Temporal order in yeast chromosome replication

Cell, Vol. 5, 263-269,

July 1975,

Copyright0

1975

Temporal Order in Yeast Chromosome

by MIT

Replication

Wylie Burke* and Walton L. Fangman Department of Genetics University of Washington Seattle, Washington 98195

Summary Previous work with bacteria has shown that a gene is maximally sensitive to mutagenesis by Nmethyl-N’-nitro-N-nitrosoguanidine (NG) at the time it is being replicated. NG was used to test for temporal order in the replication of the genome of the unicellular eucaryote, Saccaromyces cerevisiae. Yeast ceils growing exponentially were more sensitive to mutagenesis by NG than cells in which DNA synthesis had been inhibited. Further, in a synchronized population of cells, individual genetic markers exhibited maximum sensitivity to mutagenesis at distinct limited intervals within the DNA synthesis period. The peaks of sensttivity are interpreted as reflecting the times of replication of different genes. Since markers for five genes on four different chromosomes showed discrete periods of maximum sensitivity, it is likely that temporal ordering of replication exists for most genes in the yeast genome. These results imply that sites for initiation of DNA replication occur at fairly specific regions along yeast chromosomal DNA molecules, and are activated at predetermined times in the DNA synthesis period. Introduction In viruses such as h and T7, and procaryote organisms such as E. coli, the entire genome is contained in a single DNA molecule, and replication is initiated at a specific site on the molecule (Schnos and Inman, 1970; Dressler, Wolfson, and Magazin, 1972; Bird et al., 1972). In higher eucaryotes the genome is distributed among several chromosomes, and DNA replication is initiated at multiple sites along chromosomal DNA molecules (Huberman and Riggs, 1968; Callan, 1972; Blumenthal, Kriegstein, and Hogness, 1973). Multiple initiation sites have also been observed for the chromosomal DNA of the microbial eucaryote, Saccharomyces cerevisiae (Newlon et al., 1974). This yeast has 17 chromosomes and a genome size only 2-3 times that of E. coli (Whitney and Hall, 1974). The precision with which replication initiation sites are chosen each cell cycle in eucaryote cells is not known. We have used the mutagen N-methyl-N’-nitroN-nitrosoguanidine (NG) to study the specificity of the initiation process in Saccharomyces cerevisiae. *Present address: Department of Pediatrics, ton School of Medicine, Seattle, Washington

University 98195.

of Washing-

Work with E. coli has shown that NG preferentially mutagenizes DNA sequences in the process of being replicated: different genetic markers are mutated at maximal rates by NG at different times during a synchronous replication cycle, and the order in which markers are maximally mutated by NG corresponds to the order in which they are replicated, as determined by independent techniques (see (Hohlfeld and Vielmetter, 1973). We treated samples of cells taken from synchronous yeast cultures with NG to determine whether the replication of individual yeast genes is ordered within the S period. If initiation of replication is a specific process-that is, occurring at specific sites along the DNA molecules and at predetermined times during the S periodthen individual genes will replicate in an ordered manner, each gene having a precise replication time during the S period. Related experiments have been reported by Dawes and Carter (1974), and a preliminary account of this work has been presented elsewhere (Burke, Newlon, and Fangman, 1975). Results NG Mutagenesis: Replication Dependence In order to utilize NG to study the replication timing of yeast genes, it was necessary first to show that NG preferentially mutagenizes replicating DNA in yeast as it does in bacteria. This was done by comparing rates of NG mutagenesis in normally growing cells with rates in cells treated to prevent DNA synthesis. Two methods of treatment were used: inhibition of the initiation of DNA synthesis with cycloheximide, and arrest of cells of the a mating type in the Gl phase of the cell cycle with (Y factor (Bucking-Throm et al., 1973). Table 1 presents rates of reversion for three auxotrophic markers in cells incubated with NG, with or without prior treatment with cycloheximide. Cycloheximide, a protein synthesis inhibitor, has been shown to inhibit initiation of the S period in yeast (Hereford and Hartwell, 1973; Williamson, 1973). Table 1. Induction of Revertants 11 D with Cycloheximide

after

Treatment

of Strain

Nitrosoguanidine-Induced Marker

Background0 (revertants

Asynchronous Culture per 108 viable

1422Rateb

CycloheximideTreated Cells

cells)

ade-1

20

2400

150

leu-1

2

280

35

met-2

6

140

29

aValues were similar for synchronous and cycloheximide-treated cells. bTreatment was at 0°C for 30 min.
Cell 264

