VoL 9, 651-665, August 1998 Cell Growth & Differentiation 651

Alterations in Pura Levels and Intracellular Localization in thecv-1 Cell Cycle1

Hiroshi Itoh, Margaret J. Wortman,Mechael Kanovsky, Ronald R. Uson,Ronald E. Gordon, Nancy Alfano, andEdward M. Johnson2

Department of Pathology [H. I., M. J. W., M. K, R. R. U., R. E. G., N. A.,E. M. J.J and Brcokdale Center for Molecular Biology [E. M. J.J, MountSinai School of Medicine, New York, New York 10029

AbstractLevels of the single-stranded DNA-binding proteinPura, previously implicated in control of both DNAreplication and gene transcription, are altered duringthe CV-1 cell cycle. Just prior to the onset of S phase,Purer levels drop precipitously, after which they recovernearly 8-fold throughout S and C2 to peak just aftermitosis. As observed previously, Purer binds thehypophosphorylated form of the retinoblastomaprotein, Rb. Coimmunoprecipitation of Purer and Rbreveals that the complex declines as cells enter Sphase and does not reform as Purer levels recover in Sand G2. As Purer levels recover, the protein is localizedto nuclear foci containing newly replicated DNA, asdetermined by immunoelectron microscopy usingdifferent sized gold beads and antibodies against Purerand bromodeoxyuridine-labeled DNA. These foci alsocontain cyclin A, and Purer coimmunoprecipitates with

cyclin A from extracts of cells in S and G2 phases.Purer remains with these foci throughout G2, after thebulk of DNA synthesis has ceased. Changing levels ofPurer may affect Purer functions at the onset of S phaseand during progression to mitosis.

IntroductionRecent work by various investigators has implicated thecellular protein Purer as an important mediator of both geneexpression and DNA replication of certain viruses in mam-malian cells (1-5). Pura is a sequence-specific DNA-bindingprotein with affinity for the purine-rich single strand of itsDNA recognition element (6, 7). The genorne of human JCV3

Received 1/16/98; revised 5/8/98; accepted 6/18/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This work was supported by NIH Grants CA55219 and NS35000 (toE. M. J.) and National Institute of Environmental Health Sciences TrainingGrant in Experimental Pathology T32 ES07265 (to M. K J. and M. J. W).2 To whom requests for reprints should be addressed, at Department ofPathology, Box 1194, Mount Sinai School of Medicine, Now York, NY

10029. Phone: (2i2) 241-7510; Fax: (212) 534-7491 ; E-mail: johnson@msvax.mssm.edu.3 The abbreviations used are: JCV, JC polyomavirus; BPV, bovine papil-loma virus; BrdUrd, bromodeoxyuridine; RPA, replication protein A; Cdk,cyclin-dependont kinaso; LRR, loucine-rich repeat.

possesses several PUR elements within its origin of DNAreplication and in two 98-bp repeats, proximal to the origin,which modulate both early and late promoter transcription(8). A region designated the lytic control element in the JCV98-bp repeat contains a PUR element adjacent to a poly(dA-dl) tract that displays a single-stranded configuration (9).

Pura interacts at this element with another cellular protein,YB-i , and with the viral large T antigen to activate viral lategene transcription (5). Purer also interacts with T antigen, in acooperative fashion, at the JCV origin of replication (4). Inglial cells transfected to overexpress Purer, this protein was apotent inhibitor of JCV DNA replication. In contrast, trans-fection of glial cells with antisense purer cONA stimulatedJcv DNA replication (4). JCV has an extremely restrictivehost range for replication, replicating in vitro only in humanfetal glial cells (1 0, 1 1). Pura has recently been reported toact at the origin of DNA replication of another mammalianDNA virus, that of BPV (3). Purer binds to two elements in theBPV genome, one in the minimal origin of replication and onein the region designated plasmid maintenance sequencedomain 1 , each with a binding constant for Pura of nearly1010 (3). The functional significance of Pure binding to theBPV elements is not yet known. Purer has also been mph-cated in mediating the activation of certain cellular genes bythe avian retrovirus, Rous sarcoma virus (2).

Recently, it has become clear that the ability of Pura toactivate transcription at the JCV late promoter is subject tocontrol by the HIV-i protein, Tat (1). JCV is the etiologicalagent of the neurodegenerative disease progressive multifo-cal leukoencephalopathy, which is frequently encountered inAIDS patients (1 2, 13). HIV-i and JCV both infect cells in thebrain, although not necessarily the same cells. Tat is report-edly secreted by HIV-i -infected cells and is capable of beingtaken up by adjacent cells in an active form (1 4-1 6). Tatactivates JCV transcription through interaction with a Tat-responsive element near the JCV origin of replication, whichhas been identified as a PUR element (8). Tat does not itselfbind the PUR element, but it binds to Pure, strongly enhanc-ing activation of late promoter transcription (1). Tat is essen-

tial for transcription of the HIV-i genome, its effects beingexerted primarily through interaction with an RNA element(TAR) in the 5’ region of the transcript (for review see Ref. 17).The TAR sequence includes both a Tat-binding element anda nearby PUR element. Pura is capable of binding this RNA

element, and it has now been demonstrated that Tat andPurer can both affect HIV-i transcription (1 8). The binding ofPurer to Tat involves two acidic leucine-rich repeats inter-spersed in the Purer DNA-binding domain (1). The coopera-tive activation by Tat and Purer of JCV transcription exhibitscell cycle dependency, suggesting that Purcr may be a me-diator of cell cycle influence (1).

Purer is highly conserved throughout metazoan organisms(1 9). The protein was originally cloned based on its affinity for

652 Pura Localized in G1, 5, and G2

an element present in many eukaryotic origins of DNA rep-hication (7), including initiation zones in the vicinity of hamster

dhfr (20-22) and rhodopsin genes (23), human c-myc (6, 24,25) and f3-globin (26) genes, and the mouse adenosinedeaminase gene (27). Pura has a region of homology toseveral viral proteins involved in control of initiation of DNAreplication, including SV4O large T antigen (19). This homol-ogy encompasses the binding site of T antigen for the reti-noblastoma protein, Rb. We have now demonstrated thatPurrs binds the hypophosphorylated form of Rb with an af-finity at least as high as that of T antigen (28). Although littleis known regarding functions of Pura in cells not infected byviruses, ‘It �5 conceivable that its cellular functions will mirrorits multifunctional effects on viral genomes. In addition to itseffects with Tat on JCV transcription, cell cycle dependencyfor Pure function is indicated by the binding of Pure to Rb

(28). Rb phosphorylation is known to occur in G1 , prior to theonset of DNA replication (29, 30). Because Pure directlyaffects JCV DNA replication in vivo (4), it is of interest todetermine whether Pura is present at foci of replicating DNAin cells and whether the location of Pura changes during thecell cycle, particularly upon dissociation from phosphoryl-

ated Rb in G1 phase. Here, using a quantitative application ofimmunoelectron microscopy, we report that Pura levels in

Cv-1 cells fluctuate significantly during the cell cycle and thatPura associates with nuclear foci of newly replicated DNAduring S phase and remains with such foci, closely associ-ated with cyclin A, throughout S and G2. These results sug-gest a role for Pura in the onset of DNA replication and in theprogression of replicated DNA to mitosis.

