Mutagen-induced recombination in mammalian cells in vitro
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Mutation Research, 284 (1992) 37-51 37 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
Mutagen-induced recombination in mammalian cells in vitro
Dennis Hellgren Department of Clinical Genetics, Karolinska Hospital, Karolinska Institute, S-104 O1 Stockholm, Sweden
(Accepted 30 March 1992)
Keywords: Recombination; Mutagen; Mammalian cells in vitro
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.
Correspondence: (present address) Dennis Hellgren, Environ- mental Medicine Unit, CNT/NOVUM, Hiilsoviigen 7, S-141 57 Huddinge, Sweden.
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.
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 . 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.
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.
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 . This has also been found for chromosomal integration of
retrovirus vectors  and for adenovirus . 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].
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
FREQUENCY OF BACKGROUND RECOMBINATION IN MITOTIC CELLS
Cells Frequency (F) Type Type Reference or rate (R) * of recombination of vector construct
CHO 7 x 10 -5 (F) interchromosomal endogenous markers 105 Rat 3 5 x 10-6 (R) a interchromosomal TK gene lacking promoter ~ 106
4 x 10-3 (R) b intrachromosomal functional TK gene b 106 FM3A (mouse) 1.4 x 10-6 (R) intrachromosomal? endogenous repetitive 64
sequence CHO 6.8 x 10 6 (R) intrachromosomal/ tandem neo gene construct 107
interchromosomal AB1 ES cells 4.3 x 10 6 (R) c intrachromosomal duplicated hprt exon 3 108
construct 3.8x 10 3 (R) d intrachromosomal duplicated Hox-2.6 108
construct E-14TG2a 8.7 x 10-7 (F) e intrachromosomal duplicated hprt exon 3 109
ES cells construct DBA mice 0.9 X 10-7 (F) f intrachromosomal/ recombination between 110
interchromosomal retroviral LTR sequences LM205 5 10 s (R) intrachromosomal/ promoterless neo gene g 63 Human cells interchromosomal
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
* 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.
a The TK promoter was inserted downstream of the TK coding sequence. An unequal exchange between chromatids inserts the promoter in the correct position.
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
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.
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.
reviews for further aspects on recombination [15- 20].
Definitions In this review, the term homologous recombi-
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.
Extrachromosomal recombination refers to ex- changes between or within DNA molecules that are located outside the chromosomes, such as plasmids or viruses.
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.
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
INDUCTION OF EXTRACHROMOSOMAL HOMOLOGOUS RECOMBINAT ION
(A) Treatment of DNA
Substrate DNA Cells Mutagenic Induction Comments Reference treatment
Adeno 5 mutants fibroblasts UV + 23
ts SV40 viruses CV-1 UV + 24 monkey kidney cells UV - Both DNA and cells 25
MMC - were treated UV + 26
Herpes simplex Vero UV + 26 viruses LMTK + 26
AT + 26 XP-A + 26 XP variant + 26 fibroblasts, normal + 26
Herpes simplex fibroblasts, normal UV + 27 viruses fibroblasts, XPA UV + 27
fibroblasts, variant UV + 27
Herpes TK gene 2E5tk (CHO, normal) UV + 43 deletions UVL1 tk- (CHO mutant) UV + 43
SupF mutants CV-1 (African green monkey) UV + 28
(B) Treatment of cells
Cells Substrate DNA Treatment Induction Comments
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
23 23 23 23 24 25
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.
Interchromosomal recombination can occur between identical homologous chromosomes or nonidentical chromosomes.
The background frequency of recombination 'Background' recombination rates have been
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.
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.
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 . This has also been found for mice . 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 . This might be a reflection of regional differences in the recombination frequency in mitotic chromo- somes.
Induction of recombination by mutagenic agents
Extrachromosomal homologous recombination The induction of extrachromosomal recombi-
nation after mutagenic treatment has been stud- ied using viruses [23-27] and plasmids  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
which a recombination has to occur in order to create a functional product. Mutagenic agents studied include UV light and MMC (Table 2).
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.
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.
Recombination between extrachromosomal DNA and chromosomes
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 , and will therefore be discussed only briefly here.
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 .
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 . In this case the integration is due
to a nonhomologous recombination process, since no large sequence homology exists between the participating sequences at the site of integration.
Recently, Mudgett and Taylor  showed that transfer of sequences from chromosomes to plas-
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
INDUCTION OF HOMOLOGOUS RECOMBINATION OF INTEGRATED SEQUENCES
Cel l s Construct Mutagenic agent Induction Reference
Mouse LM tk aprt tandem mutated tk genes MMC + 55 TPA - 55 Mezerein - 55
Mouse L tk - tandem mutated tk genes MMC + 56
BPDE + 56 MNNG + 56 UV + 56 6Co - 56
Mouse L tk - tandem mutated tk genes 1-NOP + 57
N-Aco-AAF + 57 4-NQO + 57
KMST-6 (human) tandem mutated hygromycin UV + 81 resistance genes
XP20S(SV), XPA UVA + 81 XP2YO(SV), XPF UVA + 81
143 tk - (human) tandem mutated tk genes UV + 80 1-NOP + 80
RD tk - (human) tandem mutated tk genes UV + 80
1-NOP + 80
XP12ROSV40 tk - (XPA) tandem mutated tk genes UV + 80
1-NOP + 80
CHO tandem mutated neo genes MMS + 58 HN2 + 58
CHO-EM9 tandem mutated neo genes MMC + 59
X-rays - 59 BPDE - 59 MMS + 59 HN2 + 59 BrdUrd + 59
LM205 (human) neo gene lacking promoter TPA + 63
m-AMSA + 63 BrdUrd + 63 MNNG + 63
Mouse FM3A repeated sequence Vr MNNG + 64 MNNG + TPA + 64
Human lymphocytes HLA-A X-rays + 65
MMC + 65
cells. Linearization of the plasmid increased the frequency of chromosome-plasmid recombina- tion, whereas treatment with UV light did not.
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].
Intra- and interchromosomal recombination eL,ents The induction of exchanges between chromo-
somes in intact cells has mostly been studied at the cytogenetic level. However, a few studies have been done on whole organisms.