Cells which have started DNA synthesis, however, complete the S period in the presence of the inhibitor. For the data shown, cells were treated with cycloheximide for a period sufficient to allow completion of the S period. As can be seen, cycloheximide-treated cells show a 5-16 fold reduction in NG-induced rates of reversion, compared to growing cells. A reduction in the rate of mutagenesis is also observed for cells arrested at the Gl phase of the cell cycle, as shown in Table 2. In these cells, rates of NG-induced reversion for two markers, leu-7 and met-2, are reduced almost to background level. For the third marker, ade-7, the rate of reversion in Glarrested cells, although well above background, was always less (4-8 fold) than the rate observed in growing cells. NG Mutagenesis: Treatment of Synchronous Cultures The effects of cycloheximide and (Y factor treatment of yeast cells on NG-induced mutagenesis suggest that in yeast, as in bacteria, NG preferentially mutagenizes replicating DNA. In order to study this effect further, NG mutagenesis was performed with cells undergoing a synchronous S period. Yeast cells of a mating type were arrested in the Gl period with (Y factor, collected by filtration and suspended in fresh medium without LYfactor. Figure 1 shows that cells treated in this way undergo a synchronous DNA synthesis period as evidenced by the incorporation of 3H-uracil into DNA. The timing of appearance of budded cells in a population released from cy factor arrest can be used as an additional measure of synchrony. Data on the appearance of budded cells in the partially synchronous culture are also plotted in Figure 1. NG mutagenesis treatments were carried out with samples taken from such a synchronous population of cells. Figure 2 shows the results expected from this experiment if -the timing of replication of the gene is random, so that different cells in the population replicate the gene in question at different times, Table 2. Induction 11 D with a Factor

of Revertants

after

Treatment

of Strain

Nitrosoguanidine-induced Marker

Backgrounda (revertants

ade-1

Asynchronous Culture per 108 viable

Minutes Figure 1. DNA Synthesis and Appearance Release from 01 Factor Arrest

of Budded

Cells

after

Cells of strain 1422-I 1 D were prelabeled with SH-uracil for about 0.3 generations prior to 01 factor arrest and treated with (Y factor in the continued presence of label. After release, additional label was added at the same specific activity. For determination of percent, budded cells samples were collected into equal volumes of 10% formalin, treated with a I:30 dilution of glusulase (Endo) at 37OC overnight, then examined microscopically.

-GI

S-------I--G2-

-IA--, A+ (precise replication time)

A--A’ (random replication time)

-:

:-:

1422-

rateb 01 Factor Arrested (Gl) Cells

cells)

Time

600

IO

2,300

leu-I

1

270

4

met-2

5

140

14

aValues were similar for asynchronous bTreatment was at 0°C for 30 min.

-or if the timing of replication of the gene is precise, so that each cell replicates the gene at the same relative time within the S period.

and 01factor

Figure 2. Expected Results a Synchronous Population arrested

ceils.

for

+ Nitrosoguanidine

Mutagenesis

Cells are removed at frequent intervals from a culture undergoing a synchronous S period, treated with NG, and plated to determine the frequency of A+ revertants.

of

Temporal 265

Order

in Yeast

Chromosome

Replication

These predicted results assume that the rate of DNA synthesis remains relatively constant throughout the S period; the kinetics of incorporation of label in DNA during a synchronous culture, as shown in Figure 1, is consistent with this assumption. Figure 3 shows the rates of reversion to Ade+, Leu+, and Met+ observed in the yeast strain 142211 D during the course of a synchronous S period. All three show distinct peaks of NG-induced reversion during synchronous growth. Moreover, the peaks are separable, Ade+ revertants occurring maximally about 10 min before Leu+, and Met+ revertants about 10 min after Leu+. The mutation rate declines at the end of the first S period, and then rises again as the culture enters a second less synchronous, S period. In another experiment, shown in Figure 4, a sufficient number of samples were taken between 100 and 160 min to determine whether a separation of NG-induced peaks could be observed during the second S period as well. Rates of reversion to Ade+ and Leu+ were followed; in the second S period, as in the first, the maximal induction of Ade+ precedes the maximal induction of Leu+ cells. These results support the idea that NG mutagenesis is replication dependent. The peaks of NG-in-

