induction of extrachromosomal recombination is not limited to mammalian cells, since it is also found to occur in Xenopus laevis oocytes [29,30] in response to X-rays. However, there might be some species differences with respect to in- ducibility of recombination by various mutagenic agents since UV light, in contrast to X-rays, was found to induce extrachromosomal recombina- tion in Xenopus. This deserves further study. </p><p>Recombination between extrachromosomal DNA and chromosomes </p><p>The effect of DNA damage on the frequency of recombination between plasmids and chromo- somal DNA was the topic of a recent comprehen- sive review [31], and will therefore be discussed only briefly here. </p><p>It is clear from a number of reports that treat- ment of plasmids with DNA damaging agents prior to transfection can enhance the ability of the host cell to integrate foreign DNA into the chromosomes [32-42]. However, this does not seem to hold true for CHO cells [41,43,44]. It has been speculated that this is due to the fact that CHO cells already have an increased ability to take up and integrate DNA in the genome. This elevated integration may be related to an in- creased recombination activity in these cells, measured in protein extracts using D-loop forma- tion as an assay method [4]. </p><p>Also pretreatment of cells with DNA damag- ing agents increases the frequency with which plasmids are integrated in the genome [34- 36,38,45,46]. Transfection of cells with high molecular weight chromosomal DNA was stimu- lated by pretreatment of mouse FM3A cells with UV light [46]. In this case the integration is due </p></li><li><p>to a nonhomologous recombination process, since no large sequence homology exists between the participating sequences at the site of integration. </p><p>Recently, Mudgett and Taylor [47] showed that transfer of sequences from chromosomes to plas- </p><p>41 </p><p>mids can be stimulated after X-ray treatment of the plasmid. Chromosomally integrated bacterial ampicillin resistance sequences were referred to and replaced a mutant ampicillin gene present on a shuttle vector in African green monkey kidney </p><p>TABLE 3 </p><p>INDUCTION OF HOMOLOGOUS RECOMBINATION OF INTEGRATED SEQUENCES </p><p>Cel l s Construct Mutagenic agent Induction Reference </p><p>Mouse LM tk aprt tandem mutated tk genes MMC + 55 TPA - 55 Mezerein - 55 </p><p>Mouse L tk - tandem mutated tk genes MMC + 56 </p><p>BPDE + 56 MNNG + 56 UV + 56 6Co - 56 </p><p>Mouse L tk - tandem mutated tk genes 1-NOP + 57 </p><p>N-Aco-AAF + 57 4-NQO + 57 </p><p>KMST-6 (human) tandem mutated hygromycin UV + 81 resistance genes </p><p>XP20S(SV), XPA UVA + 81 XP2YO(SV), XPF UVA + 81 </p><p>143 tk - (human) tandem mutated tk genes UV + 80 1-NOP + 80 </p><p>RD tk - (human) tandem mutated tk genes UV + 80 </p><p>1-NOP + 80 </p><p>XP12ROSV40 tk - (XPA) tandem mutated tk genes UV + 80 </p><p>1-NOP + 80 </p><p>CHO tandem mutated neo genes MMS + 58 HN2 + 58 </p><p>CHO-EM9 tandem mutated neo genes MMC + 59 </p><p>X-rays - 59 BPDE - 59 MMS + 59 HN2 + 59 BrdUrd + 59 </p><p>LM205 (human) neo gene lacking promoter TPA + 63 </p><p>m-AMSA + 63 BrdUrd + 63 MNNG + 63 </p><p>Mouse FM3A repeated sequence Vr MNNG + 64 MNNG + TPA + 64 </p><p>Human lymphocytes HLA-A X-rays + 65 </p><p>MMC + 65 </p></li><li><p>42 </p><p>cells. Linearization of the plasmid increased the frequency of chromosome-plasmid recombina- tion, whereas treatment with UV light did not. </p><p>This type of recombination, transfer of se- quences from chromosomes to plasmids, also oc- curs in E. coli, and is stimulated by treatment with N-Aco-AAF, UV light and BPDE [48,49]. </p><p>Intra- and interchromosomal recombination eL,ents The induction of exchanges between chromo- </p><p>somes in intact cells has mostly been studied at the cytogenetic level. However, a few studies have been done on whole organisms. </p><p>Treatment of...</p></li></ul>

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