J. i!ifob. Rid.
(1977)
116, 240-260
Control of Saccharomyces cerevisiae 2pm DNA Replication by Cell Division Cycle Genes that Control Nuclear DNA Replication DENNIS
M. LrvINosroNt Department
ANI)
DORIS M. KI-PFER
of Genetics
Cniuersity of Washington &‘eattlu, Wash. Y8195, P.8.A. (Received 22 March 1977) Tire replicatron of the 2 pm DNA ofXucchnromycev cerew&ae has been examined in cell division cycle (cdc) mutants. The 2 pm DNA does not replicate at the restrictive temperature in cells bearing the c&28, cdc4, and cdc’i mutations which prevent passage of cells from the Gl phase into S phase. Plasmid replication also is prevented in a mating-type cells by c( factor, a mating hormone which prevent’s ~11s from completing an event early in G-1 phase. The 2 pm DNA ceases replication at 36°C in a mutant harboring the cd& mutation, a defect in the elongation reactions of nuclear DNA replication. Plasmid replication continues at the restrictive temperature for approximately oue generation in a cdcl3 mutant defective in nuclear division. These results show that 2 pm DNA replication is controlled by the same genes t,hat cont,rol t,hr initiat’ion and cornplrtion of nuclear IINA replication.
1. Introduction Genes necessary for the progression of a yeast cell through the events of the division cycle have been identified by temperature-sensitive. conditional lethal mutations (Hartwell et al., 1970). The gene products of the cdc28, c&4, and cdc7 genes function in a dependent sequence in the order given to promote the passage of cells from the Gl phase into S phase (Hereford & Hartwell, 1974; Hartwell, 1976). The cdc8 gene product is directly involved in nuclear DNA replication because nuclear DNA replication ceases abruptly in cd& mutants when shifted to the restrictive temperature (Hartwell, 1971). Other cdc mutations prevent the completion of events occurring after S phase, such as nuclear division and cytokinesis, and inhibit subsequent rounds of nuclear DNA replication (Hartwell et al., 1974). Except for the cdc8 mutation and the c&21 mutation, which is a temperaturrsensitive thymidine 5’-monophosphate auxotroph (Game, 1976), all other cdc mutations so far tested do not inhibit mit,ochondrial DNA replication (Cottrell et al., 1973; Cryer et al., 1973; Newlon & Fangman, 1975). Mitochondrial DNA replication also is not prevented by the presence of cycloheximide, a protein synthesis inhibitor (Grossman et al., 1969), or by CLfact,or, a yeast mating hormone (Petes t Fangman, 1973; t Permanent address: Department, of Bifpchemistjry, of Minnesotn, Minneapolis, Minn. 55455, U.S.A. 249
Medical
School, 277 Millard
Hall, University
The event’s that control DNA replication tiurin~ r t Ire ccl1 division cyelt: trright I,(, conveniently studied using the yeast 2 pm DSA plasmid. This circular, d0&1(:sbranded DNA molecule is present in approximately 100 copies per cell (Sinclair et (AZ., 1967 ; Clark-Walker & Miklos, 1974). Petes & Williamson (1975) have already observed double-branched replicative intermediates of 2 ,um DNA which are similar in appearance to simian virus 40 DNA replicative intermediates. Zeman & Lusena (1974) have presented evidence that cycloheximide inhibits 2 pm DNA replication at the same time as it prevents nuclear DNA replication. Although 2 pm DNA replication is defective in cd& and cdc21 mutants (Petes & Williamson, 1975), its replication has not been studied in other cdc mutants which control the initiation of nuclear DNA replication. In order to determine whether the replication of this presumed cytoplasmic DNA (Clark-Walker, 1972; Clark-Walker & Miklos, 1974; Livingston, 1977) requires gene products controlling nuclear DNA replication or escapes nuclear control as does mitochondrial DNA replication, we have examined 2 pm DNA replication in cdc mutants controlling inhibited cells.
the initiation
of nuclear
DNA
replication
and in CI factor-
2. Materials and Methods (a) Strains Saccharomyces cerevisiae strain A364A and the various cell division from the collection of Leland H. Hartwcll (Hartwell et a.l., 1973). (b)
Cell
cycle mutants were
growth
Cells were grown in medium containing per 1: 6.7 g yeast nitrogen base without amino acids and (NH&SOB (Difco), 0.5 g yeast extract (Difco), 20 g glucose, 10 g succinic acid, 6 g NaOH, 1 g (NH,)$OI, 20 mg adenine, 2 mg uracil, and 40 mg each of histidine, lysine, tyrosine, and leucine when required. Cells were grown at 23°C until they reached a density of approx. 2 x lo* cells/ml. The culture was then divided into an appropriate number of 120-ml samples and 0.6 mCi [6-3H]uracil (25 mCi/mmol ; New England Nuclear) was added to each sample. In most experiments the samples were incubated at 23°C for 10 min after addition of [3H]uracil. Half of the samples were removed to a 36°C waterbath shaker. The increase in temperature from 23°C to 36°C requires approximately 10 min. For strain H185.3.4 (c&28) a 38°C waterbath shaker replaced the 36°C waterbath shaker. In the experiments involving erfactor the pH of the medium was decreased to pH 3.5 by adding only 0.75 g NaOH to each 1 of medium. Sufficient a factor was added to inhibit growth for 6 h. The CLfactor was generously supplied by Russell Chan. The cell density, percentage of cells with appropriate terminal phenotype, and radioactivity of the total cellular DNA were monitored as previously described (Hartwell, 1970). (c) Spheroplast
formation
Cells were collected by centrifugation and spheroplasts were created using glusulasc (Endo) &s previously described by Peterson et al. (1972) with modifications of Newlon & Fangman (1975). Enzymatic cell wall digestion was carried out at 23°C and was terminated when greater than 99% of the cells were lysed by the addition of sodium lauryl sarcosinato (Colgate-Palmolive) to a concentration of 1% (w/v). The spheroplasts were collected by centrifugation at 3000 g for 5 min, resuspended in 5 ml of 1.0 M-sorbitol, 0.1 M-sodium
YEAST
-’- p’m
DNA
citrate buffer (pH 5.1), 0.06 M-EDTA, subjected to centrifugation resuspended in 0.75 ml of the above sorbitol solution.
