A temperature-sensitive mutation in asparaginyl-tRNA synthetase causes cell-cycle arrest in early S phase

Experimental

Cell Research 184 (1989) 53-60

A Temperature-Sensitive Mutation in Asparaginyl-tRNA Synthetase Causes Cell-Cycle Arrest in Early S Phase GILL DIAMOND, Department

HOWARD CEDAR,’ and MENASHE

of Genetics and Cellular

Biochemistry,

Hebrew

University,

MARCUS2

Jerusalem,

Israel

The Chinese hamster temperature-sensitive cell-cycle mutant ts24 was analyzed biochemicallly in order to determine the nature of this lesion. The inability of these cells to proceed through S phase at the restrictive temperature could be complemented by the addition of asparagine to the growth medium, and enzymological analysis showed that this line contains a temperature-sensitive asparaginyl-tRNA synthetase. Normal asparaginyltRNA synthetase activity was restored in cells transfected with cloned genomic DNA that overcomes the mutational defect. In corroboration with these results it was shown that a different temperature-sensitive asparaginyl-tRNA synthetase mutant isolated in another laboratory was blocked in S phase in a manner similar to that of ts24. While the mechanism by which asparaginyl-tRNA synthetase affects cell-cycle progression has not been elucidated, it can be shown that it is not mediated through alteration in overall levels of protein synthesis. @ 1989 Academic Press, Inc.

The analysis of mutant cell lines defective for cell-cycle functions can provide a wealth of information concerning the control of cell proliferation [l]. Yet, of the many temperature-sensitive (ts) cell-cycle mutants isolated to date, only a few have yielded significant information about the control of DNA replication. From its initiation at the beginning of S phase, DNA replication in mammalian cells proceeds under strict control [2]. Synthesis initiates simultaneously at many points in the genome, and clusters of replicons are replicated in a specific ordered pattern [3, 41 which can be divided into three substages [5]. During the first two substages the light Giemsa bands (G-bands) undergo replication, while the dark G-bands are replicated in the last substage [6]. Furthermore, only one round of replication is carried out per cell cycle. In order to study the mechanisms involved in S phase control, Hirschberg and Marcus [7] isolated mutant Chinese hamster cell lines specifically defective in cell-cycle advancement, including several which were unable to progress through S phase at the nonpermissive temperature. One of these mutants, ts24, when grown at the nonpermissive temperature (npt), arrests in early S phase with approximately l&15% of its genome replicated, as determined by flow microfluorometry and premature chromosome condensation analysis [8]. In an attempt to further characterize the nature of this mutation, it was shown that while total DNA replication ceases 8-10 h after transfer to the npt, the rate of nucleotide incorporation does not decrease. Further analysis demonstrated that at the point ’ To whom reprint requests should be addressed. * Deceased. 53

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of arrest, ts24 synthesizes short pieces of DNA which rapidly undergo degradation [8]. We show here that this mutation is complemented by the addition of asparagine to the growth medium and that cell-cycle arrest in S phase is attributable to a thermolabile asparaginyl-tRNA synthetase. MATERIALS