Treatment of mouse embryos with X-rays on day 9.25 post coitus (p.c.) gave rise to an in- creased number of recombinant mice . Similar results were obtained after combined treatment with EMU and limonine . However, Fisher et al. reported negative results after X-ray irradia- tion of mouse embryos 10.25-10.50 days p.c. . Thus, one cannot exclude the possibility of differ- ential sensitivity to DNA damage, with concomi- tant differences in the rate of recombination at various developmental stages of the mouse em- bryo. Different genetic markers were used in the two studies and this may also have influenced the outcome. Induction of exchanges between daugh- ter and parental DNA strands after replication has been observed using the alkaline elution tech- nique . 1-3% of the dimers formed on the parent strands by UV irradiation were found to be transferred to the daughter strands after 34-45 h.
As mentioned above, induction of recombina- tion events can readily be observed at the cytoge- netic level, both as chromosomal translocations and as sister-chromatid exchanges (SCE), after treatment with mutagenic substances . Cyto- genetic methods are, however, not suitable for mechanistic studies at the molecular level. With the ability to construct artificial systems contain- ing selectable markers such as the herpes simplex TK gene and the neomycin resistance gene, it has become easier to study the mechanisms which are involved in the induction of recombination. The results are summarized in Table 3.
Lin and Sternberg  were the first to show that a DNA damaging agent can induce homolo- gous recombination between two sequences which
are stably integrated in the genome of mam- malian cells. They used a vector containing a tandem repeat of the herpes simplex TK gene in head-to-tail orientation. Deletions were intro- duced into different parts of the coding sequence. The vector was then introduced into mouse LMtk - cells, and two cell clones with the vector construct stably integrated in the genome were isolated and further characterized. Treatment with MMC induced a higher frequency of TK- positive recombinants in both cell lines while treatment with the tumor promoters TPA and mezerein did not. Southern blots of genomic DNA from TK-positive clones confirmed that an active TK gene had been formed.
Wang et al.  also used a herpes TK gene construction which had been inserted into mouse cells. The two TK genes were mutated by inser- tion of XhoI linkers at different positions. Treat- ment of the ceils with BPDE, UV, MMC and MNNG clearly resulted in an increased frequency of TK + recombinants while ionizing radiation from cobalt-60 did not cause any increase in the number of recombinant clones per plate. They also studied the effect of the structurally related polycyclic aromatic carcinogens 1-NOP, N-Aco- AAF and N-NQO. All of these agents induced recombination but the dose-response curve, i.e., how efficient the compound was inducing recom- bination, varied for each compound. This de- pended on whether cell death, concentration of mutagen, or number of adducts per unit of DNA was used as the measure of DNA damage for that agent .
Hellgren et al. [58,59] used two tandemly ar- ranged neo genes that contained deletions, of roughly equal size, at different positions within the coding sequence to study induction of recom- bination by mutagenic agents. The vector was transfected into CHO cells, and cell lines with the vector stably integrated in the genome were iden- tified. The agents MMS and MMC, but not X-rays and HN2, were found to increase the frequency of recombinants. In contrast, the well character- ized CHO mutant EM9  showed a dose re- sponse for HN2 as well and also after treatment with BrdUrd.
Ayusawa et al.  used a different assay sys- tem. The human thymidylate synthetase gene (TS)
was introduced into mouse TK- cells through DNA mediated gene transfer, and TK cell clones were isolated. Treatment of one isolated clone, CO, with X-rays and BrdUrd resulted in an increased number of TK- cell clones. Southern blot analysis showed an apparent loss of the whole TK gene, most likely through intrachromo- somal recombination.
Murnane and coworkers [62,63] used a con- struct where the SV40 promoter/enhancer had been inserted downstream of the neo gene, thereby making it transcriptionally silent. This construct was introduced into human cells. The gene became active when a genomic rearrange- ment, resulting in a duplication of the construct, placed the promoter in front of the neo gene. A large number of mutagenic substances were tested, but only X-rays, MNNG, m-AMSA, Brd- Urd, and TPA resulted in weak induction of genomic duplication.
Endogenous genes or sequences have been used as assay systems in a few studies. Kominami  used the internal repeated sequence Vr which is present in approximately 6000 copies in the mouse genome and is located near the ribosomal DNA sequences. Treatment of mouse FM3A cells with the alkylating agent MNNG induced recom- bination. This was assayed by the appearance of new bands on Southern blots. Furthermore, the recombination frequency in this system could be increased even further if the cells were treated first with TPA and then MNNG.
Morley and coworkers used the HLA complex as a probe for the study of gene rearrangements involving recombination in human T-lymphocytes [65,66]. Treatment with MMC and X-rays in- creased the frequency of deletions at the HLA-A locus in a dose-dependent manner, from about 40% in the spontaneous mutants to 78% in the X-irradiated mutants. They argued that the high frequency of deletions among the spontaneous mutants was due to the fact that this region undergoes 'normal' deletions during production of HLA proteins.
In conclusion, damage to DNA in the mam- malian genome can induce recombination be- tween chromosomally integrated sequences, and this can be demonstrated in many different types of assay systems.
It should also be pointed out that not only DNA damage, but also other types of stress such as thymidylate deprivation, can cause an in- creased recombination activity [67,61]. Thymidy- late deprivation also resulted in increased recom- bination and this was measured in protein ex- tracts from cultures of starved cells, using homol- ogous recombination between plasmids with dif- ferent mutations in a selectable marker, as sub- strate. Incubation with extracts was followed by transformation of bacteria and scoring for the active marker .
Factors influencing homologous recombination in mammalian cells
The induction of recombination seems to be influenced by several cellular processes such as the different DNA repair systems, protein and DNA synthesis, and the cell cycle stage. In addi- tion, the type of DNA damage introduced and the type of DNA construct used in the assay also appear to have an influence.