(tl)
Extraction
“51
REPI.ICATION again,
and carefully
of 2 pwh 1)X24
A sample of 0.5 ml of the sphoroplast solution was slowly adclt:d to 0.5 ml of 0.05 31. d’mm dodecyl sulfate contained within a Tris.HC1 (pH %O), 0.01 M-EDTA, 2% (w / \ 7) so 10 mnl x 60 mm centrifuge tubo. The tube was capped with Parafilm and slowly inverted 10 tirncs. After a lo-min incubation at 23”C, 0.25 ml of 5 nz-NaCl was added to the lysat,r. and t,ho tube was once more capped with Parafihn and inverted to insure mixture of the NaCl solution. The lysates were t)lietl incubated at 0°C for 12 to 16 11before being subjected t,o cent,rifugation at 24,000 g for 60 min. Using this modification of the procedure of .Hirt. ( I!)#), the supernatant solution contains greater t,han 95:& of the 2 pm DNA but, only IO of tllr mitoclloltdrial DNA. to 20” (Jf the rNl&ar DNA alld apprOXimatd\ 5oq/, To tile supernatant solution containing tht: 2 pm I)NA was added 2.5 ml of 95qb (v/v) ct,llallol (- 20°C). The mixture of the ethanol and supernatant solution was incubated at - 20°C: for 30 min and t,hen subjrctod t,o centrifugat.ion at 24,000 g for 15 min. The result,ing supernatant, solut,ion was discarded. The pellet containing t,he 2 pm DNA as well as other nucleic acids and proteins was dissolved in 0.5 ml of 0.05 PI-TriseHCl (pH 8.0), 0.01 ;\r-EDTA, 0.05 &I-NaCl and then subjected to cent,rifugation at, 12,000 g for 5 min. The nlpernatant solution was removed and saved and tile tbxtraction was repeated. Thcl 2 supernatant solutions totalling I.0 ml were combined and 2.0 ml of 9506 ethanol (- 20°C) were added. Following incubation and collection by centrifugation as described above. tllr rclsulting pellet’ containing 2 ~111 DNA was washed witll I.0 ml cold 95% ethanol, ~ollectI~d by cerltrifugatioli at 24,000 g for 5 rnin, and tlirn dricsd ill air to remove traces M-Tris.HCl (pH ‘i-6), of c:tllsfrol. The- pelldts were t.ltcn resuspended in 100 pl of WI 0.001 M-EDTA and remaining irlsoluble debris was removed by cc,tLtrifugation at 12,000 g for 5 Inin. Thr sllpernatant solutions were removed atld troatcd for 2 11at 37°C with 50 pg of RNasc~ (I,ovitlc pancreas. 5 x rrcrystallizcd; Sigma) which had prc\viously hewn hcxattl(l a&ate. After 2 etllanol precipitations tllc, for 10 min at 80-C in a solution of 0.1 M-SOdilttn IWO\.(~~?; of DNA from thr origirlal supernatwnt solution is approsimat.cly 70:- Ilc>atirrg in 0.5 ml of wat(‘r. Scintillation fluid (A(111asol; _Vc\v Etrgland Nuc~l~ar) \I’W adtl~tl t,o t,ltfi dissolved gel section and the radioactivit,y ~r~asnrt~l. To i nsl~rr’ tllat ot~ly DNA was bc-irlg mcasnred by this method, an initial c,xperimcnt was pcrformc~d it, \vllictl grl sections \vt~ dissolved in 1.0 ml of 1.0 N-NaOH by hcat,ing to 1OO’C for 10 min. The DXA in the solntiorl was then precipitated with trichloroacc~t~ic a~cid and collected on a filter as previously described (Hartwell, 1970). This initial (,xperiment sllowcd that grewt,Pr than 957; of the radioactivity rnrasnred in gel sections by dissolving directly in xvater rcsult,ed from alkaline-stable, acid-precipitjablo DNA and not RNA or llllcleosrde precursors. T11ospecific activity of the 2 pm DNA was calcnlated I,)- adding ttlcr radioactivity content of tbrt supcrhelical and relaxed circular 2 pm DNA and dividing by tile sum of t,lt(> weights of the 2 DNA species. Thcs specific actjirity of aorrcatcrrat~rtl molocnlcs \vas riot included ill the calculations.
(f) Specific activity
of nuclear and mitochon&ial DNA A 0.25 ml sample of the splleroplast solution was combined with 0.75 ml of O-01 MTris*HCl (pH 7.6), 0.001 M-EDTA and 1.0 ml of 0.5 iw-Tris.HCl (pH 7.6), 0.1 M-EDTA, 2% (w/v) sodium lauryl sarcosinatc. The sol&on was incubated at 65°C for 10 min. Nuclear and mitochondrial DNA were separated by isopycnic centrifngation as described by Grossman et al. (1969). Fractions from the CsCl gradients conta,ining nnclear and mitochondrial DNA were combined with 2 vol. of 0.01 M-Tris-HCl (pH 7.6), 0.001 M-EDTA and then 6 vol. of cold ethanol wore added to precipitate the DNA. T11a DNA was collected by ct,ntrifugation and sub,j(bctcd t,o gel c~lect~rol)liorcsis t,c) dc>tc:rniinc it,s specific activity as doscribed abovo. determination
3. Results (a) Measurement
of 2 pm DNA
replicatkm
To follow the replication of tL pm DNA in clEc mutants, the iucrease in specific of 2 pm DNA was measured after addition of a radioactive precursor. If immediate equilibration of the radioactive label within the cellular deoxynucleoside triphosphate pools occurs, then the specific activity of the DNA will be one-half of the value of fully labeled DNA after one romld of replication, and three-quarters of the fully labeled value after two rounds of replication. Measurement of specific activity rather than total radioactivity or tot,al amount of DNA avoids the possible problems arising from unequal recoveries of DNA during preparation of extracts for measurement. In order to measure the specific activity of 2 pm DNA, a method has been used which utilizes small quantities of 2 pm DNA and is not dependent on the recovery of superhelical molecules. As shown in Figure 1, both superhelical 2 pm DNA molecules (form I) and circular 2 pm DNA molecules containing single-sbrand interruptions (form II) separate from nuclear and mitochondrial DNA when subjected to electrophoresis in an agarose gel. The amount of DNA in the bands has been estimated by ethidium bromide staining and densitometer analysis, while the radioactivity in the same band has been measured in a scintillation spectrometer (Fig. 2).
