<i>De novo</i> synthesis of budding yeast DNA polymerase alpha and <i>POL1</i> transcription at the G₁/S boundary are not required for entrance into S phase

Vol.90, pp. 10519-10523, November1993 Biochemistry

De novo

synthesis

of

budding yeast DNA polymerase a and

POLI

transcription

at

the

G1/S

boundary

are not

required for

entrance

into

S

phase

(Saccharomycescerevisiae/transcriptional control/cell cycle)

MARCO Muzi FALCONI*t, ANNA PISERI*, MARINA FERRARI*, GIOVANNA LUCCHINI*t, PAOLO PLEVANI*t,

AND MARCO FoIANI*

*Dipartimento di Genetica e di Biologia dei Microrganismi, Universita di Milano, Via Celoria 26, 20133 Milan,Italy;andtlstitutodi Genetica, Universita di

Sassari,Via Mancini5,07100Sassari, Italy

Communicatedby Thomas J. Kelly,August16, 1993

ABSTRACT ThePOLI gene, encoding DNApolymerasea (pola) inSaccharomyces cerevisiae, istransiently transcribed

duringthecel cycle at theG1/Sphase boundary. Here we show that yeastpola is present at every stageoftheceUlcycle, and its level only slightly increases following the peak of POLI

transcription. POLI mRNA synthesis driven byaGAL] pro-moter can becompletely abolished withoutaffecting the growth rateof logarithmicallygrowing yeast cultures for several cell

divisions, althoughtheamountof thepola polypeptide drops belowthephysiologicallevel.Moreover,a-factor-arrestedcells

can enter S phase and divide synchronously even if POLI

transcriptionis abolished. These results indicate that the level of yeastpola is not ratelimitingandde novosynthesis of the enzyme is notrequired for entrance into S phase.

Eukaryotic DNA polymerase a (pola), together with the tightlybound DNAprimase, playsanessential role inlagging strandsynthesis and initiation of DNA replicationat anorigin (1-3). Thegeneencoding pola in Saccharomycescerevisiae

(POLI) istransiently transcribedduringthe cellcycleatthe G1/S phase boundary (4)concomitantly with several DNA synthesisgenes(forareview,seerefs. 5 and6).Aconserved promotersequence, the Mlu Icellcycle box (MCB),

medi-ates this transcriptional control and is present twice in the POLI gene (7-9). One component of the transcription fac-tor(s) that binds to the MCB is theSWI6geneproduct,whose function is relevant forcellcycle-dependenttranscription of yeastDNAsynthesis genes (10-12). However, SWI6 deletion is notlethalbut leads to deregulated constitutive transcrip-tionof these genes (10, 11). Moreover, it has been shown that the levelofproteinsrequiredforSaccharomyces cerevisiae DNAreplication(replicationfactor A)orfor entranceinto S phase (CDC46 gene product) doesnot showanylarge fluc-tuationduring thecellcycle(13, 14), although the transcrip-tion of thecorrespondinggenes isclearly periodic(14,15).A nearly constant amountofessentialreplicationproteins has been observed also in actively cycling cells from other eukaryotes, including mammalianpola, RF-A, proliferating cell nuclear antigen (PCNA), DNAligase (13, 16-19), and Schizosaccharomyces pombe pola, PCNA, and DNAligase (20-22). However, the transcription of the corresponding genes in these organisms does not appear tobe cell cycle regulated. Thesefindingsleaveuncertain astowhether the transcriptionalactivation ofDNAreplicationgenes observed in Saccharomyces cerevisiae is required for the onset of DNAreplication inSphase.

Ourgoal in the present workwas toestablish whether the

amountofpolainSaccharomycescerevisiaeisratelimiting

Thepublicationcostsof this articleweredefrayedin partbypagecharge payment.This article must therefore beherebymarked"advertisement" in accordance with18U.S.C. §1734 solelytoindicate thisfact.

andperiodic transcription of the POLI gene is necessary for entrance into S phase. Weshow that yeast pola is present at every stage of the cell cycle and its level only slightly increases following the peak of POLI mRNA at the G1/S phase boundary. To monitor the requirement for de novo synthesis of pola, we fused the POLI gene to the repressible GALl promoter (23) andblocked POLImRNAsynthesis in asynchronously and synchronously growing cells containing this construct as theonly source of the enzyme. We observed that even when POLItranscription is abolished in G1 and the amountofpola drops below thephysiologicallevel,cells can stillundergo several division cycles. These findings indicate that pola remains functional when inherited by daughter cells, and de novosynthesis of the enzyme is not essential for initiation of DNA replication withinthe same cell cycle.