AND METHODS

Cells. Line E36 and its derivatives were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FCS), streptomycin, and penicillin, routinely at 34°C in 5% COZ. UCW 206 was obtained from John Wasmuth and grown in the same medium, supplemented with 5 X 10e4M asparagine and 2 x 10m4proline. For determining cell growth 5 x 16 cells were seeded in SO-mmpetri dishes (Sterilin) and incubated at 34°C until they attached (34 h). Half of the plates were then transferred to 40.3”C, in the presence or absence of asparagine. At various times, plates were withdrawn and the cells harvested by trypsinization and counted in a hemacytometer. Cells were prepared for the fluorescence activated cell sorter (FACS-IV, Becton-Dickinson) by the following method: Cultures were harvested by trypsinization, washed once in medium without serum, and resuspended in 1 ml of PI staining solution-50 &ml propidium iodide, 50 Q4 Tris-HCI, pH 7.5, 10 mM NaCl, 0.1% sodium citrate, 0.1% Triton X-100, 50 &ml RNase A. The mitotic index was determined on cultures treated with colcemid (0.1 ug/ml) for 15 min to arrest cells in mitosis. Cells were removed by trypsinization, treated hypotonically, fixed, dripped into slides, and stained with Giemsa as described previously [8]. The number of mitotic cells per 1000total was determined by light microscopy. Enzyme assays. Whole-cell extracts were prepared essentally as reported [9]. Briefly, cells were harvested by trypsinization, washed three times with phospate-buffered saline, resuspended in lysis buffer (10 m&f Tris-HCI, pH 7.5, 5 r&I EDTA, 1 n04 DTT), and subjected to two cycles of freezing and thawing in liquid nitrogen. The lysate was centrifugated at 15,OOOgfor 15 min and the protein concentration of the supematant was assayed by the method of Bradford [ 101. Asparagine synthetase activity was measured in 140 mM Tris-HCl, pH 7.5, 8 mM MgC&, 8 m&f ATP, 30 mM Gin, and 10 @4 Asp by following the conversion of [14C]Asp (Amersham, 220 mCi/mmol) to [14C]Asn using Dowex-l-acetate column chromatography ill]. Asparaginase activity was measured in the same manner in a reaction mixture containg 50 m&f Tris-HCI, pH 7.8, 10 nuI4 Mg acetate, 30 r&f KCl, 5 mM 2-mercaptoethanol, and 5 nuI4 [‘4C]Asn. Radioactivity was determined by scintillation counting using a toluene-based scintillation fluid containg Triton X-100. Asparaginyl-tRNA synthetase activity was determined by measuring the extent of esteritication of [14C]Asn (Amersham, 232 mCi/mmol) to tRNA, as reported [12]. The reaction mixture of 0.2 ml contained 0.1 M Tris-HCl, pH 7.7,5 mM Na*ATP, 2.5 n&I NarCTP, 40 nuI4 KCl, 10 nuI4 Mg acetate, 0.4 n&f DTT, 0.4 mg yeast tRNA, and 0.1 mM Asn. Reactions were carried out under linear assay conditions at 34°C unless otherwise specified. The extent of 14Clabel in tRNA was measured by TCA precipitation on glass fiber filters. Analysis of protein synthesis. Cells 1x 10’ were seeded in S-cm dishes and grown overnight. The medium was aspirated and replaced with 0.5 ml fresh medium containing [‘4C]leucine, 0.5 @ml (57 mCi/mmol). Plates were removed periodically, the medium was aspirated, and the plates were rinsed twice with PBS (137 mi+f NaCl, 2.7 m&f KCI, 8.1 n&f Na2HP04, 1.3 mM KH2P04) and lysed with 1 ml PBS, 0.5% SDS. Protein was precipitated with 1 ml cold TCA and collected onto glass fiber filters. Radioactivity was measured in a liquid scintillation spectrometer.

RESULTS Growth of Cells at the Nonpermissive

Temperature

The S phase mutant, ts24, was originally isolated from the Chinese hamster lung fibroblast line E36 [7]. Initial experiments with this line indicated that while it was unable to grow at the npt in DMEM, near-normal growth was observed in a-MEM, which contains additional nutrients, including all of the nonessential amino acids. By testing the effect of each individual amino acid on the growth of

Mutant

asparaginyl-tRNA

synthetase arrests cells in S phase

55

‘Ol------

Time , hours Fig. 1. Growth kinetics of ts24 and E36. Growth rates of wild-type and mutant cell lines were measured as described under Materials and Methods at 34 or 40.3”C in the presence or absence of 50 pg/ml asparagine. ts24, 34°C (0); ts24, 40.3”C (0); ts24, 40,3”C with asparagine (0); E36, 34°C (A); E36, 40.3”C (A).