Results

Purer Levels in CV-1 Cells Decline Prior to the Onset of SPhase and Recover to High Levels at Mitosis. We initiallyasked whether intracellular levels of Pura change during the

CV-i cell cycle. We chose CV-i cells for this study becausethese cells are not transformed, they possess a wild-type Rbprotein, and phosphorylation of Rb has already been char-acterized in the CV-1 cell cycle. The CV-1 cells were syn-chronized with lovastatin, which inhibits an isoprenylationstep, leading to cell proliferation (31), imposing a mid-G1block in a variety of mammalian cells (31). Cells were re-leased from the block by being placed in medium containingrnevalonic acid, which reverses the effects of lovastatin.Cv-i cell numbers, BrdUrd incorporation and mitotic indexfollowing release from lovastatin block are shown in Fig. 1A,

confirming that lovastatin imposes a suitable cell synchrony.A variety of assays were performed to monitor parameters ofthe cell cycle to assign phases to the time points taken. Inaddition, controls were performed to ensure that data werenot due to artifacts imposed by lovastatin. Incidence of mi-

totic figures, monitored by microscopy, peaked at 1 0.5 h, at

which time 28% of cells were identified in later stages ofmitosis (Fig. iA). Cell number began increasing at 10 h andhad doubled by 12 h, correlating well with observations ofmitosis. S phase, indicated by BrdUrd incorporation, beginsat 4-6 h, is near maximal at 6-7 h, and is only minimal after9 h. This correlates well with data presented later in this

paper indicating that S phase is in full progress at -6-h afterrelease from lovastatin.

To measure levels of Pure during the cell cycle, pointswere taken at various intervals after release from lovastatinblock, and cell or nuclear extracts were prepared. Severaltypes of analyses were performed. Western blots were per-formed either directly on whole cell lysates, nuclear extracts,

or cytoplasmic extracts or on immunoprecipitated samplesobtained from each of these preparations using differentanti-Purer antibodies. The results of Western blots performedeither directly or after immunoprecipitation from nuclear ex-tracts were virtually identical.

Immunoblot controls were performed to show the speci-ficity of the antibody used to detect Pura and to assess levelsof Pura in whole-cell lysates or CV-1 cells. Fig. lB shows astandard blot of several such lysates, along with a purifiedPure control. Lane P shows purified glutathione S-transfer-ase-Pura, cleaved with thrombin to release Pura. This pro-tein is a bacterially expressed full-length human Purer, and it

migrates at Mr �40,0O0. It should lack any potential postsyn-thetic modifications that might occur in mammalian cells.Lanes BVP and Sf9 are both extracts of insect Sf9 cells. TheBVP extract is from cells infected with a baculovirus vector tooverexpress human Pura, whereas the Sf9 extract is fromcontrol uninfected cells. The Purer cDNA expressed in Lanes

P and BVP is identical. It is likely, however, that the insectcells modify the expression of the Pure protein in some waybecause two bands are derived from the same cDNA, oneslightly larger and one slightly smaller than bacterially ex-pressed Pura. Lanes CV and WI show extracts of monkeyCv-i cells and human WI-38 cells, respectively. One Puraband is detected in each of these extracts. In each case, itmigrates more slowly than the Sf9-expressed Pura, at Mr

-47,000. We have shown previously that this CV-i bandmigrates at the same position as human Purer expressed intransfected CV-1 cells and detected using either anti-Purerantibody or an anti-influenza virus hernagglutinin epitope tagantibody (28). The slower migration of the CV-i and Wl-38Purer relative to the bacterially expressed Purer is most likely

due to postsynthetic modification. We have been able todetect phosphorylated serine, threonine, and tyrosine resi-dues in Purer bands using antibodies. These controls affirmthe suitability of monoclonal antibody 9C1 2 for detection ofPurer in CV-i cells.

Fig. 1C shows an immunoprecipitation from nuclear ex-tracts of synchronized CV-i cells, using anti-Purer mono-clonal antibody 9C12, blotted and probed with the sameantibody directly conjugated to horseradish peroxidase. Asingle Purer band is detected in this assay at the position ofthe band in Fig. 1B, Lane CV. It can be seen that the intensityof this band changes during the cell cycle, being highest at2-4 hr (G1) and dropping transiently after 4 h. When Westernblots are performed directly on whole-cell lysates, the Mr47,000 Purer band fluctuates in level during the cell cycle,exactly as seen in Fig. 1C. When whole-cell lysates areanalyzed using a nonconjugated primary antibody and aperoxidase-conjugated second antibody, in addition to theMr 47,000 Purer band, three less intense bands of M, 50,00060,000 are detected. These higher molecular weight bands

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Cell Growth & Differentiation 653

peroxidase-conjugated primary antibody reduces extrane-

A. CVI synchrony after lovastatin block

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Fig. 1. Changes in detectable levels of Pura protein in monkey CV-1 cells synchronized by lovastatin block. CV-1 cells were synchronized in G1 bylovastatin block and released by treatment with mevalonic acid at time 0, as described in “Materials and Methods.” A, timing of mitosis. Cells were countedmicroscopically on grids etched in tissue culture flasks. Mitotic figures were counted in Unicryl sections stained with methylene blue and azure II, asdescribed in “Materials and Methods.” Incorporation of BrdUrd (x) quantitated by immunelectron microscopy following a 1 0-mm pulse with 1 5 mr.i BrdUrd,as described in “Materials and Methods,” is presented as relative incorporation, normalized to a value of 1 .0 for t = 0 (right verticalaxis). #{149},% mitotic cells;Li, no. of cells xlcy5. B, specificity of anti-Pura monoclonal antibody 9C12. The following samples were subjected to PAGE on a 10% gel, blotted to anImmobilon-P membrane (Millipore), and probed with anti-Pura monoclonal antibody 9C12 followed by goat antimouse antibody conjugated to horseradishperoxidase (Boehringer Mannheim); detection was with the Pierce SuperSignal Chemiluminescent system. Lane P, human glutathione S-transferase-Pur�(10 ng) cleaved with thrombin to release Purcr; Lane BVP, a lysate (50 ng) of Sf9 cells infected with baculovirus vector containing cDNA to express humanPura; Lane Sf9, a control lysate (50 ng) of uninfected Sf9 cells; Lane CV, a lysate (200 �g) of monkey CV-1 cells; Lane WI, a lysate (200 �g) of human Wl-38lung fibroblasts. C, Pura levels in synchronized cells. At the indicated times, following release from lovastatin with mevalonic acid, extracts were preparedfrom aliquots of cells, as described in “Materials and Methods.” Immunoprecipitation was performed using mouse monoclonal anti-Pura antibody 9C1 2 andDynal magnetic beads coupled to sheep antimouse antibody as described. Precipitates were redissolved in SDS gel sample buffer and subjected toSDS-PAGE. After blothng, bands were probed with antibody 9C12 directly conjugated to horseradish peroxidase, so that the immunoprecipitating antibodyis not visualized here. Detection was with the Pierce SuperSignal chemiluminescence system. Lane Ab, control precipitation with magnetic beads coupledto sheep antimouse antibody alone, in the absence of anti-Pura antibody 9C12, and probed as described.

are not seen in nuclear extracts, typified by Fig iC. The use

of nuclear extracts and immunoprecipitation concentrates

the Pura band for better visualization, and the use of the

ous background. One other protein, Purp, with homology to

Purcr is known to exist (7, 32). It has been reported that Purf3

in mouse smooth muscle fibroblasts migrates at M, 44,000whereas Pura migrates at Mr 46,000 (32). Antibody 9C12

654 Pura Localized in G1, S, and G2

recognizes a region of Purer that is not represented in PurI3,and we do not see a second band in the vicinity of Purer.