Time Figure

3. Nitrosoguanidine

Samples were the frequency Ade+, 700/10*

Mutagenesis

during

a Synchronous

S Period

duced reversion occur during the S period, and are assumed to reflect the relative timing of replication of the individual genes. These data indicate individual yeast genes do not replicate in a random manner (Figure 2), but rather replicate during a limited, defined interval within the S period. The three genes, ade-I, leu-1, and met-2 are unlinked; their chromosomal locations are shown in Figure 5. Another yeast strain, 3163-Arg+, carried two NGrevertable markers on the same chromosome: leu-7 and ade-5. Both markers are on the left arm of chromosome VII, leu-7 located close to the centromere and ade-5 located more distally. Results of a synchronous culture experiment with this strain are shown in Figure 6. Peaks of reversion to Leu+ and Ade+ occur about 20 min apart, suggesting that replication of the left arm of chromosome VII takes at least 20 min to be completed. A third marker, can-l, was studied in this strain by measuring the induction of forward mutations to canavanine resistance. This marker shows a peak of mutation at about the same time as the peak of Leu+ revertants. Genetic Genetic whether

Testing of NG-Induced Mutants testing was carried out to the NG-induced prototrophic

determine revertants

(minutes) (ade-7,

taken from a synchronous culture of 1422-11D at intervals, of Ade+, Let?, and Met+ colonies. The maximum prototroph viable cells for Leu+, and 65/108 viable cells for Met+.

leu-7,

mef-2)

treated with NG (30 min at O”C), and plated to determine frequency (100%) corresponds to 2800/108 viable cells for

Cell 266

produced in these experiments were the result of back mutation at the original site or were caused by unlinked suppressors. In the former case, the peaks observed would reflect replication times for the genetic markers carried in each strain; in the latter case, the peaks would represent replication times for suppressor loci. For each marker, 15-20 revertants were taken from a sample showing a maximal mutation rate for that marker. Each revertant (a haploid) was crossed to an 01 haploid strain which was wild type for the marker in question. The diploid was sporulated and a random spore analysis performed, using 60-90 spores from each diploid: if an NG-induced revertant is a result of an unlinked suppressor, 25% of the spore products should be auxotrophic. Results of such tests are shown in Table 3A. Leu+ revertants were studied from both strains used. In each case the original mutation carried was at leu7. As indicated, no suppressors occurred among 19 Leu+ revertants from 1422-11 D, and only one occurred among 16 tested from 3163-Arg+. Testing of Ade+ revertants from both strains similarly showed no indication of suppressor induction. In the case of 1422-11 D, the original mutation was at I

I

I

I

I

I

I

Time

ade-7. In the case of 3163-Arg+, the original mutation was at a different locus, ade-5. The results for Ade+ and Leu+ revertants thus indicate that the NGinduced mutations must have occurred at or closely Table

3. Genetic

A. Ade+,

Leu+,

Revertant

Type

Leu+

(1422-l

Testing and Met+

1 D)

of NG-Induced

Revertants

Revertants Spore

Phenotypes

100%

Prototrophic

75%

19/19

o/19

Leu+ (3163-Arg+)

15/16

l/160

Ade+

20/20

o/20

(1422-l

1 D)

Prototrophic

Ade+

(3163-Arg+)

20/20

o/20

Met+

(1422-I

10/20

10/2Ob

B. Car?

1 D)

Mutants Cell Phenotypes

Colony

Tested

Car+

Mutant

(haploid)

19/19

o/19

Mutant

X Cans (diploid)

o/19

19/19

Mutant

X Cat+

19/19

o/19

aActual bActual

value observed: range observed:

I

I

(diploid)

I

Cans

78%. 68-81%.

I

I

I

I

I

I

(minutes)

Figure 4. Nitrosoguanidine Mutagenesis of a Synchronous S Period (ade-7, leu-7) Samples were taken from a synchronous culture of 1422-110 at intervals, treated with NG (30 min at O”C), and plated to determine the frequency of Ade+ and Leu+ colonies. The maximum prototroph frequency (100%) corresponds to 2100/10* viable cells for Ade+ and 550/l 08 viable cells for Leu+.

Temporal 267

Order

in Yeast

Chromosome

Replication

linked to the sites of the original mutations in each case. As indicated in Table 3A, the Met+ revertants from strain 1422-I 1 D fell into two classes. Half of them produced no Met- spores. The other half yielded 25% (range of 19-32%) Met- spores, indicating that in these revertants the Met+ phenotype is the result of a suppressor unlinked to the original mutation at met-2. Therefore the peak of NG-induced Met+ revertants results from mutations at the met-2 locus and one or more suppressor loci. This result probably accounts for the fact that the Met+ peak is broader than either the Leu+ or Ade+ peak in Figure 3. The Cat? mutants induced by NG in strain 3163Arg+ were tested by complementation, to determine whether they carried mutations at the can-7 locus. A complementation test was possible because the Cat+ phenotype was shown to be recessive: crossing of each CanR mutant to wild-type (Cans) strain resulted in a wild-type (Cans) diploid. The complementation test was performed by crossing each CanR mutant to a tester strain containing a mutation at the can-7 locus conferring recessive canavanine resistance. As summarized in Table 3B, the resulting diploids were all Cat+. Thus each NG-induced CanR mutant resulted from an event at the can-7 locus. Discussion