ts24 at the npt, we found that supplementation of DMEM with asparagine (50 ug/ml) was suffkient to overcome the mutation. Under these conditions the ts24 growth rate (Fig. 1) and the ability to form colonies [data not shown] were similar to those of the wild type. Determination

of the Lesion in ts24

Since exogenous asparagine could complement the ts24 mutation, we reasoned that the mutation might either affect the pool of asparagine in the cell (i.e., by means of a thermolabile asparagine synthetase) or affect an enzyme for which asparagine is a substrate such as asparaginyl-tRNA synthetase or asparaginase. When crude extracts of ts24 and E36 were assayed at 34 or 40.3”C in the absence of exogenous asparagine, the activity of both asparagine synthetase and asparaginase remained constant (Table 1). In contrast, the activity of asparaginyltRNA synthetase from ts24 showed a considerable drop when incubated at the TABLE 1 Activity

of enzymes at 34 and 40.3”C” Activity E36

Enzyme Asparagine synthetase Asparaginase Asparaginyl-tRNA synthetase

ts24

34°C

40.3”C

34°C

40.3”C

0.13 1.80 1.14

0.15 2.20 1.20

0.16 1.70 0.62

0.18 1.60 0.17

’ Crude extracts from E36 and ts24 were grown at 34 and 40.3”C and assayed as described. Activities of all enzymes are expressed in nmoVmg protein/h.

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Time

(minutes

1

Time

(minutes

1

Fig. 2. Temperature inactivation of asparaginyl-tRNA synthetase. (A) Crude extracts from E36, ts24, and UCW 206 grown at 34°C were heated to 40.3”C for varying lengths of time, cooled in ice, and assayed at 34°C as described. E36 (Cl); ts24 (0); UCW 206 (M); ts24, 50 pg/ml asparagine (A). (B) Temperature inactivation was carried out at 43°C as in A. E36 (0); ts24 (0); ts24Cl (m).

high temperature. The thermolability of this enzyme was confirmed by heating extracts at 40.3”C for various times, prior to assaying at 34°C. Figure 2 shows the temperature inactivation curves of the wild-type and ts24 enzymes. While the wild-type enzyme remains fully active for over 2 h at either 40.3 or 43”C, the ts24 enzyme loses activity with a half-life of 60 min at 40.3”C or 20 min at the higher temperature. This inactivation, however, could be prevented by the addition of high concentrations of asparagine. It can be assumed that this stabilizing effect is responsible for the ability of asparagine to overcome the mutation at 40.3”C in uiuo. Other temperature-sensitive asparaginyl-tRNA synthetase mutants have been isolated [ 14; J. Wasmuth, personal communication] and extracts from these cells were also tested for their stability at 40°C. As shown in Fig 2A, the activity of one of these mutants, UCW 206, is considerably more heat-labile than that of ts24 with a half-life of about 20 min. The K,,, value for the ts24 enzyme was found to be 8.3 x lo-’ M, which is about 5 times that of the wild-type enzyme (1.75~ 10P5 M) and is independent of temperature. We have previously isolated a clone from a Charon 4A human genomic library which complements the ts24 mutation and following stable transfection of the mutant with this DNA, these cells grow normally even at the npt [13]. The asparaginyl-tRNA synthetase activity from extracts of these transformed cells (ts24Cl) showed a bimodal response to heat inactivation. About 50% of the activity was sensitive at 43°C with kinetics similar to that of ts24, while the remaining activity was heat stable (Fig. 2B). This is consistent with the idea that the complementing gene contains an asparaginyl-tRNA synthetase activity and almost conclusively proves that this is indeed the defect in ts24. The Effect of a Thermolabile Asparaginyl-tRNA DNA Replication and the Cell Cycle

Synthetase on

As has been shown previously, ts24 is a cell-cycle mutant which arrests uniformly in early S [8]. In order to prove that this effect is indeed caused by the absence of