MATERIALS AND METHODS

Strains, Plasmids, Media, and Yeast Cell Synchronization. Cultures ofSaccharomyces cerevisiae strain CG378 (MATa adeS leu2-3,112 trpl-289 ura3-52) (24), grown in synthetic medium (25) supplemented with the requirednutrients, were synchronized by a-factor treatment as described (8). The CG378APOL1[pAP415]strain has been constructedby replac-ing thePOLI chromosomal copy (25) with anull poll allele carryingadeletion of the2325-bpHpa Ifragmentinternalto

thePOLIcoding region (7) in strain CG378[pAP415]. Plasmid pAP415 is anARSI TRPI CEN6plasmid carrying aPOLI fragmentspanning position -589 toposition+4946and con-taining the entire POLI promoter (8). Plasmid pMA2 was

constructed by cloningaPOLI fragment, spanning position -8toposition +4946 with respect to thetranslation initiation codon, into the BamHI site of the centromeric plasmid pBM125(carrying the URA3 selectablemarker) (23), down-stream of theGAL) promoter. Strain CG378APOL1[pMA2I [pAP415] has been constructed by transforming strain CG378APOL1[pAP415] with plasmid pMA2 and strain CG378APOL1[pMA2]wasobtained from theprevious strain by standardplasmidshuffling procedures (25). Thesestrains have been grown under induced (galactose-containing me-dium)orrepressed(glucose-containingmedium)conditionsas

detailed in thelegendstoFigs. 2and 3.

Preparation ofYeastExtractsand WesternBlotting

Analy-sis.Totalproteinextractswereusually preparedfrom4x 108 cells collected at different times from logarithmically or synchronously growing yeast cultures. Cells were washed with 20% trichloracetic acid (TCA) in order to prevent proteolysisandresuspended in 200,ul of20% TCAat room

Abbreviations: pola, polymerase a; mAb, monoclonal antibody;

DAPI,4',6-diamidino-2-phenylindole.

tTo whomreprintrequestsshould be addressed.

10520 Biochemistry: Muzi Falconi etal.

temperature. After addition of the same volume of glass

beads,cellsweredisrupted by spinninginaVortexfor2min.

Glassbeadswerewashed twice with 200 ,ul of 5% TCA and

theresulting extractwas spunfor 10 minat 3000rpmin a

Microfugeatroomtemperature.Thepelletwasresuspended in200,u of Laemmli buffer (26), neutralized by adding 100

IlI

of1 M Tris base, boiled for 3 min, and finally clarified by centrifugation as described above. Aliquots (25

pg)

of the

extract,asdetermined byaBio-Radproteinassay(26),were

analyzed bypolyacrylamide gel electrophoresis in the pres-ence of SDS, and the detection of the immunoreactive

polypeptides was carried out as described (27). Anti-pola

monoclonal antibodies (mAbs) (28) isolated from ascites fluids were used as the primary antibodies at a 1:2000

dilution.

Miscellaneous Procedures.ExtractionoftotalyeastRNA, Northernblotting analysis,and DNAprobesusedtomonitor

the POLI,PRI2, and H2A transcripts have been described (8). The nuclear localization of polawasdetermined byinsitu

indirectimmunofluorescence(29) using anti-yeast pola mAb (24) as the primary antibody and a rhodamine-conjugated

goatanti-mouseIgG(Sigma)asthesecondary antibody.The

DNAbinding dye4',6-diamidino-2-phenylindole(DAPI)was

usedtovisualize the nucleus (29). RESULTS

Levelof polainSynchronous YeastCultures. Yeastpolais a180-kDapolypeptide (p180) tightlyboundtoDNAprimase in afour-subunit complex (28, 30). Because p180 is highly

susceptibletoproteolysis (28),wemonitoredthelevelofthis

polypeptide by Westem blot analysis (Fig. lb) on crude

extracts prepared to minimize proteolysis from

a-factor-synchronized

yeast cultures

(Fig.

la).