peaks of NG-induced mutation at different times. This is interpreted to mean that different genes replicate during a limited and programmed time in the S period. Since all five markers that were studied showed the same effect, it is likely that most or all genes in the yeast genome are replicated in this temporally ordered manner. For two markers (ade7 and leu-7) the same ordering of replication seen in the S period immediately following release from 01 factor was shown to be repeated in the subsequent S period. This observation supports the idea that the timing of gene replication seen in these experiments reflects replication events as they occur in untreated populations. Density labeling techniques have previously been used to demonstrate that a temporal order of replication exists in the S period in animal cells (Mueller and Kajiwara, 1967) and in the simple eucaryote Physarum (Braun and Wili, 1969). Autoradiographic and hybridization techniques have also been used to demonstrate temporal ordering of DNA replication in eucaryotes (for example, Douglas and Walden, 1974; Flamm, Bernheim, and Brubaker, 1971). The resolution of these experiments is limited, however, both by the large size of the genomes in the organisms studied and by the techniques employed. In the most favorable case, that of Physarum, it was possible to demonstrate that DNA sequences which replicated during one tenth of the S period in one cell cycle replicated during the

In yeast cells going through a synchronous S period, different genetic markers show distinct

Ade+ ,R \J

I -4-d? can I V’

ade 5,7

leu I

40 Time

50 CM Figure

5. Linkage

Data

for Yeast

Markers

The vertical lines represent positions of mapped genes; relevant genes are designated by name. The scale is in centimorgans. Dashed lines indicate disomic linkage, and the dotted line indicates a recombination distance determined from mitotic recombination. Most of the mapping positions are taken from the data of Mortimer and Hawthorne (1973).

Figure 6. Nitrosoguanidine (ade-5, leu-1, can-l)

Mutagenesis

60 (minutes) of a Synchronous

S Period

Samples were taken from a synchronous culture of 3163-Arg+ at intervals, treated with NG (30 min at 3O”C), and plated to determine the frequency of Ade+, Leu+, and Cat+ colonies. Canavanine was used at 60 pg/ml. The maximum prototroph frequency (100%) corresponds to 130/108 viable cells for Ade+, 110/108 viable cells for Leu+, and 10,000/10~ viable ceils for CanR.

Cell 268

same fraction of a subsequent S period (Muldoon et al., 1971). The Physarum haploid genome consists of about 1.8 x 1011 daltons of DNA (Mohberg and Rusch, 1971), or at least 20 fold more DNA than the yeast genome (Whitney and Hall, 1974). Therefore the amount of DNA which has been shown to replicate in a precise temporal order in Physarum is equivalent to two yeast genomes. Thus the results presented here extend previous findings of temporal programming in eucaryotes by demonstrating an ordering of replication for individual genes within a very small genome. The observation that individual genes replicate during discrete intervals in the S period leads to the conclusion that the initiation of replication is a fairly specific process, involving defined start regions that are activated at predetermined times in the S period. In the synchronous yeast cultures, the interval over which an individual gene is mutated amounts to 20 min or more. Since new buds are initiated over a similar period of time in the synchronized cultures (Figure 1) it seems likely that a large proportion of this spread in the time of mutation results from the imperfect synchrony in the (Y factor treated cultures. However, a limited amount of variation in both timing and location of initiations may exist.

Note Added During the preparation of this manuscript, we learned of experiments by Kee and Haber (1975), who employed zonal centrifugation to separate cells from different stages of the cell cycle after NG treatment. Their results lead to the same conclusions as ours. Experimental