DNA damage It is clear from several reports that DNA dam-
age per se plays an important role in induction of recombination. Bhattacharyya et al.  found a linear correlation between the amount of cova- lently bound mutagen and the induced number of recombinants derived from intrachromosomal re- combination. Similarly a linear relationship has been found for the amount of UV and HMT damage to plasmids and the frequency of integra- tion in the genome [33,42]. However, not every type of DNA damage seems to be able to in- crease the frequency of recombination. This has been studied most extensively with respect to integration of plasmids. Pyrimidine dimers (in- duced by UV treatment) are apparently the prin- cipal agent for stimulation of integration of dam- aged plasmids [33,41]. UV induced cross-links may possibly also contribute to the effect , since HMT induced interstrand cross-links also stimulate integration of plasmids .
Modification of plasmid DNA by AAF also causes increased integration in the genome , whereas thymine glycols, apurinic sites, and sin- gle-strand nicks do not result in any stimulation
[33,41]. These results suggest that 'bulky' adducts are more efficient inducers of recombination.
The location of the damage also seems to play a role, at least for chromosomal integration. Damage in the coding sequence of the marker gene on the plasmid was found to make that region a more likely target for nonhomologous recombination. This leads to an increased proba- bility for that region to be involved in integration in the genome and disruption of the reading frame of the marker gene [40,42]. This may ex- plain why the frequency of recovered recombi- nants decreases at higher doses of mutagenic agents.
DNA repair A correlation between DNA repair and ge-
netic recombination has been documented for E. coli and S. cereuisiae [68-71], and also to a cer-
tain extent for mammalian cells. For instance, the CHO cell mutant EM9 (which is sensitive to alkylating agents) also has a decreased ability to recombine plasmids extrachromosomally through homologous recombination [72,73]. The CHO mutants in the xrs series that are sensitive to ionizing radiation  also have a decreased abil- ity to integrate DNA into the genome by nonho- mologous recombination [73,75,76]. The SCID mouse, with a defect in immunoglobulin gene recombination, has an increased sensitivity to X- rays [77-79].
Defects in excision repair do not seem to influ- ence the frequency of induced recombinants in any of the three types of system investigated. Experiments with xeroderma pigmentosum cells show that the repair defect in these cells does not inhibit the stimulation of intraplasmidal (viral) recombination after treatment of the DNA with
> v i v > I !!I I l
Can be scorexl as activated through:
+ Gene conversion
+ Intrachromsotnal recombination
+ lnterchromsomal recombination
>V V > + Gene conversion
- Inlerchrolnosomal recombination
~7 > '> V + Gene conversion
V > >V
+ lnterchromosomal recombination
Fig. 1. Hypothetical construct for recombination assay containing two mutated selectable markers in head-to-tail formation and the possible recombination events that can be detected using them. Hatched box, promoter sequence; open box, selectable marker; arrow, transcriptional orientation of the marker; triangle, position for the introduced mutation, in this case deletion; thin line,
vector sequence separating the mutated markers.
UV light . Similarly, transfection of xero- derma pigmentosum group A and F cells with mutagen damaged plasmids caused an increased number of cells to integrate DNA in the genome [33,41]. The same, no apparent effect of deficien- cies in DNA repair, was found for intrachromoso- mal recombination [80,81]. CHO cells and repair deficient CHO cell mutants reacted in the same way to treatment with DNA damaging agents again indicating that the repair defect in these cells had no direct effect on the recombination process [35,36,41,43,82]. Taken together, these experiments indicate that DNA excision repair is probably not directly involved in the stimulation of recombination that is induced by DNA damag- ing agents. However, so far, not all aspects of DNA repair have been studied in this context and there might still be other responses in vari- ous XP complementation groups and repair defi- cient cell lines which have not yet been tested in recombination assays.
Homologous recombination between extra- chromosomal viral sequences was not affected in one ataxia telangiectasia cell line studied .
It would be interesting to study to what extent DNA damage stimulates recombination in other DNA repair deficient cell hnes as Fanconi's ane- mia patients. The molecular basis for this disease is supposed to be a deficiency in repair of DNA cross-links [83-85]. It would therefore be very interesting to study how Fanconi's anemia cells recombine plasmids containing different types of DNA damage extrachromosomally as well as their ability to integrate plasmids with such damage.
DNA constructs In principle, all the different types of DNA
constructions that have been used seem to be able to participate in recombination. One vari- able that might be important in some cases is whether a DNA element has to integrate or not in order to replicate. Vos et al.  reported that DNA damage stimulated integration of an EBV- based vector that had to integrate while no stimu- lation was found for a vector that could replicate episomally. Another variable is the ability of a particular construct to respond to different types of recombination events. The construct used for
the recombination assay may influence the fre- quency of recombination for the simple reason that it is not sensitive to a particular set of events. In principle, one can think of several factors that are of importance, e.g., the type of mutations introduced into the marker and where they are located in the coding sequence. Fig. 1 shows a hypothetical example of how different locations of mutations and promoters in a tandem gene arrangement may influence the results by exclud- ing one or another of the possible recombination events from being recovered in the assay. Since most authors have developed their own types of construct when studying induction of recombina- tion, a thorough comparison of their results is not always possible. However, in these few studies where comparisons have been possible, mam- malian cells of different species apparently yield similar proportions of recombination events. Ma- her et al. found about the same proportions of gene conversions and other exchanges in mouse and other mammalian cells [56,57,81]. The type of mutations, insertion/deletion and the size of them may also influence the proportion of scored re- combination events. This, however, remains to be further investigated.
The role of protein, DNA and RNA synthesis Nagata et al.  have shown that protein
synthesis is required to demonstrate the stimula- tion of DNA mediated gene transfer in mouse FM3A cells after UV light exposure. Exposure of the cells to cycloheximide prior to transfection inhibited the UV stimulating effect. Protein bind- ing to modified nucleotides seems to occur on damaged DNA. Cotransfection experiments sug- gest that the cycloheximide sensitive stimulating activity has preference for damaged DNA. In the presence pSV2-gpt, the efficiency of transforma- tion, i.e., integration into the genome by the unmodified pSV2-neo, was reduced in favor of integration of damaged pSV2-gpt .