Mutant asparaginyl-tRNA

rA

synthetase arrests cells in S phase

57

B

Cl

DNA

G2

content

DNA

content

DNA

content

Fig. 3. Cell-cycle distribution as determined by flow microfluorometry. Cells were grown at 34 or 40.3”C for 24 h, harvested, and subjected to FACS analysis as described under Materials and Methods. (A) Analysis of E36 at 34°C (-) or 40.3”C (---); (B) analysis of ts24 at 34°C (-), 40.3”C (---), or 40.3”C in the presence of 50 kg/ml asn (. . .); (C) analysis of UCW 206 at 34°C (-), or 40.3”C (---).

asparaginyl-tRNA synthetase activity, we studied the growth properties of another mutant (UCW 206) which was originally isolated as temperature sensitive for asparaginyl-tRNA synthetase activity. When ts24 and UCW 206 are grown at 34°C (Fig. 3) and analyzed by flow microfluorometry we observe a normal cell-cycle distribution characteristic of logarithmically growing cells. In contrast, growth of ts24 at the npt (40”) leads to an accumulation of cells in early S. The asparaginyl-tRNA synthetase mutant UCW 206 exhibits a more complicated pattern of cell-cycle arrest which is also characterized by a shift from G1 to early S, but a fair proportion of the cells remain fixed in G1 even after very long exposure of this line to the higher temperature. For both mutations, growth at the restrictive temperature in the presence of asparagine gave a normal cell-cycle distribution. The results for ts24 are shown in Fig. 3B. This property is most probably due to the ability of this substrate to protect the temperature-sensitive asparaginyl-tRNA synthetase from heat inactivation. To demonstrate conlusively that UCW 206 cells behave like ts24 in that they cannot proceed past early S phase, we designed an experiment to test the fate of late G1 cells at the npt. UCW 206 cells were seeded at low density, transferred to 40.3”C, and periodically examined to determine the mitotic index. Figure 4 shows that after 10 h at the npt, the mitotic index drops sharply, indicating that cells located in Gi and early S (i.e., more than 10 h before mitosis) cannot proceed through S. This experiment also identified the mutant’s execution point, the last point in the cell cycle beyond which transfer to the npt cannot stop the cell from completing the cycle and reaching mitosis [ 151.The execution point for UCW 206 is in early S phase, 10 h prior to mitosis, a point similar to that obtained for ts24

PI. One of the major roles of asparaginyl-tRNA synthetase in the cell is to provide t-RNA for protein synthesis. It was of interest to ask whether it is the loss of this

58

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Cedar, and Marcus

0

2

4

6

8

IO 12 14 16 I8 20

Time , hours Fig. 4. Execution point analysis of UCW 206. The UCW 206 line was grown at 34°C and transferred to the restrictive temperature (40.3”C). At various times cells were harvested and their mitotic index determined.

activity which prevents ts24 from proceeding through the cell cycle. To this end, E36 and ts24 cells were assayed for their protein synthesis activity by following the incorporation of [14C]leucine. When ts24 was grown at the npt, protein synthesis proceeded at a normal rate for over 4 h before coming to a halt (Fig 5). Thus, turning off the asparaginyl-tRNA synthesis has a delayed action on protein synthesis. In contrast, transfer of ts24 to the npt while the cells are in early S causes a rapid (within 2 h) effect on progression through the cell cycle [8]. This strongly suggests that the defect in asparaginyl-tRNA synthetase causes cellcycle arrest by mechanisms which do not involve protein synthesis inhibition. DISCUSSION The fact that asparagine complements ts24 indicated that the defect in this cellcycle mutant was most likely due to a single mutation and allowed us to characterize this lesion at the biochemical level. We show here that the mutant phenotype in ts24 is due to a thermolabile asparaginyl-tRNA synthetase. This conclusion is based both on enzymatic studies and on a comparison of the cell-cycle properties of ts24 and another confirmed, independently isolated, temperature-

TimeChours) Fig. 5. Protein synthesis in ts24 and E36. Cells were transferred at zero time to either 34 or 40.3”C in the presence of [“%Jleucine and incorporation assayed at various time points. E36, 34°C (0); E36, 40.3”C (0); ts24, 34°C (0); ts24, 40.3”C (0).

