thymidylate stress induces homologous recombination activity in mammalian cells

Mutation Research, 246 (1991) 215-220 215 Elsevier

MUT 04932

Thymidylate stress induces homologous recombination activity in mammalian cells

Yuji Mi sh ina 1, Da i A y u s a w a 2, Takesh i Seno 3 and H i d e k i K o y a m a 1

1 Kihara Institute for Biological Research, Yokohama City University, Minami-ku, Yokohama 232, 2 Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113 and 3 Institute of Genetics, Mishima 411 (Japan)

(Received 15 May 1990) (Revision received 27 July 1990)

(Accepted 31 July 1990)

Keywords: Homologous recombination; Thymidylate stress; Mammalian cell; Fluorodeoxyuridine; Gene targeting

Summary

We studied whether homologous recombination activity in mammalian cells could be induced by thymidylate stress (thymidylate deprivation). In vitro recombination activity in cell extracts was measured with pSV2neo-derived plasmids. When prior to the preparation of extracts, mouse FM3A cells were grown in 5-fluorodeoxyuridine (FdUrd), an inducer of thymidylate stress, the homologous recombination activity was significantly induced, as judged from an increase in the number of neomycin-resistant bacterial colonies. Maximum induction was observed in cells treated with 1/~M FUdR for 16 h. However, 3-8 h of treatment of FM3A cells with the drug followed by an additional 8-16-h incubation in its absence was sufficient to induce the recombination activity while slightly reducing their growth rates. These results indicate that thymidylate stress induces homologous recombination activity in mammalian cells as observed in Escherichia coli and in yeast.

Thymidylate stress is known to cause rapid cell death, the so-called thymineless death, studied originally in E. coli (Cohen and Barner, 1954). Such stress is also known to increase genetic recombination events in E. coli (Gallant and Spotts- wood, 1964) and in yeast (see review, Haynes, 1985). Kunz et al. (1980) reported that starvation for thymine nucleotides in yeast induces mitotic recombination but is not mutagenic. Although these studies were done with mutants deficient in thymidylate synthase which is a key enzyme of

Correspondence: Dr. Y. Mishina, Kihara Institute for Biologi- cal Research, Yokohama City University, Nakamura-cho 2- 120-3, Minami-ku, Yokohama 232 (Japan).

thymine nucleotide synthesis, inhibition of dTMP biosynthesis by FdUrd or folate antagonists also causes thymineless death and induces interchro- mosomal recombination in yeast (Kunz et al., 1980, 1986; Barclay et al., 1982; Kunz and Haynes, 1982).

Using thymidylate synthase-negative mutants of mouse FM3A cells (Ayusawa et al., 1982), Koyama et al. (1982) and Ayusawa et al. (1983) have demonstrated that thymineless death also occurs in mammalian cells and that this death is caused by double-strand breaks in their chromosomal DNA. A similar thymineless death due to DNA double-strand breaks has been observed in wild-type FM3A cells treated with FdUrd (Yoshioka et al., 1987; Ayusawa et al., 1988).

0027-5107/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)

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Thymidylate stress has also been reported to induce genetic recombination events in mammalian cells (see review, Seno et al., 1985). For example, the frequency of sister-chromatid exchanges is increased by thymidylate starvation (Hori et al., 1984) and a genetic marker integrated into the host chromosome by transfection is deleted entirely by the same treatment (Ayusawa et al., 1986). However, little is known so far about the mechanisms of these effects.

The thymidylate stress-induced genetic recombination events mentioned above may be ex- plained by an increase in the recombinagenic, broken ends of the chromosomal DNA as recombination substrates. However, there is also the possibility that thymidylate stress affects in some way a cellular system(s) responsible for homologous recombination. Therefore, we studied whether thymidylate stress induced by FdUrd treatment would affect homologous recombination activity in mammalian cells. Using an in vitro assay system, we observed that the recombination activity was increased significantly by thymidylate stress.

Materials and methods

Cells and culture condition A subclonal line (F28-7) of mouse mammary

carcinoma FM3A cells was grown in suspension in ES medium (Nissui Seiyaku Co., Ltd., Tokyo) supplemented with 2% fetal bovine serum (Hy- clone, Utah) as previously described (Koyama and Kodama, 1982). To induce thymidylate stress, FdUrd (Sigma Chem. Co., St. Louis, MO) ranging from 0.01 to 10 ~M was added to FM3A cells in logarithmic growth phase. In experiments to study shorter times of FdUrd treatment, the drug-treated cells were centrifuged, washed with and resuspended in fresh growth medium without the drug but with 10 IaM thymidine, and then cultured. Cell number was determined with a Towa Microcell Counter (Model F-300 ) and cell viability by a dye exclusion test with 0.15% trypan blue in Ca 2+- and Mg2+-free phosphate-buffered saline (PBS-).