Experiments were performed to determine whether Purerlevels decline before or after the onset of S phase. In several

experiments, Purer levels were maximal at 2-3 h and beganto decline before 4 h, distinctly prior to the beginning ofBrdUrd incorporation. Therefore, it is likely that Purer levelsdecline in G1 , prior to the onset of S phase. In Fig. i C, Purerlevels are high at 2 h, which corresponds to mid-to-late G1.A lighter exposure suggests that Purer levels have begun todecline at 4 h and remain low at 8 h. Purer levels rebound in

late S-G2, are high at mitosis, which occurs at -iO.5 h, andremain high at i2 and i 4 h, corresponding to early G1 of thenew cycle. Beyond this time point, synchrony begins todeteriorate. The significance of relatively low levels of Purer att = 0 is not known. This point was taken while the cells wereimmersed in lovastatin, and this could have some effect onthe detection of Purer.

We have asked whether the changes in intensity of Purerbands are due to changes in levels or are due instead tomasking and unmasking of epitope sites by postsyntheticmodifications. The latter has essentially been ruled out by thefact that two different monoclonal antibodies, 5Bi 1 andi 2A4, each recognizing a different epitope on Purer, bothrecognize the same Mr 47,000 Purer band fluctuating duringthe cell cycle, as shown in Fig. i C. Therefore, it is concludedthat changes in band intensity in Fig. 1 C reflect changes inintracellular levels of Purer. We further investigated whetheror not changes in Purer levels are regulated at the level ofmRNA. Blot hybridizations were performed using mRNA,isolated and described previously (7), at points in the cell

cycle. No dramatic changes in Purer levels were detected.These results suggest that Purer levels change, at least in

part, as a result of changes in protein synthesis at the level oftranslation or proteolysis. Further experiments are necessaryto determine the mechanism by which Purer levels fluctuate.

Alterations in Purer Levels are Accompanied by a Shiftto Nuclear Foci during S and G2. Changes in Purer levels

during the cell cycle are confirmed by examination of CV-icells using light microscopy and staining with anti-Purer an-tibody 9Ci 2, followed by secondary antibody coupled tohorseradish peroxidase. Visualization of the cells, shown inFig. 2, confirms that Purer, indicated by dark staining, isnuclear, although cytoplasmic staining can also be detected.

Nuclear staining for Purer has been reported previously, usinga rabbit polyclonal antibody, in C6 glioma cells, whereas inNGiO8-i5 and SK-N-SH neuronal cell lines, staining wasboth nuclear and cytoplasmic (33). Staining of synchronousCV-i cells confirms that levels of Purer are low at 4 h afterrelease from lovastatin (not shown in Fig. 2). An asynchro-nous culture of CV-i cells is shown in Fig. 2, A and B. Fig. 2Ashows cells stained to reveal Purer, whereas cells in Fig. 2Bare stained to reveal Rb. It is clear in Fig. 2A that certain cellspossess a higher level of Purer, whereas other cells possessa relatively low level. It is also apparent that in some faintlystaining cells Purer is substantially cytoplasmic. In contrast,the level of Rb, typified in Fig. 2B, is approximately the samein all cells. Frequently, cells are seen with a punctate nuclear

staining pattern for Purer. The punctate appearance of Purer

is in contrast to the more uniform appearance of Rb. Theseobservations are consistent with a fluctuating level of Purer inthe cell cycle, compared to a more constant level of Rb. Theresults also indicate that, at some point when Purer levels arerelatively high, the protein is present in nuclear foci. We

asked whether Purer associates with such foci in early G1 , in

late S-G2, or in both, because levels are high at both of thosetimes. Evidence indicates that Purer is not present in thenuclear foci in early G1 . Fig. 2C shows two CV-i cells thathave just undergone division. Levels of Purer are very high inthese cells relative to two larger cells in different stages of thecell cycle, and the appearance of Purer is essentially uniform.This observation is consistent with Purer’s association with

Rb in early G1 , and it suggests that Purer may adopt alocalization similar to that of Rb. This hypothesis is testedbelow. Fig. 2C also confirms that, unlike the cyclins A and B,Purer remains at high levels through the completion of mito-sis. Fig. 2, D and E, shows details of the punctate localizationof Purer and the relatively uniform localization of Rb in CV-icells. Fig. 2, F and G, shows Purer staining of human HeLacells. Fig. 2F shows high levels of Purer in the vicinity ofpostmitotic chromosomes. It is not presently known whetherPurer is actually in the chromatin at mitosis. Fig. 2G showspunctate staining of a large HeLa nucleus. Rb activities inHeLa cells are altered by the presence of the human papil-loma virus E7 protein (34). It is not known at this time howsuch alteration would affect the Purer- Rb interaction.

Purer Exists in a Complex with the Rb Protein in G1Phase of the Cell Cycle. It has previously been demon-strated that Purer binds specifically to the hypophosphory-lated form of the retinoblastoma protein, Rb (28). Rb is phos-phorylated on multiple sites beginning in mid-G1 of the CV-icell cycle (29). Therefore, it was of interest to determinewhether or not Purer association with Rb is subject to cellcycle control by phosphorylation in the CV-i cell cycle. Pro-teins in immunoprecipitates were subjected to SDS-PAGE,and separated proteins were blotted and reacted with anti-Rb antibody 1 i D7, as described in “Materials and Methods.”Fig. 3, top, shows a distinct pi 10RB band specifically react-ing with the anti-Rb antibody in a cell cycle-dependent man-ner. No other bands are seen on the gel except the precip-itating antibody, at Mr �55,000. At�i immunoprecipitation ofPurer from the same time point extracts is presented in Fig. 3,bottom. As in Fig. iC, Purer levels are low at t = 0. Also as inFig. iC, in the cell cycle, Purer levels are minimal at 6 h andhigh just after mitosis. Fig. 3, bottom, clearly shows theincrease in Purer levels through G2 (7.5-i 0.5 h). It is notablethat, when the CV-i cells are blocked by lovastatin, at t = 0,