radioactivity
(b) 2 pm DNA
replication
in cdc mutants unable to initiate replication
nuclear DNA
The gene products of the cdc20, c&4 and cdc7 genes function in a dependent order during Gl phase to promote the initiation of nuclear DNA replication (Hereford & Hartwell, 1974; Hartwell, 1976). At the restrictive temperature these mutants fail to initiate rounds of nuclear DNA replication but continue to replicate mitochondrial
YEAST
2 pm
DNA
453
REPLICATION
DXA (Cot’trell et al., 1973; Cryer et al., 1973; Newlon & Fangman, 1975). Also, t’hese mutants continue to synthesize RNA and proteins at the restrictive temperature for a period of two or more generations (Hartwell, 1974). The replication of 2 pm DNA has been examined in these three mutants. Figure 3(c), (d)? (e), and (f) shows that for the equivalents of two generation times after a shift from permissive to restrictive tcmpcratjurc~ t,hc specific activity of t’he 2 pm DNA does not’ increase appreciably in
(b)
(cl
Cd)
(h)
(0
(i)
FORM II
FORM
Prc:. 1. Electrophoresis of DNA-containing extracts. Extracts of yeast were prepared by t,he method of Hirt (1967) and subjected to electrophoresis in an agarose gel. Electrophoresis is from top to bottom. Slot (e) contains purified 2 pm DNA. The fastest migrating band contains superhelical 2 pm DNA molecules (form I) and the heaviest band above it contains circular molecules with single-strand interruptions (form II). Minor bands above form II are concatenated 2 pm 11X.4 molecules (Guerineau et al., 1976). The large quantity of DNA migrating above form II in the slot5 containing the extracts ((a) to (d) and (f) to (i)) is residual nuclear and mitochondrial DNA which is not precipitated during the extraction procedure. This gel contains extracts prepared from strain A364A grown in the presence and absence of the yeast mating hormone, c( factor. Slots (f) t,hrough (i) are extracts from cells grown in the presence of a fact,or and harvested at 1, 2, 4, and 6 h, respectively, after addition of t,he fact,or. Extracts from tho control culture are found in slots (a) to ((I). Fragments produced by digesting phage h DNA with the restriction cndonucloast: Kcoli 1, a~‘(: contained in slot (j) and am used as a standard.
c&28, cdc4, and cdc7 mutants. In comparison, the specific activity of 2 pm DNA increases in the same cells at the permissive temperature. Normal plasmid replication at 36°C in the wild-type parent strain (Fi g. 3(a)) shows that temperature itself does not inhibit 2 pm DNA replication. Because of the temporal proximity of the action of the cdc7 gene product to the initiation of nuclear DNA replication (Hereford & Hartwell, 1974), two alleles of c&7 were examined (Fig. 3(e) and (f)) and both were
254
D.
$1. LIVINGHTON
ANI)
1).
M.
ICUP’PER
Distance (cm) FIG. 2. Measurement of the specific activity of 2 pm DNA. In each panel the densitometer tracing of one slot of the photographic negative (-------) (Fig. 1) has been superimposed on the rediofrom cells labeled activity analysis of t*he sectioned gel (-•-•-). (a) A nrt ly sis of the extract for 4 h in the absence of CY. factor (Fig. l(c)) ; (b) the analysis of the extract grown for a corresponding length of time in the presence of c( factor (Fig. l(h)). The amount of DNA contained within form I and form II was determined as described in Materials and Methods using the EcoRI digest of h DNA (Fig. l(j)) as a calibration standard.
found to be incapable of replicating 2 pm DNA at the restrictive temperature. Thus, cdc mutations which control the initiation of nuclear DNA replication but do not affect mitochondrial DNA replication also control 2 pm DNA replication. (c) 2pm DNA
replication
in cells arrested by u factor
Yeast of mating type u secrete a polypeptide, cc factor, which reversibly arrests division of a mating type cells (Bucking-Throm et al., 1973). The a cells are specifically arrested at the c&28-mediated step in Gl phase before the action of the c&c4 and cdc7 gene-mediated steps (Hereford & Hartwell, 1974). Nuclear DNA replication ceases in a factor-arrested cells while mitochondrial DNA replication and RNA and protein synthesis continue (Petes t Bangman, 1973; Cryer et al., 1973).
2 CLm DNA
YEAST
I 8-
(01
-- (b)
I
1
255
REPLICATION I ,
-
o-p--~-~
I
I . (d) . /
E \ 5 ‘; .z
i C--*-./-P 8-
-p----> -- (f)
(e)
2
4
2
6
4
6
Tome
FIG. 3. Replication of 2 pm DNA in cells unable to complete the cell division cycle. Cells of strain 8364A ((a) and (b)), H186.3.4 (c&28-1) (c),H135.1.1 (cd&3) (d), 124Dl (c&7-1) (e), 4008 (c&7-4) (f), 198 (cd&l) (g), and 428 (c&13-1) (h) were grown at 23°C and at zero time half of the culture was shifted to 36°C (38°C for H185.3.4). In the OLfactor experiment (b) the culture was divided in half and one portion received c( factor without any change in temperature. Ten min before the temperature shift (or addition of I* factor ) [6-3H]uracil had been added to the medium except in the experiment in (f) where the label had been added 120 min before the temperature shift. At the times indicated samples were analyzed for the radioactive specific activity of the 2 pm DNA. -e-e--, 23°C control; -O--O-, 36°C (38°C) or (Lfact,or (b).
Figure 3(b) shows that 2 pm DNA replication does not occur after addition of E factor to cells. The inhibition of 2 pm DNA replication by a factor shows that the specific arrest of division by a chemical agent at 23°C has the same effect on 2 pm DNA replication as do cdc mutations at 36°C. (d) 2 pm DNA replication
in a cdc8 and a cdcla mutant
The cdc8 gene product is unlike other cdc gene products which control nuclear DNA4 replication in that both nuclear and mitochondrial DNA replication cease abruptly when the mutant is shifted to 36°C (Hartwell, 1971; Cryer et al., 1974; Wintersberger et al., 1974; Newlon & Fangman, 1975). Petes & Williamson (1975) have observed the
256
1).
