Mol Gen Genet (1992) 236:17-24

MGG

© Springer-Verlag 1992

The REV3 gene of Saccharomyces cerevisiae is transcriptionally regulated more like a repair gene than one encoding a D N A polymerase Rakesh K. Singhal 1, David C. Hinkle 2, and Christopher W. Lawrence 1 Departments of 1Biophysics and ZBiology, University of Rochester, Rochester, NY 14642, USA Received January 15, 1992

Summary. We measured the relative steady-state levels of the mRNA transcribed from the Saccharomyces cerevisiae REV3 gene in cells at different stages of the mitotic and meiotic cycles, and after UV irradiation. This gene is thought to encode a DNA polymerase concerned only with a specific recovery function, the replication on mutagen-damaged templates that produces damagedinduced mutations. In keeping with this proposed function, the REV3 gene showed no evidence of the periodic transcription at the G1/S boundary of the mitotic and meiotic cycle that occurs with genes encoding replication enzymes. However, levels of REV3 mRNA were much increased in late meiotic cells, like those of transcripts of some other DNA repair-related genes. Steady-state levels of REV3 transcript were increased only slightly in response to UV irradiation. Key words: Mutagenesis - Translesion synthesis - UV induced transcription

Introduction The REV3 gene of baker's yeast, Saccharomyces cerevisiae, encodes an inessential product that is predicted to be a DNA polymerase because the expected protein product contains, in the correct order, the six conserved regions found in all eukaryote DNA polymerases (Morrison et al. 1989). These include the hexapeptides YGDTDS and SLYPSI, which are absolutely conserved in all such proteins and found together in no other protein class. It was argued that the REV3 gene product is concerned with translesion synthesis, that is, with the replication of mutagen- (or spontaneously) damaged chromosomal DNA, but not with any other repair or replication process (Morrison et al. 1989). Mutants carrying a deletion of the REV3 gene grow normally but, Correspondence to: C. Lawrence

like those with point mutations at this locus, exhibit much reduced frequencies of induced mutagenesis, and slightly increased sensitivity to mutagens (Lemontt 1971a; Lawrence and Christensen 1979; Morrison et al. 1989). Spontaneous mutagenesis is also reduced in rev3 mutants, but mitochondrial mutagenesis is normal (Tuite and Cox 1980; Polakowska et al. 1983). Such mutants are proficient in recombination (Lemontt 197 lb; Morrison et al. 1989) and are also likely to be proficient in excision repair (Morrison et al. 1989). We investigated the regulation of REV3 transcript levels in order to examine the above hypothesis, namely that this gene is concerned with translesion replication, an activity involved in recovery from DNA damage. If this hypothesis is in fact correct, we might expect that the transcriptional regulation of this gene would differ from that of genes encoding DNA polymerases and other proteins concerned with normal chromosomal replication. Structural genes of this latter kind are periodically transcribed at the G 1/S boundary in the mitotic cell cycle (Johnston et al. 1987), and also before meiotic DNA synthesis (Johnston et al. 1986). Such periodic transcription is not observed with genes encoding repair functions (Jones and Prakash 1991; Madura and Prakash 1990a, b; Madura et al. 1990). In addition, we studied REV3 transcript levels after UV irradiation, to look for any SOS-like phenomenon. In Escherichia eoli, levels of both DNA polymerase II, which is encoded by the lexA-regulated polB gene, and DNA polymerase III are much increased in UVirradiated cells (Bonner et al. 1988). Whether yeast possesses an SOS-like response, however, has been a matter of much debate (Lawrence 1982). We find that the REV3 gene is not periodically transcribed before the S-phase of either mitosis or meiosis. Amounts of REV3 mRNA are, however, much increased in late meiotic cells, a feature that is found with some repair genes. Further, REV3 transcript levels increase to only a small extent following UV irradiation. Finally, we also find that the REV3 transcript appears to have major 5' ends located at positions - 4 6 G and -47T.