The

p180

pola

poly-a C,, 'a

'a)

m 0--100 80 60 40 20

l

l

l

0110 140 160 180 200 b

o

i

0

peptidewas present at every stageof the cell cycle and its level almost doubled following the increase of the POLI transcript (Fig. lb: time points, 170-190 and 240-260 min). Bydensitometric scanning,wecalculated that the increase in

the level of p180atthe timepoints indicated, comparedto

thatfoundin thepreceding samples (time points,140-160and 210-230 min), was 1.68-fold and 1.48-fold, respectively. A

similar resultwasobserved by assaying pola activity during

the cell cycle inimmunoprecipitates obtained with

nonneu-tralizing anti-pola mAbs and bytesting anin-frame

POLI-lacZ fusion for jgalactosidase activity (data not shown). Therefore, the 30-fold increase of POLI mRNA levelatthe G1/S transition (Fig.lc)results,atthemost,inadoubling of

theamountof thecorrespondinggene product. The highest p180 level was found to be coincident with the periodic increase of histone H2A mRNA in S phase (Fig. lc: time points, 160-180 min; ref. 22). Althoughthefluctuation ofthe

POLI mRNA is typical of highly unstable regulated

tran-scripts, thepola polypeptideisquite stable.Consistent with thisobservation, the half-life of p180 determined by pulse-chaseexperimentsis >4 hr(unpublished observation).

Sim-ilarly, otheryeastDNAreplication factorsarestableproteins evenif thelevelof thecorresponding transcripts fluctuates

periodically during thecellcycle (13, 14). These data raise the

questionsastowhether theamountofreplication proteins is limiting andiftheirsynthesis isrequired forthenextround ofDNAreplication.

The Level ofYeastpolaIsHigher than ThatRequiredfora SingleCellGeneration, and the Enzyme Is Functional when

Inherited byDaughter Cells. To test whetherde novo

syn-thesisofpola is necessaryto supportcell division,weused

the haploid strain CG378APOL1[pMA2] carrying a lethal

deletionof thePOLIchromosomal locusandthePOLl gene underthe control of theGALl promoteron a centromeric

220 240 260 280 300 Time l l (minutes) p180 C _POLl PRI2 ;-F!(,.._____''-'-H2A CMD1

FIG. 1. The levelof thepolapolypeptideincreasesnearly2-foldfollowingtheperiodicincrease in thelevel of the POLItranscriptatthe

G1/Sboundary. Alogarithmicallygrowingculture(7x 106cells perml)of strain CG378wassynchronized by a-factor treatment. Timesof

additionand removal of a-factor are indicatedbyarrows, andsamplesweretakenattheindicated times.(a)Buddingprofile.(b) Westernblot

probedwithanti-polamAbs. Each lanecontained 25 ,ug of totalproteinextractedfrom cells at the indicated times.(c)Fivemicrograms oftotal RNAper lane was used tomonitor the fluctuation ofPOLI,