M.
LIVINGSTON
AND
11. M.
KL~PFEl
-cl---,
-A-A--, nuclear DNA; 2 pm
DNA;
mitochonclrial -A--::)-
-A-A-, DN.4;
-Y---._J
-.
mito.
activky in both nuclear and 2 pm DNA after 6 h in (a) reflects t,hc cells. The parallel increase in specific activities is another indicat,ion is controlled by cell cycle events a$ is nuclear DNA replication.
activity changes incurs a possible problem of equilibration of radioactivity into deoxynucleoside triphosphate pools. If equilibration of radioactive uracil into precursor pools does not occur in the absence of nuclear DNA replication, then the lack of radioactive incorporation into 2 ,um DNA under restrict.ive conditions might not be indicative of t’he absence of 2 pm DNA replication. The lack of radioactivity in
“5H
1). 11. LIVINGSTON
;\NI)
I).
11. liCI’1~‘151{
precursor pools under restrictive conditions is unlikel,v because mitochondrial DN;\ is equally labeled in cells capable and incapable of synthesizing nuclear DNA (Fig. 4). However, whether the same precursor’ pools are used in the synt,hcsis of nuclt~r, mitochondrial, and 2 pm DNA is unknown. Another artifact might arise from a rapid degradation of 2 pm DNA in cells held under restrict,ivc: conditions. 1n all the cxperimerits we have never failed to recover superhelical 2 pm DNA from cells held under conditions which we interpret t)o hc restrictive for plasmid replication. [inless newly synthesized molecules are preferent’ially degraded in comparison to pre-existing molecules, our results show that degradation dots not contribute to our inability to detect 2 pm DNA replication under rest’rict,ive condit,ions. Yet, nnothcr limitation of the method is that radioactivit,y cannot he detected in replicative intermediates x+-hi& do not’ form recognizable hands during elecbrophoresis, Thercforc, in order to conclude that 2 pm DNA replication does not occur under restrictive conditions, we must assume that undetectable replicative intermediates do not cont’inually accumulate. This assumption is justified by the results of Petes & Williamson (1975) which show that the maximum percentage of 2 pm DNA molecules existing as replication intermediates in either &7 or cd&3 blocked cells is Ci”;, of the total number of molecules. We conclude that the replication of the yeast 2 pm plasmid DNA is controlled by many of the same genes which control nuclear DNA replication. The initiation of nuclear DNA replication has been shown t’o occur after a series of dependent events mediated by the c&28, cdc4, and cdc7 gene product,s (Hereford & Hartwell, 1974; Hartwell, 1976). Although these gene products have not heen isolated or their exact function identified, they are known to he necessary for the duplication and separation of the nuclear spindle pole body. The cdc28 gene product permits the duplication of the spindle pole body, and in turn the separation of the duplicated spindle pole body requires the action of the c&4 gene product (Byers & Goetsch, 1973). Only after the completion of the cdc28 and c&4-mediated events can t’he cdc7 gene product act to promote nuclear DNA replication. The parallel between the control of nuclear and 2 pm DNA replication is surprising because evidence suggests that 2 pm DNA is not located in the nucleus hut is associated with a cytoplasmic structure other than the mitochondria (Clark-Walker, 1972; Clark-Walker & Miklos, 1974; Livingston, 1977). The evidence which suggests the plasmid’s cytoplasmic rather than nuclear location is that isolated nuclei do not contain 2 pm DNA (Clark-Walker & Miklos, 1974), and that the plasmid can he transmitted during mating through cytoplasmic mixing in t’he absence of nuclear fusion (Livingston, 1977). The evidence promoting the cytoplasmic location for the plasmid does not exclude the possihility t,hat 2 pm DNA molecules must travel into the nucleus to he replicated. Another possibility raised by our results is that 2 pm DNA replication may occur during the S phase of the cell cycle. Consistent with this idea is the observation that 2 pm DNA undergoes very little replication in a c&7 mutant after a shift to 36°C (Fig. 3(f)), while approximately one round of 2 pm DNA replication occurs in a c&l3 mutant after a similar temperature shift (Fig. 3(h)). The lack of replication in a mutant defective in initiation of 8 (c&7) and the single round replication in a mutant defective in nuclear division (c&13) suggest that 2 pm DNA replication normally occurs after completion of the cdc28, c&4, and c&7-mediated events which lead to the initiation of S (Hartwell, 1976). 2 pm DNA replication is clearly different from mitochondrial DNA replication
YEAST
2 CLm DNA
REPLICATIOS
259
because mitochondrial DNA replication continues in c&28, cdc4, and c&7 mutants at the restrictive temperature and in CI factor-arrested cells (Cottrell et aE., 1973 ; Cryer it al., 1973; Petes & Fangman, 1973; Newlon & Fangman, 1975). Mitochondrial DNA synthesis occurs at times in the cell cycle other than S phase (Sena et al., 1975) and continues in the presence of cycloheximide, a protein synthesis inhibitor which prevents initiation of nuclear DNA replication (Grossman et al., 1969). All three cellular DNA!: do share the common features t’hat all are dependent on the c&8 gene product which is probably directly involved in the elongation reactions of DNA replication and on the c&21 gene product which is iuvolvcd in thymidine metabolism (Hartwell, 1971; Game, 1976; Cryer et al., 1973; Wint,ersbc~rgcr pt ab.. 1974: Newlon & Fangman, 19i5: Petes & Williamson, 1975). Th(a dependence of 2 pm DXA replication on gene product’s known to mediat#e Gl events t)hat are prerequisite for t’he init’iation of nuclear DNA replication makes this plasmid an attractive molecular model to study thfb evcnbs whit+ initiate and support nuclear DNA rc$ication.
WC thank Leland H. Hartwell for his interc~st and support of this work. This project was supported by a grant from the National Inst,itute of General Medical Sciences (GM17709) to Leland H. Hartwell. One of us (D.M.L.) is a postdoctoral fellow of the National Ca~~ccr Institllt,e (CA 00592).
