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Induced recombination in the mitotic cell cycle of the yeast Saccharomyces cerevisiae

The occurrence of induced recombination in the mitotic cell cycle in yeast has been analysed using conditional cell-cycle mutants held at the restrictive temperature. The strains used were heteroallelic at gall and assaying for functional galactokinase shortly after irradiation (Johnston, 1982) allowed an unambiguous determination of the cell cycle stages in which recombination could occur. Recombination was observed in most strains, including those with the cdc36 mutation, defective in 'start ' ; the cdc4, 7 and dbf4 mutations which arrest cells in Gl; the dbfl, 2 and cdc6 mutations affecting S phase; cdcl6 and cdcl7 which block cells in G2 and also cdcl4 and 15 which arrest cells in 'late nuclear division'. Recombination can therefore occur within each of the major phases of the yeast cell cycle. This analysis has also revealed that the cdc8 mutation results in a defect in induced mitotic recombination.


INTRODUCTION
Recombination in micro-organisms is commonly detected by formation of colonies on appropriate selective media, so that recombination is actually scored some time after the process has taken place. For certain purposes this is unsatisfactory, such as in determining the occurrence of induced recombination in particular stages of the mitotic cell cycle, when the results may be obscured by subsequent recombination during incubation. Existing data in yeast therefore relate more to the stages of the cell cycle sensitive to induction of recombination, rather than to the occurrence of recombination itself (Holliday, 1965;Esposito, 1968). However, Fabre (1978) has shown that recombination can apparently occur within the Gl phase of the cell cycle by looking at recombination between heteroalleles of cdc4, a conditional mutation which arrests cells in Gl. Wild-type recombinants which appeared in a population held at the restrictive temperature must have undergone recombination within Gl.
We report here a different approach to this question which is applicable to any cell-cycle mutant, even when suitable heteroalleles are not available. Moreover, it avoids any ambiguities arising from scoring recombinants on plates by using strains heteroallelic at gall and detecting recombination by direct assay of the recombinant gene product, namely functional galactokinase (Johnston, 1982). By this means we have shown that induced heteroallelic recombination can occur within any stage of the mitotic cell cycle in yeast. We have also found that the cdc8 mutation results in a defect in induced mitotic recombination.

(ii) Strains
The cell cycle, cdc, mutants were obtained from the Yeast Genetic Stock Center, Berkeley, and were in an A364A genetic background, a adel ade2 ural his7 Iys2 tyrl gall-1. The 'start' mutant, SR661-2 a cdc36-16 trpl ural (Reed, 1980) was provided by Dr Steve Reed, University of California, Santa Barbara. The dbf mutants were isolated in this laboratory (Johnston and Thomas, 1982 a) in D273-l l a (a adel hisl trp2).
For these experiments new gall alleles were isolated in strain D273-lla by ethyl-methane-sulphonate mutagenesis (Johnston, 1977) and tested for recombination with the allele present in A364A. One of these, gall-D5, gave high frequencies of recombination with gall-1 and showed very low levels of ultraviolet-light (UV) induced reversion, and was therefore selected for further use.
The cdc homozygous diploids were constructed by crossing D273-1 la containing gall-D5 with strains carrying particular cdc mutations and irradiating the diploid cells with UV at 30 J/m 2 . Strains homozygous for the cdc mutations were then obtained by identifying temperature-sensitive colonies and checking the cells of these to ensure that the appropriate cell cycle morphology was formed at 37 °C.
The dbf mutations were first combined with gall-1 by crossing with A364A. A suitable spore clone was then crossed with D273-lla gall-D5 and a strain homozygous for the dbf mutation constructed as described above.
(iii) Induction of recombination Cells were grown in YPGG at 23 °C to approximately 10 ? /ml (mid-log phase) and then transferred to 37 °C for 3 h further incubation. They were harvested, washed twice in 0 9 % saline, resuspended at 10 7 /ml in saline and irradiated with a UV dose of 125 J/m 2 (unless otherwise stated) using a Hanovia germicidal lamp (254 nm) at a dose rate of 5 J/m 2 /s. They were then centrifuged and resuspended at 10 7 cells/ml in fresh YPGG prewarmed to 37 °C and incubated in the dark for 15 h at that temperature before being assayed for galactokinase.