PRI2,

and H2A histonegenetranscripts duringthe cellcycle (8) by Northernblot

a i08 10' (A C)1 Oi06 2 3 4 5 6 7 a 0 0 --

~~~~~o-:

g~~~~~

11111111111111111111111 0 4 8 12 16 20 24 Time (hours)

b

1

2 3 4 5 6 7 8

U~~~~

15.72 2 :. 1.'t.37taO 1370.72 0.45 0ra_18 0.12 .':'::'3Y43t5 6 7 I8_. 1572 >244 0 81 CL 0793.860487 u.

F

"'EI:

d DAPI POL1 PRI2 pl80 POL1 PR12 p180 anto-nolc mAb

FIG.2. Expression of POLIdrivenby theGALl promotergives rise toafunctionalpola polypeptidethatcanbe usedfor severalcell

divisions. (a) Growthrate of strains CG378APOL1[pMA2] (A) and

CG378APOL1[pMA2][pAP415] (o) after shift from induced (galac-tose-containing medium) to repressed (glucose-containing medium) conditions. Cellsofbothstrains were grownat28°Cunderselective conditions insynthetic mediumcontaining 2%galactosetoa

concen-trationof2 x 106cells perml. Aliquotsof 3 x 108cells weretakenfor

WesternandNorthern blottinganalysis(sample1,b and c),whereas theremainingcellswerefiltered,washed,and resuspendedat1x105

cellsperml insynthetic mediumcontaining 2%glucose (timezero). Growth ratewasmonitored bycellcounting,andaliquots of4x 108

cells werewithdrawnatthetimes indicatedbyarrows(samples2-8).

(bandc)LevelofthePOLIandPR12mRNA andamountofthep180 pola polypeptide in strain CG378A&POL1[pMA2] (b) and in strain

CG378APOL1[pMA2][pAP415] (c). Five micrograms of total RNA and25pg of totalproteins preparedfromsamples 1-8wereloadedin

eachlaneandanalyzed by NorthernandWesternblotting. The level ofp180wasevaluated by densitometric scanning and itsamountis plottedrelativetothat ofaPonceau S-stainedbandontheWestern

blotusedas aloadingcontrol. (d)The nuclear localization of pola in the CG378APOL1[pMA2] culturegrown under induced conditions

plasmid (Materials and Methods). The isogenic haploid strain

CG378APOL1[pMA2][pAP415],

containing also the normally regulated

POLI

gene, wasused as a control.

In the two strains described above, galactose-induced

POLI

transcriptiongave riseto alargeincrease in thelevel

of

POLI

mRNA (Fig. 2 b and c). Correspondingly, the amount of the p180 pola polypeptide was -16-fold higher

than thatfound inyeastcells expressingthe

POLI

geneunder

thecontrolof itsownpromoter(Fig.2band c). All p180pola polypeptidewaslocalized in itscorrectsubcellular compart-ment,thenucleus(Fig. 2d). Neither constitutive norinduced

overexpression of

POLI

leadstoanyevident growth defect

(this workandunpublishedobservations), probablybecause thein vivoactivity of pola is mediated by its stoichiometric assembly with the other polypeptides of the pola-primase complex, whose expression is normally regulated [see the level of the PRI2 genetranscript, encoding thep58 primase subunit (32), in Fig. 2b andc, and its fluctuationin Fig. 3b].

Ametabolic shift from galactosetoglucosedoesnot imme-diately affect the growthrate of strain CG378APOL1[pMA2] (Fig. 2a), although the

POLI

mRNA becomes undetectable already 10

min

aftertheshift(datanotshown).Aftertheshift, the amount of pola synthesized under induced conditions

halves every cell division (Fig. 2b), but could support cell growth even if the amount of the p180polypeptide dropped

well below the physiological level(compare the levelofp180

in lanes4-8of Fig. 2b and c). Thepoolofpolasynthesized

in galactose-induced conditions was sufficient for cells to

proceed through seven generations after shift to glucose,

before a sudden arrest (Fig. 2a) with adumbbell phenotype

(datanot shown), which is typical ofyeast cells impairedin

essential functions required for DNA replication (5). There-fore, itappears that the level ofnormally expressed polais

higherthan that required forasinglecellgeneration, and the enzyme synthesized under galactose-induced conditions is very stable andcanbeused for severalcelldivisions.

Entrance into SPhase DoesNotRequire deNovoSynthesis

ofpola. To further test whether de novo synthesis of the enzyme was necessaryfor cellstoundergo DNAreplication

within the same cell cycle, CG378APOL1[pMA2] cultures

grownin galactose mediumwere treated with a-factorunder glucose-repressed conditions. As is shown in Fig. 3a, cells

recovered synchronously from a-factortreatment, allowing

properperiodic transcription of transiently expressed genes

(seethe level of PRI2 and H2Atranscripts in Fig. 3b),while

the GALl-driven

POLI

transcription was turned off. After

two synchronous divisions (time 290

min

in Fig. 3a), cells

diluted in glucose medium were abletodivide for fivemore generations, before quickly arresting as dumbbell-shaped

cells independently on the starting cell concentration (Fig. 3c),thus showing that thearrestphenotypewasnotduetoa

lack of nutrients. Neither the

POLI

transcript northe pola

polypeptide was detectable in arrested cells, but the same cells underwent a total of seven divisions after a-factor release.Therefore, newly synthesized polaisnotrequiredfor

cellsto enterS phase, and the pool ofenzymeproducedunder

induced conditionscan be usedfor severalgenerations.

DISCUSSION

DNAreplicationiscoupledtocell cycle progression,butthe

molecular mechanisms turning replication on and off and