REFERENCE8 I~uc~kirlg-Thrown, E., Dulltze, W.. Hartwell, L. H. & Ma~lney, T. I
(1977)
116, 240-260
Control of Saccharomyces cerevisiae 2pm DNA Replication by Cell Division Cycle Genes that Control Nuclear DNA Replication DENNIS
M. LrvINosroNt Department
ANI)
DORIS M. KI-PFER
of Genetics
Cniuersity of Washington &‘eattlu, Wash. Y8195, P.8.A. (Received 22 March 1977) Tire replicatron of the 2 pm DNA ofXucchnromycev cerew&ae has been examined in cell division cycle (cdc) mutants. The 2 pm DNA does not replicate at the restrictive temperature in cells bearing the c&28, cdc4, and cdc’i mutations which prevent passage of cells from the Gl phase into S phase. Plasmid replication also is prevented in a mating-type cells by c( factor, a mating hormone which prevent’s ~11s from completing an event early in G-1 phase. The 2 pm DNA ceases replication at 36°C in a mutant harboring the cd& mutation, a defect in the elongation reactions of nuclear DNA replication. Plasmid replication continues at the restrictive temperature for approximately oue generation in a cdcl3 mutant defective in nuclear division. These results show that 2 pm DNA replication is controlled by the same genes t,hat cont,rol t,hr initiat’ion and cornplrtion of nuclear IINA replication.
1. Introduction Genes necessary for the progression of a yeast cell through the events of the division cycle have been identified by temperature-sensitive. conditional lethal mutations (Hartwell et al., 1970). The gene products of the cdc28, c&4, and cdc7 genes function in a dependent sequence in the order given to promote the passage of cells from the Gl phase into S phase (Hereford & Hartwell, 1974; Hartwell, 1976). The cdc8 gene product is directly involved in nuclear DNA replication because nuclear DNA replication ceases abruptly in cd& mutants when shifted to the restrictive temperature (Hartwell, 1971). Other cdc mutations prevent the completion of events occurring after S phase, such as nuclear division and cytokinesis, and inhibit subsequent rounds of nuclear DNA replication (Hartwell et al., 1974). Except for the cdc8 mutation and the c&21 mutation, which is a temperaturrsensitive thymidine 5’-monophosphate auxotroph (Game, 1976), all other cdc mutations so far tested do not inhibit mit,ochondrial DNA replication (Cottrell et al., 1973; Cryer et al., 1973; Newlon & Fangman, 1975). Mitochondrial DNA replication also is not prevented by the presence of cycloheximide, a protein synthesis inhibitor (Grossman et al., 1969), or by CLfact,or, a yeast mating hormone (Petes t Fangman, 1973; t Permanent address: Department, of Bifpchemistjry, of Minnesotn, Minneapolis, Minn. 55455, U.S.A. 249
Medical
School, 277 Millard
Hall, University
The event’s that control DNA replication tiurin~ r t Ire ccl1 division cyelt: trright I,(, conveniently studied using the yeast 2 pm DSA plasmid. This circular, d0&1(:sbranded DNA molecule is present in approximately 100 copies per cell (Sinclair et (AZ., 1967 ; Clark-Walker & Miklos, 1974). Petes & Williamson (1975) have already observed double-branched replicative intermediates of 2 ,um DNA which are similar in appearance to simian virus 40 DNA replicative intermediates. Zeman & Lusena (1974) have presented evidence that cycloheximide inhibits 2 pm DNA replication at the same time as it prevents nuclear DNA replication. Although 2 pm DNA replication is defective in cd& and cdc21 mutants (Petes & Williamson, 1975), its replication has not been studied in other cdc mutants which control the initiation of nuclear DNA replication. In order to determine whether the replication of this presumed cytoplasmic DNA (Clark-Walker, 1972; Clark-Walker & Miklos, 1974; Livingston, 1977) requires gene products controlling nuclear DNA replication or escapes nuclear control as does mitochondrial DNA replication, we have examined 2 pm DNA replication in cdc mutants controlling inhibited cells.
the initiation
of nuclear
DNA
replication
and in CI factor-
2. Materials and Methods (a) Strains Saccharomyces cerevisiae strain A364A and the various cell division from the collection of Leland H. Hartwcll (Hartwell et a.l., 1973). (b)
Cell
cycle mutants were
growth
Cells were grown in medium containing per 1: 6.7 g yeast nitrogen base without amino acids and (NH&SOB (Difco), 0.5 g yeast extract (Difco), 20 g glucose, 10 g succinic acid, 6 g NaOH, 1 g (NH,)$OI, 20 mg adenine, 2 mg uracil, and 40 mg each of histidine, lysine, tyrosine, and leucine when required. Cells were grown at 23°C until they reached a density of approx. 2 x lo* cells/ml. The culture was then divided into an appropriate number of 120-ml samples and 0.6 mCi [6-3H]uracil (25 mCi/mmol ; New England Nuclear) was added to each sample. In most experiments the samples were incubated at 23°C for 10 min after addition of [3H]uracil. Half of the samples were removed to a 36°C waterbath shaker. The increase in temperature from 23°C to 36°C requires approximately 10 min. For strain H185.3.4 (c&28) a 38°C waterbath shaker replaced the 36°C waterbath shaker. In the experiments involving erfactor the pH of the medium was decreased to pH 3.5 by adding only 0.75 g NaOH to each 1 of medium. Sufficient a factor was added to inhibit growth for 6 h. The CLfactor was generously supplied by Russell Chan. The cell density, percentage of cells with appropriate terminal phenotype, and radioactivity of the total cellular DNA were monitored as previously described (Hartwell, 1970). (c) Spheroplast
formation
Cells were collected by centrifugation and spheroplasts were created using glusulasc (Endo) &s previously described by Peterson et al. (1972) with modifications of Newlon & Fangman (1975). Enzymatic cell wall digestion was carried out at 23°C and was terminated when greater than 99% of the cells were lysed by the addition of sodium lauryl sarcosinato (Colgate-Palmolive) to a concentration of 1% (w/v). The spheroplasts were collected by centrifugation at 3000 g for 5 min, resuspended in 5 ml of 1.0 M-sorbitol, 0.1 M-sodium
YEAST
-’- p’m
DNA
citrate buffer (pH 5.1), 0.06 M-EDTA, subjected to centrifugation resuspended in 0.75 ml of the above sorbitol solution.