(iv) Assay for galactokinase
This has been described in detail previously (Johnston, 1982) and only the salient points will be repeated here. Cells permeabilized with dimethyl-sulphoxide were incubated in an appropriate buffer containing ATP and 14 C-labelled galactose. The extent of phosphorylation of the galactose was then determined by spotting the reaction mix on DEAE-cellulose paper which binds phosphorylated compounds. The unreacted reagents were removed by extensive washing of the paper and, after drying, the retained radioactivity was determined in a toluene-based scintillant.

(i) Experimental rationale
To examine recombination in the mitotic cell cycle, we have used diploids homozygous for cdc (Hartwell, 1974) or dbf (Johnston & Thomas, 1982a, b) mutations, both groups of conditional mutations which arrest cells at specific stages of the cell cycle. Each diploid was heteroallelic at the gall locus, which codes for galactokinase, and recombination was detected by assaying for the recombinant gene product, functional galactokinase (Johnston, 1982). All combinations of gall alleles tested give a relatively high background of enzyme (Johnston, 1982), which means that increases in recombination determined by this means appear to be somewhat low when compared to plated recombinants, nevertheless the assay is sufficiently sensitive to detect 5-10 recombinants/10 5 cells. Since the assay measures the phosphorylation of galactose and a fixed number of cells were used in each assay, levels of recombination are expressed as n moles of galactose phosphorylated/10 7 cells/h.
The GAL1 gene product is inducible and our strains were therefore normally grown in the presence of small amounts of galactose so that they were fully cnduced for galactokinase at all times, glycerol providing the source of carbon for growth. In practice, mutants were grown to mid-long phase at 23 °C, then transferred to 37 °C for 3 h to allow accumulation at their cell cycle block. To ensure that this had occurred, the cells were examined microscopically to ascertain that they had formed the characteristic terminal cellular morphology associated with their particular arrest point (Hartwell, 1974). In none of the mutants examined did even protracted incubation at 37 °C increase spontaneous recombination above background. Cells were therefore iradiated with UV to stimulate recombination and before assaying were incubated for 15 h to allow completion of recombination and full expression of functional galactokinase (Johnston, 1982). In these experiments this incubation was at 37 °C to ensure that all recombination was confined to the cell cycle stage being examined. This was confirmed by establishing that no increase in cell numbers occurred during the incubation. This also ensured that none of the cells had escaped their cell-cycle block during irradiation, which was at room temperature, as these would divide before again arresting in the cell cycle.

(ii) Recombination in mutants blocked in the cell cycle
When irradiated cells were assayed, straight-line plots were obtained, the slopes of which were taken as a measure of recombination (Fig. 1). To determine induced recombination the background present in the unirradiated cells was subtracted from the level of enzyme after irradiation. Thus the functional galactokinase produced due to recombination in the strains carrying cdc36, cdc4, dbfl, cdcl7 and 20 - Assay time (h) Fig. 1. Assay of functional galactokinase in cells of cdc36, cdc4, dbfl, cdcl7 strains and a wild type after UV-irradiation. The strains were grown to mid-log phase at 23 °C, transferred to 37 °C for 3 h, irradiated and then incubated at 37 °C for 15 h before the cells were permeabilized and assayed. Open symbols indicate the background of galactokinase before irradiation and closed symbols the enzyme produced after irradiation.
the wild type shown in Fig. 1 was 22, 62, 535, 32 and 5 3 nmol/10 7 cells/h respectively. It was found that UV-induced reversion in homoallelic diploids of the two gall alleles was only 0-05 nmol/10 7 cells/h for gall-1 and was undetectable for gall-D5, so the observed increase in enzyme must be due predominantly to recombination. As the four mutants mentioned above are defective in each of the major phases  Determined on EB-gal plates.
•(" Not determined. it can occur in non-viable cells (Johnston, 1982). Indeed this recombination in dead cells probably accounts for the apparent lack of correlation between the viability after the incubation and the amount of enzyme synthesized (Table 1). Recombination was clearly observed in all the mutants, the only exception being strains with the cdc8 mutation, which blocks cells in S phase.