These experiments suggest that DNA damage induces synthesis of a protein or proteins that have the ability to bind to DNA and that they have a preference for damaged DNA over un- damaged DNA. This binding to DNA may in- volve protein(s) characterized by several groups [87-90]. At least two activities have been demon-
strated by gel-shift assay, one that binds to cis- platinum modified DNA and the other to UV modified DNA [87,90], The activity that binds to cisplatinum modified DNA apparently binds specifically to these types of damages only while the UV damage binding activity also has affinity for cisplatinum damage . Interestingly, xero- derma pigmentosum complementation group E seems to lack one of these activities, the UV binding activity [88,89].
DNA synthesis may also influence recombina- tion. It has previously been shown that extrachro- mosomal recombination peaks during the S-phase  as described above, while integration into the genome does not seem to be cell cycle dependent .
The increase of recombination induced by mu- tagenic agents may involve two mechanisms. One is DNA repair activity and the other is transcrip- tional activity. Recent results suggest that induc- tion of mRNA synthesis is correlated with an increased recombination frequency. The increase in the transcriptional activity heightens the prob- ability for a recombination event involving the induced gene in question. It should be pointed out that these two mechanisms are not mutually exclusive, on the contrary they might very well cooperate. This is supported by results that show that DNA repair preferentially acts on transcrip- tionally active genes . In E. coli recombina- tion is a part of the SOS system for DNA repair and is mediated by the RecA protein . The role of DNA repair in recombination is therefore well characterized at least in bacteria and also to some extent in yeast .
It has recently been shown that increased mRNA synthesis at a specific locus is correlated with an increased recombination frequency of that locus in yeast [9,13,14] and also in mam- malian cells [10-12]. Moreover, mutagens have been shown to increase the transcriptional activ- ity from a number of promoters such as SV40, IAP LTR,/3-actin, aprt, spr2, collagenase, HIV-1, metallothionein and Mo-Musv LTR [93-96]. Of importance in this context is that many investiga- tors who have studied induction of recombination by mutagenic agents have used the SV40 pro- moter/enhancer to drive selectable markers in mammalian cells. The correlation between tran-
scription and recombination makes it difficult to determine whether the observed induction of re- combination is due solely to a DNA repair system or if it is influenced by an increased transcrip- tional activity. Recently, Kedar et al.  showed that MNNG induced a weak but reproducible increase in transcription from the TK promoter after treatment of cells with MNNG. This pro- moter has also been used for studies of recombi- nation after treatment with mutagens [55- 57,80,81].
The increase in mRNA transcription by DNA polymerase II has to be mediated by transcription factors. It is therefore interesting that transient increases in mRNA levels for c-los and c-jun, which together constitute the transcription factor AP-1, can be induced by mutagens [96,98-101]. Stein et al.  also showed that the degree of c-los mRNA induction correlates with the occur- rence of UV light caused pyrimidine dimers. This indicates a link between the sensing of DNA damage and the induction of transcription. These reports lend support to the hypothesis that an increase in RNA transcription can cause an in- creased recombination frequency.
Recombination pathways Several lines of evidence suggest that there are
multiple recombination pathways in mammalian cells. In E. coli and most likely also in S. cere- eisiae, multiple pathways for recombination have been demonstrated [102,103] and there is a priori no reason to assume that this should not also be the case in mammalian cells. For instance EM9 cells, which integrate plasmids into the genome with the same efficiency as the mother clone AA8 [72,73], have a decreased ability to recombine plasmids extrachromos0mally. Xrs mutants, which have a decreased ability to integrate DNA into the genome , show an apparently normal abil- ity to catalyze homologous recombination extra- chromosomally . These results indicate the existence of at least two pathways for recombina- tion in mammalian ceils; one for homologous recombination that operates between plasmids/ chromosomes or within a chromatid and is cell cycle dependent, and another nonhomologous pathway that operates when nonhomologous DNA is integrated into the genome, and is cell
cycle independent. Interestingly, both of these pathways can be stimulated by induction of DNA damage, indicating a possible functional overlap.
Taken together the reports cited above suggest that certain types of DNA damage induce a re- combinational response, probably as part of a DNA repair activity, but apparently not directly associated with DNA excision repair. Damage in plasmids, viruses, as well as chromosomal DNA can induce the response, which therefore does not seem to depend on the type of DNA taking part in the process. This holds both for model systems and for endogenous genes and works both extra- and intrachromosomally. CHO cells are a possible exception to this since damage in plasmids does not induce an increased frequency of recombination. The stimulating effect of DNA damage on integration of foreign DNA does not seem to be restricted to certain cell populations, i.e., cells that are competent to integrate DNA are not further induced to take up more DNA but rather more cells in the population are made competent to integrate DNA into the genome [34,41,42].
It is still uncertain how different types of model system affect the outcome of the induced recom- bination events and if this is the major cause for the observed differences in mechanisms between the systems. Here more research is needed, espe- cially if these types of system are going to be used as a primary screening for possible carcinogenic substances. As indicated above a number of other factors such as DNA and RNA synthesis, recom- bination pathways etc. may have an influence on the induction of recombination and the final out- come. To what extent is not yet clear and the importance of these factors merits further investi- gation.
Note added in proof
Nairn et al. (R.S. Nairn, G.M. Adair, C.B. Christman and R.M. Humphrey (1991) Ultravio- let stimulation of intermolecular homologous re- combination in Chinese hamster ovary ceils, Mol. Carcinogen., 4, 519-526) have shown that a CHO
cell line with a mutation in the ERCC2 gene may have altered recombination in response to UV damage, although the cell line is still recombina- tion-proficient.
My work has been supported by the Swedish Medical Research Council, the Swedish Cancer Society, the Karolinska Institute, the Swedish Work Environmental Fund, The Nilsson-Ehle Foundation, and the Magnus Bergvall Founda- tion. I am indebted to Professor Bo Lambert for his continuous support and interest.
1 Alt, F.W., T.K. Blackwell, R.A. De Pinho, M.G. Reth and G.D. Yancopoulos (1986) Regulation of genome rearrangement events during lymphocyte development, Immunol. Rev., 89, 5-30.