Preparation of cell extracts Whole cell extracts were prepared as described

elsewhere (Hotta et al., 1985) with slight modifica- tions. Logarithmically growing cells were har-

vested, washed 3 times with cold PBS- and resuspended in 20 mM Tris-HC1 (pH 7.5), 2 mM dithiothreitol (DTT), 0.1 mM p-aminobenzami- dine, 2 /~M phenylmethylsulfonylfluoride (PMSF: Sigma Chem. Co.), 1 mM EDTA, 5 mM spermi- dine and disrupted with a Dounce homogenizer. To the homogenate, KCI and Brij 58 (Nakarai Chem., Kyoto) were added to final concentrations of 0.5 M and 0.1%, respectively. The suspension was centrifuged at 160000 x g for 1 h, and an equal volume of saturated ammonium sulfate was added to the supernatant. Then the resulting suspension was centrifuged at 8000 x g for 20 rain. The precipitated material was resuspended in 10 mM Tr is -HCl (pH 7.5), 0.1 mM EDTA, 0.2 mM DTT (buffer A) and was loaded on a DEAE Sepharose (Pharmacia LKB Biotech., Uppsala) column. The column was washed with 10 bed volumes of buffer A containing 50 mM NaCl and then eluted with buffer A containing 500 mM NaC1. The eluted fraction was concentrated by ammonium sulfate precipitation. The precipitate was resuspended in 50 mM N-2-hydroxyethyl- piperazine-N'-ethanesulfonic acid (HEPES; Sigma Chem. Co.) (pH 7.8), 1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 10% glycerol (buffer B), and di- alyzed against buffer B.

In vitro recombination assay To detect homologous recombination activity,

we used an in vitro homologous recombination assay system as described by other workers (Kucherlapati et al., 1985; Song et al., 1985). Plasmid pSV2neo (Southern and Berg, 1982) was cleaved with restriction endonuclease Narl or NaeI at 2 sites within the neomycin-resistant (Neo r) gene, and, by ligating the products, we constructed a 248-base pair (bp)-deleted plasmid DL and a 283-bp-deleted plasmid DR, according to the method of Kucherlapati et al. (1985). For the recombination assay, a reaction mixture (100 /~1) containing 400 ng of each of the mutant plasmids, 35 mM HEPES (pH 7.8), 5 mM ATP, 0.2 mM each of dNTPs, 1 mM DTT, 5% (w/v) polyvinylalcohol type II (Sigma Chem. Co.) and cell extract (3-10 txg of protein) was incubated at 30°C for 2 h (Hotta et al., 1985). After the reaction, the DNA was extracted with phenol and approximately 100 ng of the DNA was transfected

into recombination-deficient ( recA-) E. coli strain HB101 or DH1, so that the resulting Neo r colonies were scored. In each experiment, the reaction was done in duplicate and 4 kanamycin plates were used for each reaction. The number of Neo r colonies increased linearly with the amount of the cell extracts and saturated at 30 #g of protein. The actual number of Neo r colonies varied 5-fold between assays; however, in each assay the results were consistent and reproducible. Both mutant plasmids were linearized by digesting at the ligated site with either NarI or NaeI before use, to enhance the reaction of homologous recombination (Song et al., 1985).

Results

Effect of thymidylate stress Thymidine-5'-monophosphate is synthesized

through methylation of dUMP by thymidylate synthase with methylene-tetrahydrofolate in mam- marian cells. FdUrd, a thymidine analog, is metabolized into fluorodeoxyuridine-5'-monophosphate, which strongly inhibits thymidylate

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Incubation time 2 4

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24 24 24 30 h Fig. 1. Induction of homologous recombination activity by treatment of FM3A cells with various concentrations of FdUrd. Cells were treated with FdUrd at the indicated concentrations for 24 or 30 h, harvested and cell extracts were prepared. 400 ng of each linearized recombination substrate (DL and DR) was incubated with 10 gg protein of the extracts, and the resulting recombined molecules were detected by formation of neomycin-resistant (Neo r) E. coli colonies, as described in Materials and methods. The average of duplicate reactions is

presented. The error bar shows variation from the average.