(iii) The cdc8 mutation results in defective recombination
Cells with the cdc8 mutation failed to show any recombination as measured by increased synthesis of galactokinase (Table 1). This could be due either to the mutant being blocked at a particular stage of the cell cycle preventing recombination, or to a direct involvement of the gene product in recombination. The latter interpretation is supported by the occurrence of recombination in other mutants blocked in S (Table 1); and in addition, when cells were irradiated without the 3 h preincubation at 37 °C but were placed at 37 °C immediately aftewards, they failed available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672300025659 Downloaded from https://www.cambridge.org/core. IP address: 207.241.234.82, on 10 Jul 2020 at 16:08:44, subject to the Cambridge Core terms of use, to recombine (Fig. 2 A). This suggests the cells do not have to be at their cell-cycle block for the defect in recombination to be apparent.
The viability of the mutant after irradiation and incubation is very low (Table 1), which could account for the lack of recombination. However in an experiment using a dose of only 60 J/m 2 the viability was 79 % after irradiation and 1 % after the incubation. This is comparable to several of the mutants in Table 1 which recombined successfully, including dbf4, which produced 80 % of the wild-type level of enzyme, yet again cdc8 mutants showed no recombination (Fig. 2 B). Furthermore GAL1 cells of cdc8 mutants grown with glycerol as sole carbon source and induced after irradiation by addition of galactose were able to synthesize new galactokinase at 37 °C, even 3 h after the irradiation (Fig. 3), indicating that the intracellular machinery was functional. Incidentally, when we performed this control with cells of a cdc9 mutant defective in DNA ligase (Johnston & Nasmyth, 1978), after only 25 J/m 2 irradiation damage to the cells was such that very little galactokinase was induced at 37 °C, invalidating this particular approach with cdc9 mutants.
Finally, Fabre & Roman (1979) developed a method for assessing the effect on recombination of a gene containing a conditional lethal mutation by means of conventional plating experiments. This employs heteroalleles within the gene of interest, both alleles being temperature-sensitive, and recombination is detected by the appearance of colonies at the restrictive temperature. We have performed these experiments with cdc8 to confirm that it confers a recombination defect. A cdc8-141 /cdc8-172 heteroallelic diploid was irradiated with a low dose of UV having no effect on viability of the mutant (Fig. 4, inset) Fig. 3. Induction of galactokinase by galactose in a cdc8 GAL1 strain. A 0AL1 recombinant of the cdc8 gall diploid was obtained and was grown in YEP containing 2 % glycerol to mid-log phase at 23 °C when 1 % galactose was added to part of the culture and incubation continued at 23 °C. The remaining cells were irradiated with 60J/m 2 and divided into 3 aliquots. The first aliquot was resuspended in YEP containing 2 % glycerol and 1 % galactose and was incubated at 23 °C; the second aliquot was resuspended in the same medium but was incubated at 37 °C; and the third aliquot was resuspended in the same medium without galactose, was incubated at 37 °C, and galactose to 1 % final concentration was added 3 h later. The cells were harvested aftera total of 12 hincubation, permeabilized and assayed for galactokinase. O O. Background galactokinase; # # , unirradiated incubated at 23 °C; • D. irradiated incubated at 23°C;x x, irradiated incubated at 37 °C; A A. irradiated incubated at 37 °C, induced after 3 h incubation. incubated at 23 °C and transferred to 37 °C at intervals. Only those cells recombining to produce wild-type CDC8 gene were able to form colonies at the high temperature and as expected for a temperature-sensitive protein involved in recombination, the longer the incubation at 23 °C the greater the number of colonies at 37 °C (Fig. 4).