2 Lehrman, M.A., J.L. Goldstein, D.W. Russell and M.S. Brown (1987) Duplication of seven exons in LDL recep- tor gene caused by Alu-Alu recombination in a subject with familial hypercholesterolemia, Cell, 48, 827-835.
3 Nicholls, R.D., N. Fischel-Ghodsian and D.R. Higgs (1987) Recombination at the human alpha-globin cluster: sequence features and topological constraints, Cell, 49, 369-378.
4 Kenne, K., and S. Ljungquist (1987) RecA-like activity in mammalian cell extracts of different origin, Mutation Res., 184, 229-236.
5 Kucherlapati, R.S., J. Spencer and P.D. Moore (1985) Homologous recombination catalyzed by human cell ex- tracts, Mol. Cell. Biol., 5, 714-720.
6 Wong, E.A., and M.R. Capecchi (1987) Homologous re- combination between coinjected DNA sequences peaks in early to mid-S phase, Mol. Cell. Biol., 7, 2294-2295.
7 Miller, D.G., M.A. Adam and A.D. Miller (1990) Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection, Mol. Cell. Biol., 10, 4239-4242.
8 Young, C.S.H., G. Cachianes, P. Munz and S. Silverstein (1984) Replication and recombination in adenovirus-in- fected cells are temporally and functionally related, J. Virol., 51,571-577.
9 Grimm, C., P. Schaer, P. Munz and J. Kohli (1991) The strong ADH1 promoter stimulates mitotic and meiotic recombination at the ADE6 gene of Schizosaccharomyces pombe, Mol. Cell. Biol., 11, 289-298.
10 Nickoloff, J.A., and R.J. Reynolds (1990) Transcription stimulates homologous recombination in mammalian cells, Mol. Cell. Biol., 10, 4837-4845.
11 Schlissel, M.S., and D. Baltimore (1989) Activation of immunoglobulin kappa gene rearrangements correlates
with induction of germline kappa gene transcription, Cell, 58, 1001-1007.
12 Blackwell, K., M.W. Moore, G.D. Yancopoulos, H. Sub, S. Lutzker, E. Selsing and F.W. Alt (19861 Recombina- tion between immunoglobulin variable region gene seg- ments is enhanced by transcription, Nature, 324, 585-589.
13 Stewart, S.E., and G.S. Roeder (1989) Transcription by RNA polymerase I stimulates mitotic recombination in Saccharomyces ceret~isiae, Mol. Cell. Biol., 9, 3464-3472.
14 Thomas, B.J., and R. Rothstein (1989) Elevated recombi- nation rates in transcriptionally active DNA, Cell, 56, 619- 630.
15 Bollag, R.J., A.S. Waldman and R.M. Liskay (1989) Ho- mologous recombination in mammalian cells, Annu. Rev. Genet., 23, 199-225.
16 Roth, D., and J. Wilson (1988) Illegitimate recombination in mammalian cells, in: R. Kucherlapati and G.R. Smith (Eds.), Genetic Recombination, American Society for Microbiology, Washington, DC, pp. 621-653.
17 Wang, J.C., P.R. Caron and R.A. Kim (1990) The role of DNA topoisomerase in recombination and genome stabil- ity: a double-edged sword?, Cell, 62, 403-406.
18 Subramani, S., and B.L. Seaton (1988) Homologous re- combination in mitotically dividing mammalian cells, in : R. Kucherlapati and G.R. Smith (Eds.), Genetic Recom- bination, American Society for Microbiology, Washing- ton, DC, pp. 549-573,
19 Kucherlapati, R. and P.D. Moore (19881 Biochemical aspects of homologous recombination in mammalian so- matic cells, in: R. Kucherlapati and G.R. Smith (Eds.), Genetic Recombination, American Society for Microbiol- ogy, Washington, DC, pp. 575-595.
20 Meuth, M. (1989) Illegitimate recombination in mam- malian cells, in: D.E. Berg and M.M. Howe (Eds.), Mo- bile DNA, American Society for Microbiology, Washing- ton, DC, pp. 833-860.
21 Donis-Keller, H. et al. (1987) A genetic linkage map of the human genome, Cell, 51, 319-337.
22 Wahl, G.M., B.R, de Saint Vincent and M.L. DeRose (1984) Effect of chromosomal position on amplification of transfected genes in animal cells, Nature, 307, 516-520.
23 Van der Lubbe, J.L.M., H.J.M. Rosdorff and A.J. van der Eb (1989) Homologous recombination is not enhanced in UV-irradiated normal and repair-deficient human fibro- blasts, Mutation Res., 217, 153-161.
24 Dubbs, D.R., M. Rachmeier and S. Kit 11974) Recombi- nation between temperature sensitive mutants of simian virus 40, Virology, 57, 161-174.
25 Gentil, A., A. Margot and A. Sarasin (1983) Effect of UV-irradiation on genetic recombination of simian virus 40 mutants, in: E.C. Friedberg and P.C. Hanawalt (Eds.), Cellular Responses to DNA Damage, Liss, New York, pp. 385-396.
26 DasGupta, U.B., and W.C. Summers (19801 Genetic re- combination of herpes simplex virus, the role of host cell and UV-irradiation of the virus, Mol. Gen. Genet., 178, 617-623.
27 Hall, J.D., J.D. Featherstone and R.E. Almy 11980) Evi-
dence for repair of ultaviolet light-damaged herpes sim- plex virus in human fibroblast by a recombination mecha- nism, Virology, 105, 490-500.
28 Proti~, M.. E. Roilides, A.S. Levine and K. Dixon (1988) Enhancement of DNA repair capacity of mammalian cells by carcinogen treatment, Somat. Cell. Mol. Genet. 14, 351-357.
29 Sweigert, S.E., and D. Carroll (1990) Repair and recom- bination of X-irradiation plasmids in Xenopus laet,is oocytes, Mol. Cell. Biol., 10, 5849-5856.
30 Hays, J.B., E.J. Ackerman and Q. Pang (19901 Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laet~is oocytes, Mol. Cell Biol., 10, 3505-3511.