Procedures

Strains and Growth Conditions For NG mutagenesis experiments, the haploid strains were 142211 D (a, /e&f, &e-l, his-2, met-2, lys-7, frp-5, ura-3, gal-l) and 3163-Arg+ (a, leu-7, met-l, ade-5, ura-3, trp-3), an Arg+ derivative of X3163-4C, both provided by D. Hawthorne. With the exception of his-2, the markers in 1422-IID are not suppressed by known supersuppressors (D. Hawthorne, personal communication). Cells were grown in YM-1 medium [5 g Difco yeast extract, IO g Difco bactopeptone, 6.7 g Difco yeast nitrogen base (with amino acids), IO g succinic acid, 6 g NaOH per I] supplemented with 50 mg adenine, 50 mg uracil, and 20 g glucose per I. For radioactive labeling, uracil-6-3H was added to IO pg/ml uracil. Haploid 01 strains used for mating with NG-induced mutants, in genetic analysis of the mutants, were: 5178-I C (a, gal-2, sue, ma/, ura-7, ade-l), and 2950-38 [CT, GAL, sue, ma/, ura-4, lys-7), provided by D. Hawthorne; 2262-2A (II, ade-7, ura-7, his-5, lys-17, leu-2, gal-l), provided by L. Hartwell; X-57 [a, his-7-2, fyr-7-7) provided by H. Roman: 2912-38 (a, arg-4-76, his-5-2, ura-4, adeI), provided by R. Mortimer, and 01, 131-20 (a, ade-2, ura-3, cyhR2, car%7, leu-1) provided by A. Hopper.

The tester Revertant

strains

were

used

or Mutant

as follows:

Jester

Leu+ (from Ade+ (from

1422-I 1422-l

1 D) 1 D)

5178-1C

Met+ (from

1422-11

D)

Leu+ (from Ade+ (from

3163-Arg+) 3163-Arg+)

2262-2A x-57

CanR (from

3163-Arg+)

Strain

2950-38

x-57 2912-38

and N 131-20

(Y Factor Arrest and Synchronous Cultures a factor was concentrated from (Y strain culture fluid by the column procedure of Duntze et al. (1973) except that before final elution of the factor with 0.01 M HCI-80% ethanol, the column was washed with 0.001 M HCI-80% ethanol. The column eluant was concentrated by evaporation in the cold. 01factor was assayed by monitoring the accumulation of unbudded cells in an asynchronous population to which it was added. For 01 factor arrest and synchrony experiments, 01factor was used at a concentration which maintained 95% unbudded cells for at least 30 min longer than a generation time. For 01 factor arrested populations, 01 factor was added to an asynchronous culture at 2.5 X 107 cells/ml. Cells were collected after a time period equivalent to one generation of growth. For synchronous cultures, arrested cells were collected by filtration, washed with fresh medium, and resuspended in fresh medium. Cells were sonicated after (Y factor treatment to break up cell clumps. When incorporation of radioactive uracil was determined in cultures, samples were taken into cold TCA (10% final TCA concentration) containing 1 mg/ml uracil, centrifuged in the cold, and resuspended in 0.6 N NaOH containing 1 mg/ml uracil. Samples were incubated at 37°C overnight, then chilled and precipitated with an equal volume of 20% TCA, 0.6 N HCI. Filtering and counting of radioactive samples were as previously described (Petes and Fangman, 1972). Mutagenesis Cell samples taken for N-methyl-N’-nitro-N-nitrosoguanidine (NG) mutagenesis were chilled and then collected by centrifugation or filtration. The samples were washed with cold sterile distilled water, and then resuspended in 0.2 M sodium acetate (pH 5.0) containing 1 mg/ml NG (K & K Co.). Cells at 5 x iO*/ml were aerated during mutagenesis. After incubation, cells were collected by filtration, washed with cold sterile distilled water, and resuspended in sterile distilled water for plating. Cells were spread on agar (2%) plates made of Y-minimal medium (Newlon et al., 1974), supplemented with 20 g glucose and 50 mg each of the appropriate nutrients per liter. For experiments with strain 1422-11 D, mutagen treatment was carried out at 0°C for 30 min. For strain 3163-Arg+, it was necessary to carry out the mutagen treatment at 30°C for 30 min in order to obtain a measurable rate of reversion to Ade+. Less than 20% of the cells were killed by the mutagen treatment at O”C, and less than 50% at 30°C. Under the conditions used, induction of mutations by NG continues linearly for at least 60 min at 30°C and for at least 6 hr at 0°C. DNA synthesis, as measured by incorporation of radioactive uracil into DNA, is inhibited by addition of NG at the concentration used for mutagenesis. Genetic analysis of revertants was carried out by standard techniques (Mortimer and Hawthorne, 1973). Acknowledgments This work was supported by a grant from the National Institute of Health and the University of Washington Graduate School Research Fund. W. B. was supported by a National Science Foundation predoctoral fellowship and by a Public Health Service Training Grant. We thank Dr. Herschel Roman for helpful criticism of the manuscript, and Loretta Lenny for technical assistance. Received

April

4, 1975;

revised

May 1, 1975

Temporal 269

Order

in Yeast

Chromosome

Replication

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