18 Materials and methods

Yeast strains and plasmids. The yeast strains used were CL1265-7C (MATe arg4-17 leu2-3, 112 his3- 1 trpl ura352) and its rev3 deletion derivative AMY32 (Morrison et al. 1989), 4910-3-3a (MATa his7 ural edc7-4 barl-1), g857, which was obtained by mating g833-1B (MATa leu2 eanl hisl-1 trp2) with g833-2D(MATc~ hom3-10 hisl-7 ade2), and g721-2 (MATa/MATa leu2-1/LEU2 eanl/CAN1 hom3-10/HOM3 hisl-1/hisl-7 trp2/TRP2 ade2/ADE2). Plasmids pDS3 and pDS4 were constructed by ligating a 9.8 kb KpnI fragment, which carries the REV3 gene together with its promoter and terminator sequences, from pJA6 (Morrison et al. 1989) into the KpnI site of YEplac 112 (Gietz and Sugino 1988), a multicopy yeast shuttle vector that carries TRP1, the 2 gm replication origin and a pUC19 multicloning site. In pDS3, the REV3 fragment is in the minus orientation, and in pDS4 is in the plus orientation, relative to transcription from the E. coli lac promoter. Plasmids used to produce antisense R N A probes for detecting yeast mRNAs on Northern blots were constructed by inserting a fragment carrying each yeast gene into the Bluescript KS + vector (Stratagene, La Jolla, Calif.). In each case, the yeast fragment was oriented to allow transcription of antisense R N A from the T7 R N A polymerase promoter. In pKS-rev, a 4.6 kb fragment of REV3 DNA, extending from an upstream XbaI site to a blunt end (produced by deletion with Bal31) 266 bp from the 3' end of the open reading frame, was inserted between the XbaI and EcoRV sites of the vector. In pKS-pol and pKS-ura, a 3.5 kb PstI fragment from the 5' end of the POLl gene (Pizzagalli et al. 1988) or a 1.1 kb HindIII fragment from the URA3 gene was inserted into the PstI or HindIII site, respectively. Measurement of transcript levels. Relative amounts of REV3, POLl, or URA3 m R N A were measured by the Northern blotting procedure (McMaster and Carmichael 1977). Total R N A was extracted (Madura et al. 1990) from samples containing about 109 cells, and 40 gg aliquots (typically the yield from 8 x 107 cells) were fractionated by agarose gel electrophoresis and transferred to Gene-Screen nylon membranes (NEN) for hybridization. In each experiment, a duplicate agarose gel was stained with ethidium bromide to visualize the rRNA and confirm that equal amounts of R N A had been loaded. Radiolabelled R N A probes for each m R N A were prepared in 20 gl reactions containing 1 ~tg of CsC1 gradient-purified plasmid D N A (pKS-rev, pKS-pol, pKS-ura), 250gCi of [~-32p]UTP (3000Ci/mmol), 0.5 mM each of ATP, GTP, and CTP, 40 mM TRISHC1, pH 8.0), 8 mM MgClz, 25 mM NaC1, 2 mM spermidine, 10 mM dithiothreitol, 10U placental ribonuclease inhibitor (Life Technologies), and 20 U of T7 R N A polymerase. After 60 rain at 37 ° C, 1 ~1 of yeast tRNA (5 mg/ml) and 6 gl of RNase-free DNaseI (1 rag/ ml) were added and incubation was continued for 15 min. Typically, 60% of the radioactivity was incorporated into insoluble RNA. Reactions were terminated by adding 3.5 ~1 each of 200 mM EDTA and 10% SDS,

and the R N A probe used for hybridization at 3 x 106dpm/ml without further treatment. Hybridization and washing were carried out as described by Hurley et al. (1989), except that denatured salmon sperm D N A replaced yeast R N A in the hybridization mixture and hybridization was carried out for 40 h at 45°C in a Seal-a-Meal bag. Autoradiography was carried out with Kodak X-AR5 film at - 7 0 ° C, using two intensifying screens and band intensities were measured by densitometry using an Ultroscan XL densitometer (LKB).