31 Vos, J.-M.H., and P.C. Hanawalt (1989) Effect of DNA damage on stable transformation of mammalian cells with integrative and episomal plasmids, Mutation Res., 220, 205-220.
32 Spivak, G.., A.K. Ganesan and P.C. Hanawalt (1984) Enhanced transformation of human cells by UV-irradiat- ed pSV2 plasmids, Mol. Cell. Biol., 4, 1169-1171.
33 Spivak, G., S.A. Leadon, J.-M. Vos, S. Meade, P.C. Hanawalt and A.K. Ganesan (1988) Enhanced transform- ing activity of pSV2 plasmids in human cells depends upon the type of damage introduced into the plasmid, Mutation Res., 193, 97-108.
34 Perez, C.F., M.R, Botchan and C.A. Tobias (19851 DNA-mediated gene transfer efficiency is enhanced by ionizing and ultraviolet irradiation of rodent cells in vitro. 1. Kinetics of enhancement, Radiation Res., 1/14, 2011-213.
35 Perez, C.F., and L.D. Skarsgard (1986) Radiation en- hancement of the efficiency of DNA-mediated gene transfer in CHO UV-sensitive mutants, Radiation Res., 106, 401-407.
36 Rubin, J.S. (1988) Effect of gamma rays on the efficiency of gene transfer in DNA repair-proficient and -deficient cell lines, Somat. Cell. Mol. Genet., 14, 613-621.
37 Chu, G., and P. Berg 11987) DNA cross-linked by cis- platin: a new probe for the DNA repair defect in xero- derma pigmentosum, Mol. Biol. Med., 4, 277-290.
38 Debenham, P.G., and M.B.T. Webb (1984) The effect of X-rays and ultaviolet light on DNA-mediated gene trans- fer in mammalian cells, Int. J. Radiat. Res., 46, 555-568.
39 Barbis, D.P., R.A. Schultz and E.C. Friedberg (1985) Isolation and partial characterization of virus-trans- formed cell lines representing the A, G, and variant complementation groups of xeroderma pigmentosum, Mutation Res., 165, 175-184.
40 Leadon, S.A., A.K. Ganesan and P.C. Hanawalt (1987) Enhanced transforming activity of ultraviolet-irradiation pSV2-gpt is due to damage outside the gpt transcription unit, Plasmid, 18, 135-141.
41 Van Duin, M., A. Westerveld and J.H.J. Hoeijmakers (1985) UV stimulation of DNA-mediated transformation of human cells, Mol. Cell. Biol., 5, 734-741.
42 Vos, J.-H.H., and P.C. Hanawalt (1989) DNA interstrand cross-links promote chromosomal integration of a se- lected gene in human cells, Mol. Cell. Biol., 9, 2897-2905.
43 Nairn, R.S., R.M. Humphrey and G.M. Adair (1988) Transformation of UV-hypersensitive Chinese hamster ovary cell mutants with UV-irradiated plasmids, Int. J. Radiat. Biol., 53, 249-260.
44 Herskind, C., J. Thacker and O. Westwergaard (1985) Radiation-induced inactivation of single isolated genes mediated by secondary radicals, Radiat. Protect. Dosime- try, 13, 153-156.
45 Postel, E.H. (1985) Enhancement of genetic transforma- tion frequencies of mammalian cell cultures by damage to the cell DNA, Mol. Gen. Genet., 201, 136-139.
46 Nagata, Y., H. Tagaki, T. Morito and M. Oishi (1984) Stimulation of DNA-mediated transformation by UV ir- radiation of recipient (mouse FM3A) cells, J. Cell. Phys., 121,453-457.
47 Mudgett, J.S., and W.D. Ta}lor (1990) Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells, Mol. Cell. Biol., 10, 37-46.
48 Luisi-DeLuca, C., R.D. Porter and W.D. Taylor (1984) Stimulation of recombination between homologous se- quences on plasmid DNA and chromosomal DNA in Escherichia coli by N-acetoxy-2-acetylaminofluorene, Proc. Natl. Acad. Sci. USA, 81, 2831-2835.
49 Abbott, P.J. (1985) Stimulation of recombination between homologous sequences on carcinogen-treated plasmid DNA and chromosomal DNA by induction of the SOS response in Escherichia coli K12, Mol. Gen. Genet., 201, 129-132.
50 Panthier, J.-J., J.-L. Gu6net, H. Condamine and F. Jacob (1990) Evidence for mitotic recombination in wei /+ heterozygous mice, Genetics, 125, 175-182.
51 Fahrig, R. (1984) Genetic mode of action of cocarcino- gens and tumor promoters in yeast and mice, Mol. Gen. Genet., 194, 7-14.
52 Fisher, G., D.A. Stephenson and J.D. West (1986) Inves- tigation of the potential for mitotic recombination in the mouse, Mutation Res., 164, 381-388.
53 Fornace Jr., A.J. (1983) Recombination of parent and daughter strand after UV-irradiation in mammalian cells, Nature, 304, 552-554.
54 Takehisa, S. (1982) Induction of sister chromatid ex- changes by chemical mutagens, in: S. Wolff (Ed.), Sister Chromatid Exchange, Wiley, New York, pp. 87-147.
55 Lin, F.-L., and N. Sternberg (1984) Homologous recombi- nation between overlapping thymidine kinase gene frag- ments inserted into a mouse cell genome, Mol. Cell. Biol., 4, 852-861.
56 Wang, Y., V.M. Maher, R.M. Liskay and J.J. McCormick (1988) Carcinogens can induce homologous recombina- tion between duplicated chromosomal sequences in mouse L cells, Mol. Cell. Biol., 8, 196-202.
57 Batthacharyya, N.P., V.M. Maher and J.J. McCormick (1989) Ability of structurally related polycyclic aromatic carcinogens to induce homologous recombination be- tween duplicated chromosomal sequences in mouse L cells, Mutation Res,, 211,205-214.
58 Hellgren, D., H. Luthman and B. Lambert (1989) In- duced recombination between duplicated neo genes sta- bly integrated in the genome of CHO cells, Mutation Res., 210, 197-206.