Purer is not associated with Rb. This might reflect the rela-tively low levels of Purer at t = 0 and may or may not be dueto the presence of lovastatin. In any case, at 1 .5 h afterrelease from lovastatin, when cells are in G1 , Rb is stronglycoprecipitated with the anti-Purer antibody. As cells enter Sphase, after 4.5 h, the amount of Rb coprecipitating withPurer decreases. This may be due to phosphorylation of Rbbut may also reflect the decrease in Purer levels. However, inlate G2 at iO.5 h, a time when Purer levels are high (Fig. iC,Fig. 3, bottom), Rb does not coprecipitate with Purer. It hasbeen reported previously that Rb, after the onset of S phase,

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Cell Growth & Differentiation 655

Fig. 2. Comparison of intranuclear distributions ofPura and Rb in monkey CV-1 cells and human HeLacells. A and B, asynchronous monkey CV-1 cellsprobed either with mouse monoclonal antibody9C12, to visualize Pura (A), or with rabbit antibody0.47, to visualize Rb (B). Secondary antibodies wereeither goat antimouse or goat antirabbit coupled tohorseradish peroxidase. Development of stainingwith diaminobenzidene (brown, but visualized asdark staining) was as described previously (64).Magnification, x 1200. C, a pair of CV-1 cells justhaving undergone mitosis (darkly staining nuclei) ad-jacent to two cells at a different stage of the cellcycle (faintly staining nuclei). Staining was with anti-Pura antibody 9C12 as described. Magnification, x3000. 0 and E, details ofCV-1 cells stained for eitherPura (D) or Rb (E). F and G, human HeLa cellsstained with anti-Pura antibody 9C12 as described.Darkly staining mitotic cells are shown in F (magni-fication, x 1200). G, punctate staining of Pura in aninterphase nucleus.

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Pura

remains predominantly hyperphosphorylated until mitosis(29, 30). Therefore, the association of Purer with Rb, which isdiminished in vitro by Rb phosphorylation (28), appears to be

similarly diminished by Rb phosphorylation in vivo. After

mitosis, Purer does not immediately associated with Rb, al-

though Purer levels remains high (compare Fig. 3, top and

bottom). In most experiments, synchrony begins to deterio-

rate subsequent to the first mitosis. In several experiments,

however, reassociation of Pura with Rb was detected at 1 6 h,

which still corresponds to early G1 . It is notable that thegreatest decrease in Purer levels occurs between 3 and 4.5 h,

whereas the greatest decrease in Rb associated with Purcr

occurs between 4.5 and 6 h.

Purer Colocalizes with Newly Replicated DNA and with

Cyclin A in Distinct Subnuclear Chromatin Foci. DNA rep-

hication in eukaryotes occurs at discrete nuclear foci (35), and

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Fig. 3. Coimmunoprecipitationof Rb with Pura in the CV-1 cellcycle. Immunoprecipitation wasperformed on CV-1 cell extractsusing anti-Pura monoclonal anti-body 9C12 and Dynal magneticbeads coupled to sheep anti-mouse antibody as describedpreviously (28) and in “Materialsand Methods.” Precipitates wereredissolved in sample buffer andsubjected to SDS-PAGE. Top, af-ter blotting, bands were probedwith anti-Rb monoclonal anti-body 1 1 D7 followed by horserad-ish peroxidase-conjugated goatantimouse antibody. Detectionwas with the Dupont Renais-sance system. Times (in h) afterlovastatin release are indicated(lane numbers). Mitosis occurredat -1 1 h (M). Lane Ab, aliquot ofpurified antibody 9C12. The po-sition of the Mr 1 10,000 Rb pro-tein is indicated by p1 1 0RB#{149}Theposition of the major band fromthe precipitating antibody 9C12is indicated at left. Bottom, im-munoprecipates from the sametime point samples were probedwith anti-Pura mouse mono-clonal antibody 9C12 directlyconjugated to horseradish pen-oxidase. Detection was asabove.

these foci have now been well characterized by confocal (36)

and electron microscopy (37, 38), including immunelectron

microscopy (38, 39). Because Pura is present in nuclear foci

(Fig. 2) and Pura has been implicated in DNA replication (3,

4, 6, 40), we tested whether the protein is located at foci of

newly replicated DNA. CV-i cells were pulsed with i5 MM

BrdUrd for 5 mm and treated with anti-BrdUrd and anti-Purer

antibodies for immunoelectron microscopic colocahization,

as described. In Fig. 4 BrdUrd is represented by large beads

and Pura by small beads. It can be seen that foci of BrdUrd

incorporation colocalize with Pura. One such structure is

shown at high magnification in Fig. 4A. It consists of a

chromatin density of -�300 nm, circular in cross-section, with

chromatin strands radiating outward. In this typical example,

a Pur� bead is seen to be associated with multiple cychin A

beads on chromatin strands emanating from the dense body.

The parameters of these structures (round, occasionally elip-

toid, or kidney-shaped; iOO-500 nm in diameter; and con-

taming incorporated BrdUrd) are characteristic of structures

reported previously as replication foci in HeLa cells based onelectron microscopy (38). In that study, it was estimated thatsuch structures, termed “factories, “ each contain -20 rep-

lication forks. Approximately 30% of replication foci labeled

with BrdUrd in our study contained Purer beads. It should be

noted that pulsing with BrdUrd labels nascent DNA strands

at active replication forks and that a large percentage of

these may be distant from sites of initiation.To affirm that the structures seen containing Purer and

BrdUrd are replication foci, the association of Purer with

several proteins reported to be in such foci, including RPA

subunits, proliferating cell nuclear antigen, and cychin A,

was examined. A full compendium of these results is notpresented here, but results of colocahization with cyclin A

are presented because they indicate an activity of Pura

that occurs during and after DNA synthesis. Cychin A hasbeen reported previously to be present at sites of DNA

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p:..Fig. 4. Colocalization of Purer in CV-1 cell nuclear foci with BrdUrd-pulse-labeled DNA or with cyclin A using immunoelectron microscopy. Cells were fixed,sectioned, and treated with antibodies to localize Pura either with BrdUrd or with cyclin A. A, colocalization with DNA pulse-labeled with BrdUrd.Asynchronous CV-1 cells were pulsed for 5 mm with 1 00 �M BrdUrd and then fixed, sectioned, and treated with antibodies for simultaneous localizationof Pura and BrdUrd, as described in “Materials and Methods.” In this case, Purn is represented by 1 0-nm gold beads, and BrdUrd is represented by 30-nmbeads. Shown is a high-magnification section of the nucleus containing BrdUrd-labeled filamentous material in a circular, dense chromatin structure of-300-nm diameter. Magnification is indicated by the gold beads: large beads, 30 nm; small beads, 10 nm. B, colocalization with cyclin A. Shown is a lowermagnification micrograph of Purer and Cyclin A colocalized in a similar chromatin structure to that in S phase (6 h; A). Magnification is indicated by the goldbeads: large beads, 30 nm (Pura); small beads, 10 nm (cyclin A).