Mappin9 of the REV3 mRNA 5' terminus. The 5' terminus of the REV3 transcript was mapped by S1 nuclease digestion of hybrids between m R N A and a 5' end labeled D N A fragment (Berk and Sharp 1977; Weaver and Weissman 1979; Yang et al. 1989), and also by extension, using reverse transcriptase, of a labeled oligonucleotide primer (Yang et al. 1989). Labelled DNA for the S1 nuclease method was prepared by cutting pDS3 with NcoI, followed by dephosphorylation with calf intestine alkaline phosphatase, labelling with [3Zp]ATP, and release of a 5' fragment of the REV3 gene by digestion with BamHI. A 20-met primer, complementary to nucleotides 28 through 47 of the REV3 gene was used for primer extension. A dideoxy sequencing reaction, using this primer with pDS3 DNA, provided size standards for both methods. Other methods. Mitotic cells of strain 4910-3-3a were synchronized by growth at 23 ° C in YPD medium to a density of ca. 1.5 x 10 7 cells/ml, incubation for 3 h in the presence of a-factor (10 ng/ml), followed by removal of a-factor by filtration and washing with YPD. Sporulation of strain g857, and similar treatment of the MATa/ MATa diploid g721-2 (Madura and Prakash 1990a, b), entailed growth in prespore medium to a concentration of about 4× 107cells/ml, collection and washing by filtration, and resuspension at a concentration of about 2 x 10 7 cells/ml in sporulation medium, followed by incubation at 30 ° C with vigorous aeration. For UV irradiation, cells of strain CL1265-7C, grown in YPD to a concentration of about 2 x 10 v cells/ml, were collected and washed with water by centrifugation, and resuspended at a measured concentration of 1 x 108 cells/ml in 0.85% KC1. Then 50 ml aliquots of this suspension in 100 mm plastic petri dishes, without lid, were exposed to 210 J/m 2 (low UV, 87% + 1.7% survival) or 418 J/m 2 (high UV, 57%:t:0.3% survival) of 254nm UV, at a fluence rate of 1 J/m 2 per second with continuous stirring. Cells at this high concentration significantly selfshield and scatter UV, and it is important to measure concentrations accurately, use low fluence rates, and stir well. Preliminary experiments established that the two fluences used were equivalent to irradiating dilute suspensions with 29 and 58 J/m 2, respectively. Irradiated ceils were collected by centrifugation, resuspended in YPD medium at a concentration of about 4 x 107 cells/ ml, and incubated at 30 ° C. Samples from this culture, taken at successive times, were washed with water and frozen at - 70 ° C. All irradiations and incubations were carried out under yellow light or in darkness.

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Fig. 2. Relative amounts of REV3 and POLl mRNA in a-factor synchronized mitotic cells of strain 4910-3-3a, and proportion of budded cellsin the cell population. Amounts of each transcript were normalized to those in cells immediately before a-factor release (+a-factor, 3 h, Fig. 1). Data are the average of two experiments

Results

REV3 is not periodically transcribed during the mitotic cell cycle Genes whose products are concerned with normal chromosomal replication show high levels of transcription at the G1/S boundary, but not at other times within the mitotic cycle (Johnston et al. 1986). We therefore examined the steady-state levels of REV3 transcript in mitotic cells of strain 4910-3-3a that had been synchronized by release from a-factor arrest. Strain 4910-3-3a was used because the presence of the barl-1 mutation makes it particularly susceptible to a-factor, and because good synchrony can be established (Madura and Prakash 1986). Total RNA, extracted from samples of the synchronous culture, was subjected to electrophoresis through 1% agarose gels, the separated species transferred to membranes and hybridized with 32p-labelled R N A probes for the REV3, POLl, and URA3 genes and

relative amounts of m R N A estimated by densitometry of autoradiograms. The proportion of budded cells in successive samples from the synchronous culture was also measured. Relative amounts of m R N A were normalized to those in cells held for 3 h in the presence of a-factor, that is, in cells immediately before release from arrest and the start of synchronous growth. REV3 and URA3 m R N A levels decreased to 30% and 20% of their initial amounts during this 3 h period, and POLl m R N A levels decreased to 5 % of this amount. An illustrative autoradiogram for REV3 and POLl is given in Fig. 1, and normalized quantitative data, the averages from two experiments, given in Fig. 2. As shown by the proportion of budded cells and by the periodic transcription of the POLl gene (Fig. 2), good synchronous growth was maintained in the cultures for two to three mitotic cell cycles; further, the cycle times in the two replicate experiments were essentially identical. During this period, the proportion of budded cells cycled between less than 3 % to about 90%, with low values at 0, 2, and 4 h. The amount of P O L l m R N A increased 13- to 16-fold above the initial level, and was maximal at 0.5, 2.0, and 3.5h; bearing in mind the interval between time points sampled, these data are consistent with periodic transcription at the G 1/S boundary. An analysis of variance showed that the, differences in transcript level were significant (P=0.01-0.001). In contrast, REV3 m R N A levels showed no evidence of periodic transcription, and though the level appeared higher in the later cell cycles, this was not statistically significant ( P > 0.05). As expected, URA3 m R N A levels also showed no evidence for periodic transcription (data not shown). We found normalizing relative m R N A amounts to the amount of URA3 transcript, as a means of correcting for variation in the amount of R N A loaded onto the gel, no better, and sometimes worse, than careful quantitation of the R N A loaded coupled with replication of the experiment. With neither method, however, is there any evidence for the periodic transcription of the REV3 gene. REV3 transcript levels increase substantially late in meiosis, but not in the meiotic G1 In addition to being transcribed at high levels in late G1 of the mitotic cycle, genes concerned with normal chro-