59 Hellgren, D., S. Sahl~n and B. Lambert (1989) Mutagen- induced recombination between stably integrated neo gene fragments in CHO and EM9 cells, Mutation Res., 226, 1-8.
60 Thompson, L.H. et al. (1982) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand- break repair, and an extraordinary baseline frequency of sister-chromatid exchange, Mutation Res., 95, 427-440.
61 Ayusawa, D. et al. (1986) Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells, Mol. Cell. Biol., 6, 3463-3469.
62 Murnane, J.P. (1986) Inducible gene expression by DNA rearrangements in human cells, Mol. Cell. Biol., 6, 549- 558.
63 Murnane, J.P., and M.J. Yezzi (1988) Association of high rate of recombination with amplification of dominant selectable gene in human cells, Somat. Cell Mol. Genet., 14, 273-286.
64 Kominami, R. (1990) A sensitive assay for detecting mu- tations from unequal homologous recombination without phenotypic selection, Mutation Res., 243, 133-139.
65 Janatipour, M., K.J. Trainor, R. Kutlaca, G. Bennett, J. Hay, D.R. Turner and A.A. Morley (1988) Mutations in human lymphocytes studied by an HLA selection system, Mutation Res., 198, 221-226.
66 Turner, D.R., S.A. Grist, M. Janatipour and A.A. Morley (1988) Mutations in human lymphocytes commonly in- volve gene duplication and resemble those seen in cancer cells, Proc. Natl. Acad. Sci. USA, 85, 3189-3192.
67 Mishina, Y., D. Ayusawa, T. Seno and H. Koyama (1991) Thymidylate stress induces homologous recombination activity in mammalian cells, Mutation Res., 246, 215-220.
68 Smith, G.R. (1988) Homologous recombination in prokaryotes, Microbiol. Rev., 52, 1-28.
69 Friedberg, E.C. (1988) Deoxyribonucleic acid repair in the yeast Saccharomyces cerevisiae, Microbiol. Rev., 52, 70-102.
70 Schiestl, R.H., and S. Prakash (1988) RAD1, an excision repair gene of Saccharomyces cerevisiae, is also involved in recombination, Mol. Cell. Biol., 8, 3619-3626.
71 Schiestl, R.H., and S. Prakash (1990) RadlO, an excision repair gene of Saccharomyces cerevisiae, is involved in the RAD1 pathway of mitotic recombination, Mol. Cell. Biol., 10, 2485-2491.
72 Hoy, C.A., J.C. Fuscoe and L.H. Thompson (1987) Re- combination and ligation of transfected DNA in CHO mutant EM9, which has high levels of sister chromatid exchange, Mol. Cell. Biol., 7, 2007-2011.
73 Wahls, W.P., and P.D. Moore (1990) Relative frequencies of homologous recombination between plasmids intro- duced into DNA repair-deficient and other mammalian somatic cell lines, Somat. Cell Mol. Genet., 16, 321-329.
74 Jeggo, P.A. (1990) Studies on mammalian mutants defec- tive in rejoining double-strand breaks in DNA, Mutation Res., 239, lk16.
75 Hamilton, A.A., and J. Thacker (1987) Gene recombina- tion in X-ray-sensitive hamster cells, Mol. Cell. Biol., 7, 1409-1414.
76 Jeggo, P.A., and J. Smith-Ravin (1990) Decreased stable transfection frequencies of six X-ray-sensitive CHO strains, all members of the xrs complementation group, Mutation Res., 218, 75-86.
77 Fulop, G.M., and R.A. Phillips (1990) The scid mutation in mice causes a general defect in DNA repair, Nature, 347, 479-482.
78 Biedermann, K.A., J. Sun, A.J. Giaccia, L.M. Tosto and J.M. Brown (1991) Scid mutation in mice confers hyper- sensitivity to ionizing radiation and a deficiency in DNA double-strand break repair, Proc. Natl. Acad. Sci. USA, 88, 1394-1397.
79 Hendrickson, E.A., X.-Q. Qin, E.A. Bump, D.G. Schatz, M. Oettinger and D.T. Weaver (1991) A link between double-strand break-related repair and V(D)J recombi- nation: the scid mutation, Proc. Natl. Acad. Sci. USA, 88, 4061-4065.
80 Bhattacharyya, N.P., V.M. Maher and J.J. McCormick (1990) Effect of nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene, Mol. Cell. Biol., 10, 3945- 3951.
81 Tsujimura, T., V.M. Maher, A.R. Godwin, R.M. Liskay and J.J. McCormick (1990) Frequency of intrachromoso- mal homologous recombination induced by UV irradia- tion in normally repairing and excision repair-deficient human cells, Proc. Natl. Acad. Sci. USA, 87, 1566-1570.
82 Nairn, R.S., R.M. Humphrey and G.M. Adair (1988) Transformation depending on intermolecular homolo- gous recombination is stimulated by UV damage in trans- fected DNA, Mutation Res., 208, 137-141.
83 Fujiwara, Y., M. Tatsumi and M.S. Sasaki (1977) Cross- link repair in human cells and its possible defect in Fanconi's anemia cells, J. Mol. Biol., 113, 635-649.
84 Fujiwara, Y. (1982) Defective repair of mitomycin C crosslinks in Fanconi's anemia and loss in confluent nor- mal human and xeroderma pigmentosum cells, Biochim. Biophys. Acta, 699, 217-225.
85 Matsumoto, A., J.-M.H. Vos and P.C. Hanawalt (1989) Repair analysis of mitomycin C-induced DNA crosslink- ing in ribosomal RNA genes in lymphoblastoid cells from Fanconi's anemia cells, Mutation Res., 217, 185-192.
86 Vos, J.-M.H., E.L. Wauthier and P.C. Hanawalt (1989) DNA damage stimulates human cell transformation by integrative but not episomal Epstein-Barr virus-derived plasmids, Mol. Carcinogen, 2, 237-244.