preventing rereplication within the samecell cycleare still

poorly understood(forarecentreview, seeref. 33).

Thecoordinated periodic transcription of DNA synthesis

genes in budding yeasthas been generally interpretedas a

(sample1 in b) was determined by in situ indirect immunofluores-cence. DAPI was used to visualize the nucleus in the same cells.

a

1'00

a/) CD m &0 80 H 60 I 40 H 20 0 (L factor addition a afactor lreease J 1. I I I I I I I I I I ---T ---7 II I'' 0110140 160u 180 240 300

1

1

1

1

10

1

1

I1

1

1

1

Time (minutes) POLl op;*o ,le 4.4 4t..* 11W prw 4

SMIp

f * s> Z * w--a-PRI2 H2A CMD1

C DILUTION STARTING ARREST NoOF ARREST

CONCENTRATION CONCENTRATION GENERATIONS PHENOTYPE 150 1:100 1:200 1:400 SX105 25xI05 125xt05 625x104 t8xt07 8,2xl06 4.Ox106 2.0xIO6 5 5 5 5 dumbbell dumbbell dumbbell dumbbell

FIG. 3. Denovosynthesis of pola is not required forentranceinto Sphase. ACG378APOL1[pMA2]culturewasgrownin synthetic medium

containing2% galactoseto aconcentration of 7x 106cellsper ml.Glucose(2%finalconcentration) and a-factorwereaddedattimezero.After 120min,cellswerefiltered, washed, and resuspended insyntheticmediumcontaining2%glucose andaliquotsof the culturewereanalyzed atthetimes indicated.(a)Buddingprofile.(b)Fivemicrogramsof total RNAwasanalyzedon aNorthernblot.(c) Twogenerationsaftera-factor

release(sample 290 minat aconcentration of 2.5x107cellsperml),aliquotsof the synchronized cultureweredilutedasindicated in 2% glucose

syntheticmedium, and thecell number andmorphologywerefolloweduntil cells stoppeddividing.

meanstoprovideatimely supply of the necessary proteins (6, 34). However, in othereukaryoticorganisms, the transcrip-tion of DNAreplication genes is not soseverely controlled during the cellcycle (3, 16, 18, 22). Ourfindingthatpola is

astableproteinand that theG1/SincreaseofPOLI mRNA

results, at the most, in a doubling of the amount ofprotein, is in agreement with thatobservedfor other DNAreplication enzymes, both in yeast and higher eukaryotic cells (13, 16-22). Moreover, the effect ofblocking POLItranscription inlogarithmically and synchronously growing cells clearly indicates that

pola

is present in an excess amountin yeast cells, andit canbe inherited and usedby daughter cellsto

proceed through S phase even in the absence ofapool of newly synthesized enzyme. Therefore, the onset ofDNA synthesis isnot coupled to thetranscriptionalactivation of the POLI gene atthe G1/Sphase boundary. Periodic tran-scriptionmightstill be relevanttoprovideatimely supplyof limitingorlabilekey protein factors requiredto enterSphase (35-38). Moreover, coordinated activation of the DNA syn-thesis genes inbudding yeast mightrepresent afine-tuning control mechanism that optimizesthe efficiencyof Sphase rather than its timing (33, 34). Such a mechanism likely represents aselective advantage foratotipotential, rapidly growing unicellulareukaryote.

On the other hand, the in vivo function of stable DNA replication proteins mightbe modulatedbyseveralregulatory mechanisms, including posttranslational modifications

car-ried out by one of the numerous kinases or phosphatases found ineukaryotic cells (39, 40),orassociation withsome key regulatory factors. Our recent finding that the p86 protein,which istightlyboundtothep180 pola polypeptide

(30, 41), is posttranslationally modified in a cell cycle-dependent manner (unpublished data) is in agreement with this hypothesis. Moreover, pola and the p86 homolog in

human cells have beenfound to behyperphosphorylated at G2/M(42). Ourobservation that pola can be inherited from mothertodaughter cells supports thepossibility that regu-latory mechanismspossibly controllingentranceinto Sphase mightnot necessarilybe restricted to theG1/Sphase

tran-sition.Inthisview, the switchescontrollingentranceinto the Sphasemightbelinkednotonlytopassagethrough"start"

ortherestrictionpointin G1(33) but alsotoothercellcycle events, suchasmitosis. This mechanism wouldprovidethe

cell with alarger time interval to modulate the activity of

DNA synthesis factors, their intracellular localization, or

properassembly. This hypothesis is in agreement with the implicationsof the"licensingfactor" model(43),suggesting thatnuclearmembrane breakdown isrequired forentrance

into the nucleus ofa positive inducer of DNA replication synthesized during the previous cell cycle, thus coupling mitoticevents with initiation of DNAreplication.

M.M.F.and A.P.contributedequallytothis work. Wethank A. Hinnebusch(NationalInstitute of Child Health and Human Devel-opment, National Institutes ofHealth, Bethesda, MD) forhelpful discussions,M.Bianchi(UniversityofMilan)for criticalreadingof the manuscript, F. Cotelli (University ofMilan) for help in the immunofluorescence detection ofpola,and D. Mezzanzanica

(Isti-tuto dei Tumori, Milan) and E. Brocchi (Istituto Zooprofilattico, Brescia) for preparation of ascites fluids. This work has been

partially supported bygrantsfrom theTargetProjectsBiotechnology

andBioinstrumentation and GeneticEngineering,Centro Nazionale

Ricerche Italy, and by Grant SC1-0479-C(A) from the European Economic Community.

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