Cell Growth & Differentiation 657

distribution, these may be associated with replicating tensive studies will be presented elsewhere. Cell extracts

synthesis during S phase (41-44). Cyclin A clearly in-

creases in level and undergoes transport to the nucleus at

the onset of S phase (45, 46). However, whereas certain

investigators report that cyclin A assumes a punctate ap-pearance with replication foci during S phase (41 , 43),others report either lack of a punctate appearance (44) or

progression from a more to a less punctate appearance as

S phase progresses (42). Studies below address the issueof whether or not cychin A remains with the nuclear foci

during S phase. Here, we document that Purer and cyclin

A colocahize to structures with size, shape, and BrdUrd

incorporation characteristic of replication foci or factories,

as characterized previously (38). Purer was colocalized

with cychin A in synchronous CV-i cells by immunoelec-

tron microscopy. Fig. 4B shows a structure, at lower mag-

nification than that in Fig. 4A, in which beads localizing

Purer and cychin A are contiguous. Although simultaneous

colocahization of three molecules, Purer, cychin A, and

BrdUrd, could not be carried out, it was possible to local-

ize each of these molecules with the two others. Virtually

all BrdUrd incorporated was in structures such as those in

Fig. 4, together with either cychin A or Purer. Tables i and

2 describe quantitatively the association of Purer with ei-ther BrdUrd or cychin A in the CV-i cell cycle. It should be

noted that in late G2, 20-30% of Purer-cyclin A colocaliz-

ing beads are not seen in the distinct chromatin bodies

characterized as replication foci. Despite their nonfocal

,. I

� ;A

.4

Table 1 Quthe CV-1 cell

antitative colocalization of Pura with Rbcycle by immunoelectron microscopy

and cyclin A, in

TNo. of Purn % Purnbeads per contiguous

cell nucleus�’ to AbC

% Purrcontiguousto cyclin AC

24

68

10

1214

4489 ± 326 52.12897 ± 122 18.9

1143 � 163 8.73877 t 82 11.08774 � 530 22.0

6121 ± 326 14.82938 �: 114 51.0

37.263.985.4

84.0

63.842.748.2

a Time in h after release of CV-1 cells from lovastatin block by mevalonic

acid.b Fifty nuclei of approximately equal diameter (6 �m) were surveyed for

beads at each time point for colocalization of Pura with either Rb or cyclinA. This diameter was taken to be the central cross-sectional diameter.Numbers of beads per nucleus are based on a mean nuclear volume of 38

Mm3 and a mean sectional of thickness of 80 nm.C Beads within 1 0 nm of each other were scored as contiguous.

DNA, but they may also represent complexes associated

with some other functional entity.

Purer Can Be Immunoprecipitated as a Complex with

Cyclin A in S and G2. Given that Purer colocalizes with newly

replicated DNA and with cychin A, we examined the ability of

Purer to coimmunoprecipitate with cyclin A. We also exam-

med the ability of Purer in cells to interact with a variety ofother cyclins and Cdk protein kinases. Results of these ex-

658 Pura Localized in G1, 5, and G2

Table 2 Association of Pura with newly replicated DNA in S phase

No. of Purerbeads per

cell nucleusa

No. of BrdUrdbeads per

cell nucleu?

% Puracontiguousto BrdUrdb

4,2854,9383,5503,1831,183c1,061C4,1228,488

8,2843,061

449

5711,4281,469

14,73115,01712,446

1 877

2,285571

0.10.43.47.7

86.288.571.310.0

8.94.0

a Asynchronous CV-1 cells were pulsed for 10 mm with 15 m� BrdUrd.

Cells were then fixed, sectioned, and treated with antibodies for thesimultaneous detection of Purer and BrdUrd as described in “Materialsand Methods” and in the legend to Fig. 4. Ten representative nuclei of6-sm diameter are presented. Numbers of beads per nucleus were do-termined as described for Table 1.b Beads within 10 nm of each other were scored as contiguous.C As seen by comparison of the number of Pura beads to those in Table

1, maximal BrdUrd incorporation (S phase) corresponds to a cell cycle

time of -6 h after release from lovastatin.

were obtained at different cell cycle points and subjected toimmunoprecipitation with either anti-Purer monoclonal anti-body 9Ci2 or anti-cychin A rabbit polyclonal antibody. Fig. 5,bottom, is a control showing that extract protein concentra-tions for these time points were approximately equivalent.

Proteins were eluted from immunoprecipitates, separated bySDS-PAGE, blotted, and probed with antibodies to eitherPurer or cychin A. Fig. 5, top, shows levels of cyclin A in CV-icell lysates not subjected to immunoprecipitation. It can beseen that cyclin A levels are high at 2 h and then decrease at4 h. A high level at 2 h, which represents mid-G1 , has notbeen reported previously. This cyclin A level in mid-G1 ismost likely not an artifact of the lovastatin block because thet = 0 time point shows virtually no cyclin A but may representa unique property of CV-i cells as synchronized here. Whenimmunoprecipitation is performed with the anti-Purer mono-clonal antibody, a secondary antibody is used in probing forcyclin A, and this band is seen in Fig. 5, Lane AA, at Mr

50,000. A prominent p60 cyclin A band is detected, essen-tially paralleling the cychin A band seen at the top. This bandis not seen in the antibody alone control or at t = 0. At i 0 hand 12 h, this cychin A band disappears, and a prominentlower molecular weight band, presumably a cyclin A break-down product, appears below the antibody band. Whenanti-cyclin A antibody is used, significant levels of Purer arecoprecipitated at 7 and i 0 h corresponding to S and G2phases, whereas lower levels of Purer are coprecipitated at2 h and i 2 h (Fig. 5, third from top). The coimmunoprecipi-tations using either anti-cyclin A or anti-Purer antibodies ap-proximately parallel each other, with maximum coprecipita-tion occurring at 7 h and virtually no coprecipitation

occurring at i2 h. At 2 h, the antibodies detect a moreprominent cyclin A band than Purer band, whereas at 10 h,the Purer band is more prominent. It is likely that other pro-teins are present with Purer and cychin A at different times inthe cycle, and it is conceivable that such proteins couldaffect efficiency of precipitation of the entire complex. In any

case, all data are in agreement that, at 7 h, which corre-sponds to late S phase, Purer maximally coprecipitates withanti-cyclin A antibody, and cychin A maximally coprecipitateswith anti-Purer antibody. At this time, as seen in Fig. 3, Pureris not associated with Rb. The data present a paradox: levelsof a Purer-cychin A association are highest (Fig. 5, second andthird from top) at a cell cycle time when overall nuclear levelsof Purer are approximately lowest (Fig. i C; Fig. 3, bottom).

The most likely explanation for this observation is that, al-though Purer levels are low at 7 h, much of what remains isassociated with cyclin A.