87 Chu, G., and E. Chang (1990) Cisplatin-resistant cells express increased levels of a factor that recognizes dam- aged DNA, Proc. Natl. Acad. Sci. USA, 87, 3324-3327.
88 Patterson, M., and G. Chu (1989) Evidence that xero- derma pigmentosum cells from complementation group E
are deficient in a homolog of yeast photolyase, Mol. Cell. Biol. 9, 5105-5112.
89 Hirschfeld, S., A.S. Levine, K. Ozato and M. Proti~5 (1990) A constitutive damage-specific DNA-binding pro- tein is synthesized at higher levels in UV-irradiated pri- mate cells, Mol. Cell. Biol., 10, 2041-2048.
90 Chao, C.C.K., S.-L. Huang and S. Lin-Chao (1991) Cross-resistance to UV radiation of a cisplatin-resistant human cell line: overexpression of cellular factors that recognize UV-modified DNA, Mol. Cell. Biol, 11, 2075- 2080.
91 Wong, E.A., and M.R. Cappechi (1985) Effect of cell cycle position on transformation by microinjection, So- mat. Cell Mol. Genet., 11, 43-51.
92 Elespuru, R.K. (1987) Inducible responses to DNA dam- age in bacteria and mammalian cells, Environ. Mol. Mu- tagen., 10, 97-116.
93 Bohr, V.B., D.H. Phillips and P.C. Hanawalt (1987) Het- erogeneous DNA damage and repair in the mammalian genome, Cancer Res., 47, 6426-6436.
94 Kleinberger, T., J.B. Flint, M. Blank, S. Etkin and S. Lavi (1988) Carcinogen induced trans activation of gene ex- pression, Mol. Cell. Biol., 8, 1366-1370.
95 Gibbs, S., F. Lohman, W. Teubel, P. van de Putte and C. Backendorf (1990) Characterization of the human spr2 promoter: induction after irradiation or TPA treatment and regulation during differentiation of cultured primary keratinocytes, NAR, 18, 4401-4407.
96 Lin, C.S., D.A. Goldthwait and D. Samols (1990) Induc- tion of transcription from the long terminal repeat of Moloney murine sarcoma provirus by UV-irradiation, X-irradiation, and phorbol ester, Proc. Natl. Acad. Sci. USA, 87, 36-40.
97 Stein, B., B. Rahmsdorf, A. Steffen, M. Litfin and P. Herrlich (1989) UV-induced DNA damage is an interme- diate step in UV-induced expression of human deficiency virus type 1, collagenase, c-fos, and metallothionein, Mol. Cell. Biol., 9, 5169-5181.
98 Kedar, P.S., S.G. Widen, E.W. Englander, A.J. Fornace and S.H. Wilson (1991) The ATF/CREB transcription factor-binding site in the polymerase beta promoter me- diates the positive effect of N-methyl-N'-nitro-N-nitro- soguanidine on transcription, Proc. Natl. Acad. Sci. USA, 88, 3729-3733.
99 Hollander, M.C., and A.J. Fornace Jr. (1989) Induction of los RNA by DNA-damaging agents, Cancer Res., 49, 1687-1692.
100 Hallahan, D.E., V.P. Sukhatme, M.L. Sherman, S. Viru- dachalam, D. Kufe and R.R. Weichselbaum (1991) Pro- tein kinase C mediates X-ray inducibility of nuclear sig- nal transducers EGR1 and JUN, Proc. Natl. Acad. Sci. USA, 88, 2156-2160.
101 Sherman, M.L., R. Datta, D.E. Hallahan, R.R. Weichsel- baum and D.W. Kufe (1990) Ionizing radiation regulates expression of the c-jun protooncogene, Proc. Natl. Acad. Sci. USA, 87, 5663-5666.
102 Devary, Y., R.A. Gottlieb, L.F. Lau and M. Karin (1991)
Rapid and preferential activation of the C-jun gene dur- ing the mammalian UV response, Mol. Cell. Biol., 11, 2804-2811.
103 Smith, G.R. (1989) Homologous recombination in E. coli: multiple pathways for multiple reasons, Cell, 58, 807-809.
104 Roeder, G.S., and S.E. Stewart (1988) Mitotic recombina- tion in yeast, Trends Genet., 4, 263-267.
105 Wasmuth, J.J., and L. Vock ttall (1984) Genetic demon- stration of mitotic recombination in cultured Chinese hamster cell hybrids, Cell, 36, 697-707.
106 Stringer, J.R., R.M. Kuhn, J.I,. Newman and J.C. Meade (1985) Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells, Mol. Cell. Biol., 5, 2613-2622.
107 HeUgren, D., S. Sahl~n and 13. Lambert (1990) Unequal recombination is a rare event in homologous recombina- tion between duplicated nec gene fragments in CHO cells, Mutation Res., 243, 75-80.
108 Hasty, P., R. Ramirez-Solis, R. Krumlauf and A. Bradley (1991) Introduction of a subtle mutation into the Hox-2.6 locus in embryonic stem cells, Nature, 350, 243-246.
109 Valancius, V., and O. Smithies (1991) Testing an 'in-out' targeting procedure for making subtle genomic modifica- tions in mouse embryonic stem cells, Mol. Cell. Biol., 1, 1402-1408.
110 Seperack, P.K., M.C. Strobel, D.J. Corrow, N.A. Jenkins and N.G. Copeland (1988) Somatic and germ-line reverse mutation rates of the retrovirus-induced dilute coat-color mutation of DBA mice, Proc. Natl. Acad. Sci. USA, 85, 189-192.
11l Subramani, S., and J. Subnitz (1985) Recombination events after transient infection and stable integration of DNA in mouse cells, Mol. Cell. Biol., 5, 659-666.
112 Smith, A.J.H., and P. Berg (1984) Homologous recombi- nation between defective neo genes in mouse 3T6 cells, Cold Spring Harbor Symp. Quant. Biol., 49, 171-181.
113 Janatipour, M., K.J. Trainor, R. Kutlaca, G. Bennett, J. Hay, D.R. Turner and A.A. Morley (1988) Mutation in human lymphocytes studied by an HLA selection system, Mutation Res., 198, 221-226.