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mRNA in the nonsporulating MATa/MATa strain g721-2 exposed to sporulation medium. Amounts of transcript were normalized to those in cells held in presporulation medium. Data are averages from two experiments

mosomal replication are also actively transcribed in the G1 preceding meiotic divisions (Johnston et al. 1986). Repair genes are not specifically transcribed in meiotic G1, but some exhibit high levels o f m R N A later in meiosis (Jones and Prakash 1991 ; Jones et al. 1990; Madura and Prakash 1990a, b). We therefore measured the amounts of REV3 and P O L l m R N A in synchronously sporulating meiotic cultures, using diploid strain g857, in which 98 % of the cells sporulate and sporulation is fast and synchronous (Madura and Prakash 1990a). Amounts of m R N A were normalized to the amount in the presporulation culture immediately before transfer of the cells to sporulation medium, and the experiment was carried out twice. The time-course for sporulation, indicated by the proportion of asci, was virtually identical in the two replicates. As shown by an autoradiogram (Fig. 3) and by the the average quantitative data from the two experiments (Fig. 4), the level of P O L l transcript increased significantly ( P = 0.05-0.01) early in meiosis, with a maximum increase of about 14-fold after the cells

had been incubated in sporulation medium for 1.5 h. This result is consistent with earlier work by others (Johnston et al. 1986), and the cells are likely to be in the premeiotic Gl-phase at this time. P O L l mRNA levels subsequently decline and, with the autoradiogram exposures used, are undetectably low from the 6th. onward. In contrast, REV3 m R N A increases to only a small extent early in sporulation, a change that is not significant, but increases substantially and significantly (P = 0.001) after the cells have been incubated in sporulation medium for more than 3 h. A maximum increase of about 18-fold above presporulation levels was observed after 5 h in sporulation medium, and the amount of m R N A remained high up to 9 h, the latest timepoint sampled. The level of URA3 m R N A steadily decreased to one-third the initial level throughout the sporulation period (data not shown). The increased amounts of REV3 transcript late in sporulation are likely to be specifically dependent on meiosis, because they are not found in a M A Ta/MA Ta diploid incubated in the same conditions (Fig. 4). REV3 m R N A levels increase to only a small extent in response to UV irradiation Since REV3 function is required for D N A damagemediated mutagenesis (Lawrence 1982; Lawrence and Christensen 1979; Morrison et al. 1989), we were interested in determining whether REV3 transcript levels were increased in response to UV irradiation, as would be expected if this gene were regulated like E. coli D N A polymerases II and III (Bonner et al. 1988). Additionally, some RAD genes and also P O L l have been shown to exhibit a transient increase in transcription following UV irradiation (Johnston et al. 1987; Jones and Prakash 1991; Jones et al. 1990; Madura and Prakash 1986, 1990b; Madura et al. 1990). We therefore examined the response of REV3 and P O L l m R N A levels to UV irradiation, using either a fluence that resulted in 82% 4- 1.7% survival (low UV) or one that resulted in 57%4-0.3% survival (high UV) (Figs. 5, 6). Amounts of mRNA were normalized to the average amounts in cells that had been mock irradiated, but otherwise handled identically. Average values were used because transcript amounts did not vary significantly in most of these controls; the P O L l controls in the low UV experiment were just significantly heterogeneous (P=0.05), however. Averages from a

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ment. Although these UV responses were statistically significant (P = 0.05-0.01, P = 0.01-0.001, respectively), the mRNA levels were barely 50% higher than the control at any time except at 1 h in the low UV experiment, suggesting that the increases may not be biologically significant. The small increases seen are not related to the heat shock system, because we were unable to demonstrate any increase in REV3 mRNA in heat-shocked cells (data not shown).

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Fig. 6. Relative amounts of REV3 and POLl mRNA in UVirradiated cells of strain CL1265-7C. Open symbols, high UV (57% survival), closed symbols, low UV (82% survival). Amounts of transcript were normalized to the average amounts in mock-irradiated control ceols (dashed line). Data are the averages of four experiments (high UV) or three experiments (low UV)

complete set of controls for each time point sampled were used in the low UV experiments, and a set from 0 through 1.5 h in the high UV experiments. In keeping with results of earlier work (Johnston et al. 1987), the amount of POLl transcript was found to increase significantly in response to UV irradiation (high UV, P

The REV3 gene of Saccharomyces cerevisiae is transcriptionally regulated more like a repair gene than one encoding a DNA polymerase.

We measured the relative steady-state levels of the mRNA transcribed from the Saccharomyces cerevisiae REV3 gene in cells at different stages of the m...
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