Mutant

asparaginyl-tRNA

synthetase arrests cells in S phase

59

sensitive asparaginyl-tRNA synthetase mutant. Finally, a genomic DNA clone which complements the ts24 mutation confers on the cell normal asparaginyltRNA synthetase activity. It has previously been reported that the inhibition of aminoacyl-tRNA synthetases, including that for asparagine, caused rapid cell death, probably due to the loss of protein synthesis capability [16]. Amino acid starvation or other alterations in the process of protein synthesis have been shown to arrest cell growth after mitosis, but prior to the onset of DNA synthesis [17]. It is therefore somewhat surprising that the two asparaginyl-tRNA synthetase mutants we have studied do not exclusively arrest at this stage of the cell cycle. In general the degree of protein synthesis inhibition caused by any given aminoacyl-tRNA synthetase mutant is important for determining the point at which it interrupts the cell cycle [18]. In the case of ts24, temperature inactivation of the enzyme occurs at a relatively slow rate in vitro and due to substrate protection may occur even more slowly in uiuo. Thus, in this mutant, there may be enough enzyme activity present following mitosis to allow the cell to carry out all of the general functions requiring this enzyme. These same cells, however, cannot continue past early S, presumably due to the lack of some specific function provided by the asparaginyltRNA synthetase. In mutant UCW 206, which is considerably more sensitive to heat inactivation, many cells indeed get trapped in Gi, but those which overcome this obstacle cannot proceed past early S in a manner similar to that seen for ts24. These data suggest that in normal cells some specific factor is necessary for advancement out of early S. It is not clear, however, what role is played by asparaginyl-tRNA synthetase. One possibility is that cell-cycle advancement is dependent on the presence of a labile protein factor, whose synthesis has a critical requirement for charged Asn-tRNA. While this may be the case, experiments directed at understanding the kinetics of protein synthesis inhibition on ts24 suggest that effects on the cell cycle occur well in advance of the changes in protein synthesis which take place following transfer of the cell to the npt. It is thus unlikely that asparaginyl-tRNA synthetase acts through this pathway. An alternate explanation for the mutant phenotype in ts24 is that the charged asparaginyl-tRNA itself plays a role in cell-cycle control. It has been shown that charged Asn-tRNA is involved in the regulation of asparagine synthetase expression in CHO cells [9], and charged His-tRNA may play a role in the regulation of protein synthesis [19]. Charged tRNAs in mammalian cells have been shown to be essential for the conjugation of ubiquitin to proteins [20, 211, a process which has been implicated in cell-cycle regulation [22]. It has also been reported [23] that chromatin proteins undergo N-terminal modification by aminoacyl transfer from charged tRNA in mammalian cells and this may be part of a mechanism which controls protein degradation [24]. In bacteria, various charged tRNAs also take part in the control of gene expression [25] by various mechanisms, including attenuation [26], and have been directly implicated in the process of DNA replication. The dna Y gene which is essential for bacterial replication has, in fact, been identified as arginine-tRNA [27]. Although tRNA provides an attractive messenger for controlling cell cycle, it

60

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Cedar, and Marcus

should be noted that cells have a substantial pool of each charged tRNA. It is unlikely that a large decrease in its level can occur within the short time that it takes for cells to become arrested following transfer to the npt. In light of the arguments presented above we favor the possibility that asparaginyl-tRNA synthetase has an alternate function which is required for progression through early S. In the mutant ts24 the switch to 40.3”C probably causes a rapid inactivation of this function and thus leads to arrest. This gene has recently been sequenced in our laboratory, but a computer search for homology revealed no clues to the additional functions provided by this enzyme. We thank J. Wasmuth for kindly supplying the UCW 206 cell line and T. Callahan for her assistance in the enzymological studies. We are also grateful to R. Goitein, M. Gottesman, R. Kulka, and N. Bashan for many helpful discussions. This work was supported by grants from the U.S.-Israel Binational Science Foundation and the NIH.

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