DISCUSSION
UV-induced reversion of the gall alleles used in these experiments was very low and the increase in detectable galactokinase after irradiation must have been due to recombination. We believe this recombination was confined to the particular  Fig. 4. Allelic recombination within the cdc8 gene as a function of time at 23 °C before transfer to 37 °C. Mid-log phase cells of cdc8-141 /cdc8-182 heteroallelic diploid were spread on plates, irradiated with 1-5 J/m 2 and placed at 23 °C. At intervals plates were transferred to 37 °C for a further 3 days incubation before scoring recombinants. O~ O, Unirradiated; # # , irradiated. Inset: survival of the cdc8 heteroallele at 37 °C without irradiation ( # # ) and after irradiation with 1-5 J/m 2 (O O)cell-cycle stage under examination for two interrelated reasons. First, there was no increase in cell numbers during the post-irradiation incubation at 37 °C, so few, if any, of the cells have escaped the cell-cycle block. Secondly, the levels of UV-induced enzyme were high, ranging from 43% to 120% of the wild type and it is unlikely that this amount of recombination could be the result of a small, undetectable, number of cells escaping their block and recombining subsequently.
Assuming that production of galactokinase indicates completion of recombination the technique used suggests that mutants blocked in all the major phases of the cell cycle, namely 'start', Gl, S and G2/mitosis, are capable of recombining. Only diploids homozygous for cdc8 failed to recombine; however, three other mutants blocked in the S phase gave high levels of recombination, suggesting that the defect in cdc8 strains may be a specific consequence of the mutational lesion. In support of this, cells irradiated before accumulation at their cell-cycle block and then immediately transferred to 37 °C did not recombine. Moreover, controls available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0016672300025659 Downloaded from https://www.cambridge.org/core. IP address: 207.241.234.82, on 10 Jul 2020 at 16:08:44, subject to the Cambridge Core terms of use, suggested that the cdc8 defect was unlikely to be due to an inability to produce new enzyme after irradiation, or simply to a loss of viability, since non-viable cells are able to recombine (Johnston, 1982). The CDC8 gene product is believed to be a single-stranded DNA binding protein (Arendes, Kim & Sugino, 1983) and activities of this sort could well have a role in recombination, indeed an E. coli mutant defective in a similar protein is deficient in recombination (Glassberg, Meyer & Kornberg, 1979). Thus the recombination defect caused by cdc8 is probably due to a direct involvement of the gene product in recombination rather than being a secondary consequence of its cell cycle block.
Previous studies relating to recombination in the cell cycle have involved the use of synchronized cells treated with recombinogens at various points in the cell cycle (Holliday, 1965;Esposito, 1968;Davies, Tippins & Parry, 1978). This has provided information on stages of the cycle sensitive to induction of recombination, rather than demonstrating the actual occurrence of recombination in different parts of the cell cycle. The only existing experiments to provide clear data on this point are those of Fabre (1978), who showed, using cdc4, that recombination could occur within Gl, in agreement with our results.
In experiments using cell-cycle mutants the various phases of the cycle are prolonged, and the amount of recombination observed may not accurately reflect the extent to which it would occur in a normal cell cycle. In fact, existing evidence suggests that spontaneous recombination is initiated in Gl (Esposito, 1978), although sister chromatid exchange obviously must occur in S or G2. We observed most recombination in cdc4 strains, blocked in Gl, and in dbfl and dbf2 strains, both blocked in S phase. However, precise quantitation in our experiments was difficult. The numbers of plated recombinants obtained varied somewhat from mutant to mutant, and there was also considerable variation in viability of the mutants after irradiation and incubation at 37 °C, although loss of viability in itself does not prevent recombination (Johnston, 1982).
The results imply that homologous chromosomes are able to pair at any stage of the cell cycle. This may be a consequence of centromere attachment, as suggested for Schizosaccharomyces pombe by the more frequent occurrence of spontaneous mitotic gene conversion in centromere-linked genes than in unlinked genes (Minet, Grossenbacher-Grunder & Thuriaux, 1980). Gall is in fact loosely linked to its centromere so possibly the recombination we observed is a reflection of this. Alternatively it could reflect a more complete pairing of homologous chromosomes. Our results provide no information on the nature of this association but simply demonstrate that it can occur at least over much of the mitotic cell cycle in yeast.
We have attempted to extend these observations to the meiotic cycle but have been unable to detect an increase in galactokinase at any stage of meiosis. Even a strain constitutive for galactokinase production and able to sporulate in the presence of inducing levels of galactose, showed no increase in enzyme. The expected increase in gal + plated recombinants occurred, however, suggesting that synthesis of galactokinase may be repressed in meiosis. Possibly the use of a different locus is required or a different technique for detecting recombination.