Immunoelectron Microscopic Colocal’uzation of Purerwith Rb and Cychin A at Different limes in the CV-1 CellCycle. Because immunoprecipitations have indicated thatPurer is associated in complexes with either Rb or cyclin A atdifferent cell cycle times, we sought to detail the timing andlocations of these complexes by immunoelectron micros-copy. Figs. i , 3, and 5 demonstrate that the indicated anti-bodies for Purer, Rb, and cyclin A, respectively, are appro-priate for electron microscopic nuclear locahizations because

a Western blot with each antibody reveals predominantly asingle band for each respective protein. Cells were fixed withparaformaldehyde at various times after release from lovas-tatin block, embedded, and sectioned. Sections were incu-bated sequentially with a rabbit-derived primary antibody toone protein of interest followed by protein A coupled to1 0-nm gold beads and then with a mouse-derived primaryantibody to another protein of interest followed by goat an-timouse antibody coupled to 30-nm gold beads. Quantitativestatistical analyses of visual localizations, obtained by ana-lyzing 50 nuclei at each time point, have been performed.These data, presented in Tables i and 2, can be summarizedby noting that in G1 (2 h after release from lovastatin), whenPurer levels are high, >50% of beads representing Purer areassociated with beads representing Rb. In contrast, in Sphase (6 h), when Purer levels are low, -85% of beadsrepresenting Purer are associated with beads representing

cyclin A, whereas only ‘�-9% of beads representing Purer areassociated with beads representing Rb. Extensive controlswere performed to ensure antibody specificity and lack ofcross-reactivity between first and second sets of reagents. Itis not feasible to show a full complement of control micro-graphs because these outnumber the data micrographs, buta quantitative analysis of controls for the 2-h cell cycle pointcolocalization of Purer and cyclin A is presented in Table 3.These controls clearly demonstrate that contiguous associ-ation of gold beads is not artifactually produced by antibody-

antibody interaction or secondary agent cross-reactivity.Colocalizations of Purer and cychin A were compared with

those of Purer and Rb throughout the CV-i cell cycle. Elec-tron micrographs in Fig. 6 document a differential associa-tion of Purer with either cyclin A or Rb. For Fig. 6, A, C, andE, the first antibody was rabbit polyclonal to cyclin A, fol-lowed by protein A coupled to i 0-nm beads. For Fig. 6, B, D,and Fthe first antibody was rabbit polyclonal Rb, followed byprotein A coupled to 10-nm gold beads. The second anti-body for each was mouse monoclonal anti-Purer 9Ci2 fol-owed by goat antimouse coupled to 30-nm beads. Themicrographs in Fig. 6, A, and B, were taken at 4 h (G1), those

Hrs after release

0 2 4 7 10 12

cycAp6o-

AA

CycAp6O-

ppt ab -

Pura p47-

cyclln A

Co-IPPura - cyclin A

�$.. Co-IP,., CycIInA-Purct

kDa MW

66- �

a� � � .�s 4m� �

43- �

Mit

Ext.

Cell Growth & Differentiation 659

Fig. 5. Coimmunoprecipitation of Puraand cyclin A in the CV-1 cell cycle. Top,levels of cyclin A in a blot of whole CV-1cell extracts (representing 2 x 1 o� cellsper lane) probed with anti-cyclin Amonoclonal antibody BF683 (Santa CruzBiotechnology). Numbers at top aretimes after release from lovastatin block.Mitosis occurs at 1 0.5 h. Middle (secondand third panels from top), immunopre-cipitations. Preparation of extracts andimmunoprecipitation was as describedin “Materials and Methods.” Lane M,primary antibody alone. Molecularweight markers (Sigma), not visible here,were stained with Coomassie blue onthe blotted membrane after probing.Purcx-cyclin A, precipitation with anti-Pura antibody 9C1 2 and Westem blot-ting with anti-cyclin A monoclonal anti-body BF683, followed by horseradishperoxidase-conjugated goat antimousesecond antibody. ppt ab, position of theprecipitating antibody 9C12. p60, posi-tion of cyclin A. Cydin A-Pura, precipi-tation with anti-cyclin A monoclonalantibody BF683 (Santa Cruz Biotechnol-ogy) and Western blotting with anti-Puraantibody 9C12 directly conjugated tohorseradish peroxidase. The Pure bandat Mr 47,000 is indicated. The precipitat-ing antibody is not seen under theseconditions. Detection was with thePierce SuperSignal chemiluminescencesystem. Bottom, control for extract pro-tein concentrations (Ext.), Equal aliquots(40 .d) of extract prior to immunoprecipi-tation were subjected to electrophoresisto demonstrate equivalent protein con-centrations. The region of the gel shownis that encompassing both Pura and cy-din A (top and middle).

in C and 0 were taken at 6 h (S), and those in E and F were

taken at 8 h (G2). Purer, cychin A, and Rb each change in their

nuclear locahizations during the cell cycle, and structural

aspects of these changes are visually apparent.

In mid-G, (Fig. 6, A and B), Purer is localized in dense

chromatin regions at the nuclear periphery and the immedi-

ate extranuclear region, with the highest concentrations vis-

ible just inside the nuclear membrane. In Fig. 6A, the nuclear

membrane (nm) is sectioned on an oblique angle and ap-

pears thick. Purer (represented by large antibody-conjugated

beads) is seen associated with the membrane. At this timepoint (4 h), cychin A beads are primarily detected near thenuclear membrane. In early to mid-G1 , cychin A beads are not

frequently seen contiguous to Purer beads, as in Fig. 6A. In

Fig. 6B, most Purer (large beads) and most Rb (small beads)

are seen at the inner surface of the nuclear membrane. In this

case, in contrast to the localization of Purer and cychin A, Purer

beads are frequently seen contiguous to Rb beads. These

results are consistent with earlier reports that Rb is bound to

nuclear lamins A and C (47, 48). In CV-i cells, the Rb-Iamin

complex is located at the inner surface of the nuclear mem-

brane exclusively during early G1 (47).

At 6 h, most Purer beads appear to be dissociated from Rb

beads (Fig. 6D). The Purer beads are frequently in foci, which are

usually extranucleolar but may also be in the nucleolus, as seen

in Fig. 6D (no). In contrast to the dissociation from Rb, in S

phase, the majority by far of Purer beads (>85%) are seen

contiguous to cyclin A beads. These contiguities are indicated

in Fig. 6C (arrows). At this time, cychin A is frequently localized

in foci distributed throughout the nucleus. Fig. 6E is a typical

representation of the focal distribution of cychin A. In late S

phase, the cychin A foci coalesce to comprise larger, distinct

chromatin bodies of 200-500 nm. These persist throughout G2

phase. It can be seen in Fig. 6Ethat Purer colocalizes with cyclin

A in these chromatin bodies and in the chromatin strands

emanating from these bodies in G2. RB is not generally cob-calized in these structures in G2, and at this time only a minor

percentage of Rb beads are contiguous to Purer beads (Fig. 6F;Tables 1 and 2). It is conceivable that Purer is binding to a

partially dephosphorylated form of Rb at this time. Rb is phos-

phorylated on multiple serine and threonine residues. Although

maximum hypophosphorylation of Rb does not occur prior to

mitosis, dephosphorylation of some residues has been ob-

served in G2 of CV-i cells (29).

The chromatin bodies containing cyclin A and Purer grow

progressively larger throughout G2 phase, occasionally exceed-

ing 500 nm in cross-section diameter, and then become un-

recognizable as distinct entities at the time of chromosome

condensation. Cyclin A levels, as estimated by overall number

of visible beads, remain high until chromosome condensation.