(tl)
Extraction
“51
REPI.ICATION again,
and carefully
of 2 pwh 1)X24
A sample of 0.5 ml of the sphoroplast solution was slowly adclt:d to 0.5 ml of 0.05 31. d’mm dodecyl sulfate contained within a Tris.HC1 (pH %O), 0.01 M-EDTA, 2% (w / \ 7) so 10 mnl x 60 mm centrifuge tubo. The tube was capped with Parafilm and slowly inverted 10 tirncs. After a lo-min incubation at 23”C, 0.25 ml of 5 nz-NaCl was added to the lysat,r. and t,ho tube was once more capped with Parafihn and inverted to insure mixture of the NaCl solution. The lysates were t)lietl incubated at 0°C for 12 to 16 11before being subjected t,o cent,rifugation at 24,000 g for 60 min. Using this modification of the procedure of .Hirt. ( I!)#), the supernatant solution contains greater t,han 95:& of the 2 pm DNA but, only IO of tllr mitoclloltdrial DNA. to 20” (Jf the rNl&ar DNA alld apprOXimatd\ 5oq/, To tile supernatant solution containing tht: 2 pm I)NA was added 2.5 ml of 95qb (v/v) ct,llallol (- 20°C). The mixture of the ethanol and supernatant solution was incubated at - 20°C: for 30 min and t,hen subjrctod t,o centrifugat.ion at 24,000 g for 15 min. The result,ing supernatant, solut,ion was discarded. The pellet containing t,he 2 pm DNA as well as other nucleic acids and proteins was dissolved in 0.5 ml of 0.05 PI-TriseHCl (pH 8.0), 0.01 ;\r-EDTA, 0.05 &I-NaCl and then subjected to cent,rifugation at, 12,000 g for 5 min. The nlpernatant solution was removed and saved and tile tbxtraction was repeated. Thcl 2 supernatant solutions totalling I.0 ml were combined and 2.0 ml of 9506 ethanol (- 20°C) were added. Following incubation and collection by centrifugation as described above. tllr rclsulting pellet’ containing 2 ~111 DNA was washed witll I.0 ml cold 95% ethanol, ~ollectI~d by cerltrifugatioli at 24,000 g for 5 rnin, and tlirn dricsd ill air to remove traces M-Tris.HCl (pH ‘i-6), of c:tllsfrol. The- pelldts were t.ltcn resuspended in 100 pl of WI 0.001 M-EDTA and remaining irlsoluble debris was removed by cc,tLtrifugation at 12,000 g for 5 Inin. Thr sllpernatant solutions were removed atld troatcd for 2 11at 37°C with 50 pg of RNasc~ (I,ovitlc pancreas. 5 x rrcrystallizcd; Sigma) which had prc\viously hewn hcxattl(l a&ate. After 2 etllanol precipitations tllc, for 10 min at 80-C in a solution of 0.1 M-SOdilttn IWO\.(~~?; of DNA from thr origirlal supernatwnt solution is approsimat.cly 70:- Ilc>atirrg in 0.5 ml of wat(‘r. Scintillation fluid (A(111asol; _Vc\v Etrgland Nuc~l~ar) \I’W adtl~tl t,o t,ltfi dissolved gel section and the radioactivit,y ~r~asnrt~l. To i nsl~rr’ tllat ot~ly DNA was bc-irlg mcasnred by this method, an initial c,xperimcnt was pcrformc~d it, \vllictl grl sections \vt~ dissolved in 1.0 ml of 1.0 N-NaOH by hcat,ing to 1OO’C for 10 min. The DXA in the solntiorl was then precipitated with trichloroacc~t~ic a~cid and collected on a filter as previously described (Hartwell, 1970). This initial (,xperiment sllowcd that grewt,Pr than 957; of the radioactivity rnrasnred in gel sections by dissolving directly in xvater rcsult,ed from alkaline-stable, acid-precipitjablo DNA and not RNA or llllcleosrde precursors. T11ospecific activity of the 2 pm DNA was calcnlated I,)- adding ttlcr radioactivity content of tbrt supcrhelical and relaxed circular 2 pm DNA and dividing by tile sum of t,lt(> weights of the 2 DNA species. Thcs specific actjirity of aorrcatcrrat~rtl molocnlcs \vas riot included ill the calculations.
(f) Specific activity
of nuclear and mitochon&ial DNA A 0.25 ml sample of the splleroplast solution was combined with 0.75 ml of O-01 MTris*HCl (pH 7.6), 0.001 M-EDTA and 1.0 ml of 0.5 iw-Tris.HCl (pH 7.6), 0.1 M-EDTA, 2% (w/v) sodium lauryl sarcosinatc. The sol&on was incubated at 65°C for 10 min. Nuclear and mitochondrial DNA were separated by isopycnic centrifngation as described by Grossman et al. (1969). Fractions from the CsCl gradients conta,ining nnclear and mitochondrial DNA were combined with 2 vol. of 0.01 M-Tris-HCl (pH 7.6), 0.001 M-EDTA and then 6 vol. of cold ethanol wore added to precipitate the DNA. T11a DNA was collected by ct,ntrifugation and sub,j(bctcd t,o gel c~lect~rol)liorcsis t,c) dc>tc:rniinc it,s specific activity as doscribed abovo. determination
3. Results (a) Measurement
of 2 pm DNA
replicatkm
To follow the replication of tL pm DNA in clEc mutants, the iucrease in specific of 2 pm DNA was measured after addition of a radioactive precursor. If immediate equilibration of the radioactive label within the cellular deoxynucleoside triphosphate pools occurs, then the specific activity of the DNA will be one-half of the value of fully labeled DNA after one romld of replication, and three-quarters of the fully labeled value after two rounds of replication. Measurement of specific activity rather than total radioactivity or tot,al amount of DNA avoids the possible problems arising from unequal recoveries of DNA during preparation of extracts for measurement. In order to measure the specific activity of 2 pm DNA, a method has been used which utilizes small quantities of 2 pm DNA and is not dependent on the recovery of superhelical molecules. As shown in Figure 1, both superhelical 2 pm DNA molecules (form I) and circular 2 pm DNA molecules containing single-sbrand interruptions (form II) separate from nuclear and mitochondrial DNA when subjected to electrophoresis in an agarose gel. The amount of DNA in the bands has been estimated by ethidium bromide staining and densitometer analysis, while the radioactivity in the same band has been measured in a scintillation spectrometer (Fig. 2).