217

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Incubation time (h) Fig. 2. Time-course of the induction of homologous recombination activity in FM3A cells treated with FdUrd. Cells were treated with 1 /~M FdUrd for various lengths of time, and whole extracts were prepared. 400 ng of each linearized recombination substrate (DL and DR) was incubated with 10 ~tg protein of the extracts as described in Materials and methods. The average of duplicate reactions is presented. The error bar

shows variation from the average.

synthase and causes thymidylate deprivation in FM3A cells (Yoshioka et al., 1987; Ayusawa et al., 1988). In order to examine the effect of thymidylate stress on homologous recombination, FM3A cells were cultured in FdUrd prior to the preparation of the lysates. As shown in Fig. 1, when cells were treated with various concentrations of FdUrd for 24 or 30 h, the recombination activity was significantly increased. The cells treated with 1 /~M FdUrd for 24 h exhibited a 2.1-fold elevation of the activity, as assayed by the number of bacterial Neo r colonies. A longer time of treatment (30 h) with 0.1 /~M FdUrd reduced the activity. Conversely, concentrations of more than 1 /~M were extremely toxic for the cells; e.g., approximately 90% of the cells incubated with 10 /~M FdUrd for 24 h was stained with trypan blue (data not shown).

We then investigated the effect of treatment time with FdUrd on the induction. FM3A cells were grown for various times with 1 txM FdUrd, harvested and assayed for recombination activity. As shown in Fig. 2, the recombination activity increased gradually with incubation time and reached a maximum level after 16 h; this level was 3.7 times higher than that found in the extracts from untreated cells at time 0. In this condition,

2 1 8

58% of the cells were viable by t rypan blue stain- ing.

Effect of shorter times of FdUrd treatment Although we observed an increased recombina-

t ion activity upon thymidylate stress, t reatment

with 1 # M F d U r d for 16 h was still too severe for

F M 3 A cells to survive, because the treated cells lost their ability to grow normal ly when washed

and transferred to fresh med ium without the drug

(data not shown). To obta in recombinagenic, liv- ing cells, we investigated shorter times of F d U r d t reatment which permit ted a significant increase of homologous recombina t ion activity. F M 3 A cells were grown in 1 IaM F d U r d for either 3 or 8 h, washed out to remove the drug, and were again cul tured in fresh growth med ium with 10 /~M thymidine. As shown in Fig. 3, the recombina t ion

activity was increased even after these shorter times of F r U r d treatment . In the 3-h-treated cells, the activity increased 3.6-fold at 12 h (or 9 h after

the removal of the drug), but then decreased to the basal level at 16 h. On the other hand, in the

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0 4 8 12 16 20 24

Incubation time (h) Fig, 3. Effect of shorter times of FdUrd treatment with homologous recombination activity. FM3A cells were treated with 1 /*M FdUrd for 3 (closed circles) or 8 h (double circles). After the treatment, the cells were centrifuged and resuspended in fresh growth medium without the drug. Thymidine was added at a final concentration of 10 t~M to rescue the cells from thymidylate stress. The cells were further cultured to the indicated times, and cell extracts were prepared. 10 b~g protein of the extracts were used in the in vitro recombination assay, as described in Materials and methods. The average of duplicate reactions is presented, The error bar shows variation from

average.

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Fig. 4. Growth curves of FdUrd-treated FM3A cells. Cells were grown in ES medium supplemented with 2% fetal bovine serum, exposed to 1 ~M FdUrd at a density of 5.2×105 cells/ml (upward arrow), and incubated. 8 h later, the cells were centrifuged, washed with and resuspended in fresh growth medium without FdUdr but with 10 ~M of thymidine (down- ward arrow). The cells were further cultured and passaged at a density of 8.7 × 105 cells/ml. The growth curve of untreated cells is represented by open circles, thai of FdUrd-treated cells by closed circles, and that of the treated cells followed by washing and subculturing in the drug-free medium by open

triangles.