At some point, not precisely discernible in these studies, be-tween the time of chromosome condensation and the appear-

Purer Localized in G1, S, and G2

Table 3 Control mm unoolectron microsc opic reactions

lncubatio n orders G old bead quantitationL�

Reaction1st AbC 1st secondary 2nd Ab 2nd secondary

No. ofPura beads

(nucleus)

No. of cyclinA beads(nucleus)

% Puracontiguous to

cyclin A

Standard order

Pura controlblocked

Cyclin A controlblocked

Cyclin A 1st Abomitted

Purcr 1st Abomitted

Purer and cyclin Ablockedsimultaneously

Rabbit anti-cyclmnA

Rabbit anti-cyclinA

Rabbit anti-cyclinA blocked withcontrol protein

Rabbit anti-cyclinA

Rabbit anti-cyclinA blocked withcontrol protein

Protein A-b nmgold

Protein A-i 0 nmgold

Protein A-b nmgold

Protein A-b nmgold

Protein A-i 0 nmgold

Protein A-i 0 nmgold

Mouse anti-PuramAb

Mouse anti-PurermAb blockedwith purifiedGST-Pura

Mouse anti-PuramAb

Mouse anti-PuramAb

Mouse anti-PurermAb blockedwith purifiedGST-Pura

Goat antimouso-30 nm gold

Goat antimouse-30 nm gold

Goat antimouso-30 nm gold

Goat antimouse-30 nm gold

Goat antimouse-30 nm gold

Goat antimouso-30 nm gold

4,526 ± 219

62

3,869 ± 438

4,380 ± 365

55

657 ± 21 9

17,885 ± 2,190

1 7,374 ± 1 971

1 314 ± 438

584 ± 292

21 ,535 ± 1 533

2,409 ± 584

36.0

1.6

3.1

<0.1

1.8

2.7

a Standard order of incubation of first and second antibodies for localization of Pura and cyclin A in synchronous CV-i cells by immunogold electronmicroscopy is shown, along with five different control incubations. Anti-Pura antibody was blocked with purified GST-Pura. Anti-cyclmn A antibody wasblocked with control peptide sc-i60 P from Santa Cruz Biotechnology.b Visual quantitation of gold beads for a time point 2 h after release from lovastatin is presented. Shown are the average numbers of anti-Pura gold beadsper nucleus and anti-cyclin A gold beads per nucleus for 50 nuclei, each -6 �sm in diameter, taken to be the central cross-sectional diameter. Numbersof beads per nucleus wore calculated based on a mean nuclear volume of 38 �.tni� and a mean sectional thickness of 80 nm. % Pura contiguous cyclinA refers to the percentage of anti-Purer gold beads contiguous to anti-cyclmn A gold beads. Contiguous beads were scored here as beads within 10 nm ofeach other.C Ab, antibody; mAb, monoclonal Ab; GST, glutathione S-transferase.

ance of daughter cells, cyclin A levels decrease to nearly zero.It has been reported previously that, although cyclin A may beassociated with condensing chromosomes in HeLa cells earlyin mitosis, by metaphase, the cyclin is not associated withcondensed chromosomes, and by anaphase, cyclin A largelydisappears from the cell (46). The precise disposition of thePurer molecules in relation to condensed chromosomes re-

mains to be determined. Under the conditions of electron mi-croscopic fixation and staining we have used, the mitotic chro-

mosomes are noted primarily by the absence of electron-densestaining. Nonetheless, high levels of Purer can be seen widely

distributed near, but not in, these lightly staining areas. In thelight micrograph of Fig. 2F, high levels of Purer are seen in thevicinity of postmitotic chromosomes in HeLa cells. This is con-sistent with the assessment of Fig. i that Purer levels are high atthis time in CV-i cells. In contrast to the localization of Purer, Rbappears to be uniformly distributed throughout the cell at mi-

tosis, as detected by immunoelectron microscopy, and is onlyrarely associated with Purer.

Quantitation of lmmunoelectron Microscopic Localize-tion of Purer, Associated Proteins, and Replicating DNA.Considerable quantitative information can be obtained from

micrographs in which proteins are visualized using antibodiesconjugated to gold beads. Scrutiny of Tables i-3, indicatesthat, in G1 phase of the CV-i cell cycle, -4500 beads repre-senting Purer are seen per nucleus. Controls in Table 3 clearlyshow that background levels of beads are minimal, either whenPurer or cyclin A are blocked or when the first antibody is

omitted. The electron microscopic data indicate a preferentialassociation of Purer molecules either with Rb or with cyclin A atdifferent times in the cell cycle. Because there has been no

observed association between cyclin A and Rb (49), ‘it is likelythat these associations are exclusive. One caveat would be thatthe antibody we have used may be blocked from recognizing asubclass of Purer molecules bound either to cyclin A or Rb. Ifthat were true, such a subclass is not likely to represent a majorportion of Purer molecules because it can be calculated that thelevels of Purer beads visualized during the cell cycle by electronmicroscopy are consistent with the levels of Purer detected byimmunoblotting. The association of Purer with cyclin A and Rbis influenced by the changing levels of Purer. A majority of Purermolecules associated with Rb at 4 h are either destroyed or arerendered undetectable by antibodies as cells progress into Sphase. As Purer levels rise nearly 8-fold from S (6 h)through lateG2 (10 h), as seen in Table i, the majority of new nuclear Purermolecules are associated with cyclin A. It can be seen in Table3 that, in G1, at 2 h after release from lovastatin, the number ofcyclin A beads detected is nearly 4-fold the number of Purerbeads detected. That is a relatively high point for Purer levelsand a low point for cyclin A levels. Therefore, in S and early G2,as cyclin A levels rise and Purer levels fall, cyclin A molecules

would most likely far outnumber Purer molecules. Thus, al-

though many Purer molecules are sequestered with cyclin A inS and early G2 (Table i), Purer would only direct a subset ofcyclin A molecules to its locale.

The BrdUrd pulse-label experiment of Table 2 confirmsthat Purer levels are low in S phase, that Purer colocalizes withnewly replicated DNA in S phase, and that these events arenot an artifact of a method of synchronizing cells. This pulse-labeling was performed on asynchronous cells. Rows show-ing cells indicating a maximum of BrdUrd incorporation areindicated with Footnote c in Table 2. These cells, taken to be

a” i,.,,

� E.

.. I

� .�1i

.� �-

I.,,

I. �

p“4%’ 4�

I

..�.1

;�

1tq

p�..#{188}

‘�

.,�

‘ � _a �.� ..i

I �Ia t: .

q , �‘

a�1Il�&�,a� � I� � .;.

� . �:�_

- . . a,�4t ...j

“s-fl �

� ‘ #{149}�#{149}‘� ‘

“.4.

$� � � � � � � �

3� �-�# p C A

point is near the peak of S phase.

Cell Growth & Differentiation

,‘

� �#

� ,.� i....