radioactivity
(b) 2 pm DNA
replication
in cdc mutants unable to initiate replication
nuclear DNA
The gene products of the cdc20, c&4 and cdc7 genes function in a dependent order during Gl phase to promote the initiation of nuclear DNA replication (Hereford & Hartwell, 1974; Hartwell, 1976). At the restrictive temperature these mutants fail to initiate rounds of nuclear DNA replication but continue to replicate mitochondrial
YEAST
2 pm
DNA
453
REPLICATION
DXA (Cot’trell et al., 1973; Cryer et al., 1973; Newlon & Fangman, 1975). Also, t’hese mutants continue to synthesize RNA and proteins at the restrictive temperature for a period of two or more generations (Hartwell, 1974). The replication of 2 pm DNA has been examined in these three mutants. Figure 3(c), (d)? (e), and (f) shows that for the equivalents of two generation times after a shift from permissive to restrictive tcmpcratjurc~ t,hc specific activity of t’he 2 pm DNA does not’ increase appreciably in
(b)
(cl
Cd)
(h)
(0
(i)
FORM II
FORM
Prc:. 1. Electrophoresis of DNA-containing extracts. Extracts of yeast were prepared by t,he method of Hirt (1967) and subjected to electrophoresis in an agarose gel. Electrophoresis is from top to bottom. Slot (e) contains purified 2 pm DNA. The fastest migrating band contains superhelical 2 pm DNA molecules (form I) and the heaviest band above it contains circular molecules with single-strand interruptions (form II). Minor bands above form II are concatenated 2 pm 11X.4 molecules (Guerineau et al., 1976). The large quantity of DNA migrating above form II in the slot5 containing the extracts ((a) to (d) and (f) to (i)) is residual nuclear and mitochondrial DNA which is not precipitated during the extraction procedure. This gel contains extracts prepared from strain A364A grown in the presence and absence of the yeast mating hormone, c( factor. Slots (f) t,hrough (i) are extracts from cells grown in the presence of a fact,or and harvested at 1, 2, 4, and 6 h, respectively, after addition of t,he fact,or. Extracts from tho control culture are found in slots (a) to ((I). Fragments produced by digesting phage h DNA with the restriction cndonucloast: Kcoli 1, a~‘(: contained in slot (j) and am used as a standard.
c&28, cdc4, and cdc7 mutants. In comparison, the specific activity of 2 pm DNA increases in the same cells at the permissive temperature. Normal plasmid replication at 36°C in the wild-type parent strain (Fi g. 3(a)) shows that temperature itself does not inhibit 2 pm DNA replication. Because of the temporal proximity of the action of the cdc7 gene product to the initiation of nuclear DNA replication (Hereford & Hartwell, 1974), two alleles of c&7 were examined (Fig. 3(e) and (f)) and both were
254
D.
$1. LIVINGHTON
ANI)
1).
M.
ICUP’PER
Distance (cm) FIG. 2. Measurement of the specific activity of 2 pm DNA. In each panel the densitometer tracing of one slot of the photographic negative (-------) (Fig. 1) has been superimposed on the rediofrom cells labeled activity analysis of t*he sectioned gel (-•-•-). (a) A nrt ly sis of the extract for 4 h in the absence of CY. factor (Fig. l(c)) ; (b) the analysis of the extract grown for a corresponding length of time in the presence of c( factor (Fig. l(h)). The amount of DNA contained within form I and form II was determined as described in Materials and Methods using the EcoRI digest of h DNA (Fig. l(j)) as a calibration standard.
found to be incapable of replicating 2 pm DNA at the restrictive temperature. Thus, cdc mutations which control the initiation of nuclear DNA replication but do not affect mitochondrial DNA replication also control 2 pm DNA replication. (c) 2pm DNA
replication
in cells arrested by u factor
Yeast of mating type u secrete a polypeptide, cc factor, which reversibly arrests division of a mating type cells (Bucking-Throm et al., 1973). The a cells are specifically arrested at the c&28-mediated step in Gl phase before the action of the c&c4 and cdc7 gene-mediated steps (Hereford & Hartwell, 1974). Nuclear DNA replication ceases in a factor-arrested cells while mitochondrial DNA replication and RNA and protein synthesis continue (Petes t Bangman, 1973; Cryer et al., 1973).
2 CLm DNA
YEAST
I 8-
(01
-- (b)
I
1
255
REPLICATION I ,
-
o-p--~-~
I
I . (d) . /
E \ 5 ‘; .z
i C--*-./-P 8-
-p----> -- (f)
(e)
2
4
2
6
4
6
Tome
FIG. 3. Replication of 2 pm DNA in cells unable to complete the cell division cycle. Cells of strain 8364A ((a) and (b)), H186.3.4 (c&28-1) (c),H135.1.1 (cd&3) (d), 124Dl (c&7-1) (e), 4008 (c&7-4) (f), 198 (cd&l) (g), and 428 (c&13-1) (h) were grown at 23°C and at zero time half of the culture was shifted to 36°C (38°C for H185.3.4). In the OLfactor experiment (b) the culture was divided in half and one portion received c( factor without any change in temperature. Ten min before the temperature shift (or addition of I* factor ) [6-3H]uracil had been added to the medium except in the experiment in (f) where the label had been added 120 min before the temperature shift. At the times indicated samples were analyzed for the radioactive specific activity of the 2 pm DNA. -e-e--, 23°C control; -O--O-, 36°C (38°C) or (Lfact,or (b).
Figure 3(b) shows that 2 pm DNA replication does not occur after addition of E factor to cells. The inhibition of 2 pm DNA replication by a factor shows that the specific arrest of division by a chemical agent at 23°C has the same effect on 2 pm DNA replication as do cdc mutations at 36°C. (d) 2 pm DNA replication
in a cdc8 and a cdcla mutant
The cdc8 gene product is unlike other cdc gene products which control nuclear DNA4 replication in that both nuclear and mitochondrial DNA replication cease abruptly when the mutant is shifted to 36°C (Hartwell, 1971; Cryer et al., 1974; Wintersberger et al., 1974; Newlon & Fangman, 1975). Petes & Williamson (1975) have observed the
256
1).