8-h-treated cells, elevation of the activity was observed after the same time lag of 4 - 5 h as above,

and, 16 h later (8 h after the removal of the drug), reached a level 4 .5-4 .6-fold higher than the basal one. It is notewor thy that this high activity was main ta ined dur ing a relatively long period of 8 h,

In this experiment , we also examined the abil i ty of the FdUrd- t r ea t ed cells to grow after transfer to the drug-free, fresh medium. As shown in Fig. 4, the average doubl ing t ime of the unt rea ted F M 3 A cells was calculated to be 13.2 h dur ing the incuba t ion t ime of up to 196 h. By contrast , the cells treated with 1 / , M F d U r d for 8 h exhibited a 20% greater doubl ing t ime of 16.0 h at the first passage after the t rea tment and transfer into normal medium. These cells, however, resumed the same growth rate as the unt rea ted cells dur ing the

2nd passage and thereafter were able to grow normally. These data indicate that 8 h of FdUrd treatment of FM3A cells followed by the additional 8-16-h incubation in normal medium was enough to induce homologous recombination activity while slightly reducing their growth rates.

Discussion

In this study, we observed induction of homologous recombination activity in FM3A cells exposed to FdUrd. FdUrd inhibits thymidylate synthase to cause thymidylate stress (Seno et al., 1985). It has been found in mammalian cells that thymidylate stress induces various genetic recombination events such as increased frequency of sister-chromatid exchanges (Hori et al., 1984) and segregation of an exogenous genetic marker integrated into the chromosome by transfection (Ayusawa et al., 1986). Our present data provide some evidence that these induced recombination events are due to an increase in homologous recombination activity.

Treatment of FM3A cells with FdUrd rapidly stops cellular DNA synthesis by causing the imbalance of dNTP pools peaking at 8 h and then results in DNA double-strand breaks at about 10 h, followed by a loss of cell viability (Yoshioka et al., 1987; Ayusawa et al., 1988). From these ob- servations, induction of genetic recombination events by thymidylate stress may be caused by increasing the number of broken ends of chromosomal DNA which may serve as recombination substrates. Alternatively, thymidylate stress may stimulate directly some cellular recombination system. The data presented here suggests the latter possibility, because 3 or 8 h of FdUrd treatment was sufficient to increase the activity (Fig. 3).

Before the double-strand breaks occur in the DNA, an endonuclease(s) activity is induced (Yoshioka et al., 1987; Ayusawa et al., 1988). Such endonuclease activity would enhance the in vitro homologous recombination reaction. However, this activity is specific for single-strand DNA and has no exonuclease activity (Wataya, personal com- munication). In addition, we used 2 linearized plasmids as recombination substrates. Therefore, we rule out the involvement of the endonuclease activity in our recombination assay system.

219

FdUrd treatment synchronizes mammalian cells in S phase by thymidylate starvation. Wong and Capecchi (1987) reported that the activity of homologous recombination between plasmids introduced into rat cells is higher in early to middle S phase than in other phases. However, we do not infer that recombination activity in FM3A cells was induced since the cells were arrested in S phase by exposure to FdUrd (Yoshioka et al., 1987). As FM3A cells double in about 12 h and the S phase is 8 h (data not shown), most of the population of treated cells would be arrested at the G1-S boundary to S phase during 3 h and 8 h of FdUrd treatment. If induction of the recombination activity simply depended on the cell cycle, similar induction curves following release from the treatment would be observed in both conditions (Fig. 3). Therefore, we suggest that thymidylate stress by FdUrd treatment causes activation or induction of a protein(s) directly responsible for homologous recombination such as RecA protein induced in an SOS function in E. coli (Clark and Margulies, 1965).

Wang et al. (1988) and Bhattacharya et al. (1989) reported that carcinogen-treated mouse L cells show higher homologous recombination activity than untreated cells. These and our data indicate that we may alter liomologous recombination activity in mammalian cells as in prokaryotes or lower eukaryotes. Recently, the method for gene targeting was developed to disrupt or to correct chromosomal genes of mammalian cells with introduced DNA (Smithies et al., 1985; Mansour et al., 1988). Our present results suggest that an adequate pretreatment of recipient cells with FdUrd may allow us to obtain highly recom- binogenic, viable cells to perform gene targeting experiments at greater efficiency.

Acknowledgements

We thank Drs. Michio Matsuhashi, Masaaki Wachi, and Tosaku Kanda for their helpful suggestions during this study. Support for this work was partly provided by grants-in-aid from the Ministry of Education, Science and Culture, and the Agency of Science and Technology in Japan.

220

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thymidylate stress induces homologous recombination activity in mammalian cells

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