M.
LIVINGSTON
AND
11. M.
KL~PFEl
-cl---,
-A-A--, nuclear DNA; 2 pm
DNA;
mitochonclrial -A--::)-
-A-A-, DN.4;
-Y---._J
-.
mito.
activky in both nuclear and 2 pm DNA after 6 h in (a) reflects t,hc cells. The parallel increase in specific activities is another indicat,ion is controlled by cell cycle events a$ is nuclear DNA replication.
activity changes incurs a possible problem of equilibration of radioactivity into deoxynucleoside triphosphate pools. If equilibration of radioactive uracil into precursor pools does not occur in the absence of nuclear DNA replication, then the lack of radioactive incorporation into 2 ,um DNA under restrict.ive conditions might not be indicative of t’he absence of 2 pm DNA replication. The lack of radioactivity in
“5H
1). 11. LIVINGSTON
;\NI)
I).
11. liCI’1~‘151{
precursor pools under restrictive conditions is unlikel,v because mitochondrial DN;\ is equally labeled in cells capable and incapable of synthesizing nuclear DNA (Fig. 4). However, whether the same precursor’ pools are used in the synt,hcsis of nuclt~r, mitochondrial, and 2 pm DNA is unknown. Another artifact might arise from a rapid degradation of 2 pm DNA in cells held under restrict,ivc: conditions. 1n all the cxperimerits we have never failed to recover superhelical 2 pm DNA from cells held under conditions which we interpret t)o hc restrictive for plasmid replication. [inless newly synthesized molecules are preferent’ially degraded in comparison to pre-existing molecules, our results show that degradation dots not contribute to our inability to detect 2 pm DNA replication under rest’rict,ive condit,ions. Yet, nnothcr limitation of the method is that radioactivit,y cannot he detected in replicative intermediates x+-hi& do not’ form recognizable hands during elecbrophoresis, Thercforc, in order to conclude that 2 pm DNA replication does not occur under restrictive conditions, we must assume that undetectable replicative intermediates do not cont’inually accumulate. This assumption is justified by the results of Petes & Williamson (1975) which show that the maximum percentage of 2 pm DNA molecules existing as replication intermediates in either &7 or cd&3 blocked cells is Ci”;, of the total number of molecules. We conclude that the replication of the yeast 2 pm plasmid DNA is controlled by many of the same genes which control nuclear DNA replication. The initiation of nuclear DNA replication has been shown t’o occur after a series of dependent events mediated by the c&28, cdc4, and cdc7 gene product,s (Hereford & Hartwell, 1974; Hartwell, 1976). Although these gene products have not heen isolated or their exact function identified, they are known to he necessary for the duplication and separation of the nuclear spindle pole body. The cdc28 gene product permits the duplication of the spindle pole body, and in turn the separation of the duplicated spindle pole body requires the action of the c&4 gene product (Byers & Goetsch, 1973). Only after the completion of the cdc28 and c&4-mediated events can t’he cdc7 gene product act to promote nuclear DNA replication. The parallel between the control of nuclear and 2 pm DNA replication is surprising because evidence suggests that 2 pm DNA is not located in the nucleus hut is associated with a cytoplasmic structure other than the mitochondria (Clark-Walker, 1972; Clark-Walker & Miklos, 1974; Livingston, 1977). The evidence which suggests the plasmid’s cytoplasmic rather than nuclear location is that isolated nuclei do not contain 2 pm DNA (Clark-Walker & Miklos, 1974), and that the plasmid can he transmitted during mating through cytoplasmic mixing in t’he absence of nuclear fusion (Livingston, 1977). The evidence promoting the cytoplasmic location for the plasmid does not exclude the possihility t,hat 2 pm DNA molecules must travel into the nucleus to he replicated. Another possibility raised by our results is that 2 pm DNA replication may occur during the S phase of the cell cycle. Consistent with this idea is the observation that 2 pm DNA undergoes very little replication in a c&7 mutant after a shift to 36°C (Fig. 3(f)), while approximately one round of 2 pm DNA replication occurs in a c&l3 mutant after a similar temperature shift (Fig. 3(h)). The lack of replication in a mutant defective in initiation of 8 (c&7) and the single round replication in a mutant defective in nuclear division (c&13) suggest that 2 pm DNA replication normally occurs after completion of the cdc28, c&4, and c&7-mediated events which lead to the initiation of S (Hartwell, 1976). 2 pm DNA replication is clearly different from mitochondrial DNA replication
YEAST
2 CLm DNA
REPLICATIOS
259
because mitochondrial DNA replication continues in c&28, cdc4, and c&7 mutants at the restrictive temperature and in CI factor-arrested cells (Cottrell et aE., 1973 ; Cryer it al., 1973; Petes & Fangman, 1973; Newlon & Fangman, 1975). Mitochondrial DNA synthesis occurs at times in the cell cycle other than S phase (Sena et al., 1975) and continues in the presence of cycloheximide, a protein synthesis inhibitor which prevents initiation of nuclear DNA replication (Grossman et al., 1969). All three cellular DNA!: do share the common features t’hat all are dependent on the c&8 gene product which is probably directly involved in the elongation reactions of DNA replication and on the c&21 gene product which is iuvolvcd in thymidine metabolism (Hartwell, 1971; Game, 1976; Cryer et al., 1973; Wint,ersbc~rgcr pt ab.. 1974: Newlon & Fangman, 19i5: Petes & Williamson, 1975). Th(a dependence of 2 pm DXA replication on gene product’s known to mediat#e Gl events t)hat are prerequisite for t’he init’iation of nuclear DNA replication makes this plasmid an attractive molecular model to study thfb evcnbs whit+ initiate and support nuclear DNA rc$ication.
WC thank Leland H. Hartwell for his interc~st and support of this work. This project was supported by a grant from the National Inst,itute of General Medical Sciences (GM17709) to Leland H. Hartwell. One of us (D.M.L.) is a postdoctoral fellow of the National Ca~~ccr Institllt,e (CA 00592).
REFERENCE8 I~uc~kirlg-Thrown, E., Dulltze, W.. Hartwell, L. H. & Ma~lney, T. I