.. ,

‘� . *

�: �t � -#{149}�“

. �#{149}1;_.� ‘#{149}-�

�

�

� ‘�v:�:. �..

i���; �‘ ‘ �

-- - . .r’ e

� ‘�s� .�‘

� � �,J

Fig. 6. Immunoelectron microscopic localization of Purer, Rb, and Cyclin A in the Cv-1 cell cycle. Synchronized CV-1 cells were fixed, sectioned, andtreated with antibodies for immunogold localization of Purer simultaneously with either Rb or cyclin A, as described in “Materials and Methods.” In each case,the larger 30-nm beads were used to localize Pura and the smaller 1 0-nm beads were used to localize either cyclin A (A, C, and E) or Rb (B, D, and F). A

and B, cells taken at 4 h post-lovastatin release (G1); C and D, cells taken at 6 h (5); E and F, cells taken at 8 h (G2). Solid arrows, Pura-cyclin A complexes;open arrows, Pura-Rb complexes; nm, nuclear membrane; no, nucleolus. Magnification is indicated by the gold beads: large beads, 30 nm; small beads,1 0 nm. Quantification for micrographs is presented in Tables 1-3; controls are presented in Table 3.

661

in S phase, have minimal levels of Purer, showing -1000beads per nucleus, whereas >85% of these beads are co-

localized with BrdUrd. The rows of Table 2 that are indicated

with a footnote are very similar in Purer numbers to the 6-h

point in Table 1 , providing further evidence that this time

DiscussionThe results presented indicate that the intranuclear localiza-

tion of Purer is governed, at least in part, by its association

with Rb. It is notable that levels of Purer are low in early S

phase (Figs. 1 and 3), concomitant with phosphorylation ofRb in CV-1 cells (29). Although any role for Rb in restriction

Puro Localized in G1, 5, and G2

of replication prior to the G1 start point remains to be eluci-

dated, it is frequently generalized that hyperphosphorylationof Rb, occurring in late G1 , promotes the release of certainproteins, such as E2F (50-52), that are positively involved inthe onset of S phase. In this regard, these observations raisethe question of whether the loss of Purer is obligately coupled

to the onset of S phase. At this time, we cannot exclude thepossibility that Purer is stabilized by it association with Rb.Fig. 3, however, indicates that a major decline in Purer levelsoccurs at 4.5 h, prior to the most significant decline inPurer-Rb association at 6 h.

Work is in progress to determine whether Purer levelsdecline in the cell cycle due to targeted protein degradation.Purer possesses certain sequence motifs that might suggesta predisposition to cell cycle-dependent proteolysis. Twoacidic, 27- and 28-amino acid LRRs in Purer are similar toseveral LRRs in �45Skp2 a cyclin A-binding protein in trans-formed human cells (53). These repeats (described in Ref. 1)

are characteristic of a class of LRRs found in a variety ofproteins of diverse functions. In the case of Purer, the second

of the two LRRs contains a very good so-called PEST se-quence, thought to predispose proteins to rapid degradation(54). Consistent with this notion is the presence in Purer of

amino acid motifs similar to the cyclin destruction boxes ofboth human cyclin A (Purer sequence, RDALAKLIDDY) andBi/B2 (Purer sequence, RFYLDVKQN). The destructionboxes are necessary for cyclin degradation by a ubiquitin-dependent pathway (55). Mutation of the invariant R at thefirst position of the destruction box prevents ubiquitination of

cyclins, thought to occur at K residues downstream (55). It isnot known at this time whether or not Purer becomes ubiq-

uitinated, although it does possess requisite K residues, orwhether or not ubiquitination contributes to the observedfluctuation in levels seen in the cell cycle. In other respects,

Purer bears little resemblance to any of the cyclins. For ex-ample, no known cyclin is a sequence-specific DNA-bindingprotein, as is Purer.

During S and G2 Purer coimmunoprecipitates with cyclin A(Fig. 5) and colocalizes with cyclin A in the nucleus (Figs. 4and 6; Table 1). These results, however, do not necessarilyimply a molecular binding of Purer and cyclin A. Conceivably,

the primary interaction of Purer could be with other protein(s)

associated with cyclin A, including Cdk2. Experiments are inprogress to determine whether this is the case. At this time,we have not detected any phosphorylation of Purer by cyclinNCdk2 kinase. Purer possesses 28 serine and threonineresidues, but none of them match a consensus sequenceconsidered favorable for Cdk2-catalyzed phosphorlyation.

PUR elements are present in virtually every mammalianchromosomal or viral origin of DNA replication thus farmapped. The GGN repeat is also feature of certain transcrip-tional promoters and, notably, of human telomeric DNA re-peats, which include the purine-rich element as a 3’ single-strand overhang. Purer could help regulate phosphorylationof Cdk2 substrate proteins by directing the cyclin A/Cdk2complex to PUR elements on the DNA. Neither cyclin A norCdk2 is known to bind DNA. In addition to not being asubstrate for the Cdk2 protein kinase, Purer is not an inhibitorof Cdk2 kinase activity, either alone or in the presence of the

Purer DNA recognition element. In contrast, the observationthat Purer may stimulate cyclin AJCdk2 activity will be dealtwith in another publication. Therefore, any quarternary com-plex involving Purer, cyclin NCdk2, and DNA would remaincapable of promoting the phosphorylation of Cdk2 sub-strates in its vicinity. The substrates of Cdk2 that might affectDNA replication have not been identified, but in addition toHi , they could include both RPA (56, 57) and DNA polym-erase er (58). RPA is present in prereplication complexes,where, in Xenopus eggs, it assembles postmitotically (59). Inmammalian cells, RPA is present in nuclear foci in S phase(44). Although the Mr 34,000 subunit of RPA is phosphoryl-ated by Cdk kinases, such phosphorylation is not requiredfor replication because elimination of Cdk2 phosphorylationsites in RPA does not affect replication (56, 57). Phospho-rylation of the p1 80 and p68 subunits of DNA polymeraseer-primase are cell cycle-regulated and may play a role incontrol of initiation (58). Intriguingly, cyclin A/Cdk2-catalyzedphosphorylation of the two pol-er-primase subunits inhibitsinitiation of replication at the SV4O origin in vitro (60). Manyother proteins may currently be considered potential sub-strates for Cdk kinases. A fraction of cyclin A/Cdk2 exists ina quarternary complex with proliferating cell nuclear antigenand p2i�1��F1 (6i). It is possible, although speculative atthis point, that Purer could localize all or part of this entirecomplex to specific chromosomal sites. Any effects of Pureron the localization, assembly or stability of this complexcould be functionally significant with regard to regulation ofthe cell cycle.

The known functions of Purer would be highly susceptibleto modulation by changes in Purer levels or sequestration. Ithas recently been reported that one allele of the PURA gene,located at human chromosome band 5q3i , is deleted inmany cases of myelodysplastic syndrome, a preleukemiccondition, and in acute myelogenous leukemia (62). Findingsof such gene disruptions highlight the potential importance

of changes in Purer levels in the cell cycle. If Purer intracellularlevels are critical for an aspect of cell cycle progression, it isconceivable that a gene dosage effect could lead to aberrantregulation.

The results presented strongly suggest an active role forPurer in mediating cell cycle events in late S-M, the periodwhen newly replicated DNA progresses to mitosis and whenlevels of Purer, in association with this DNA, increase to theirmaximum. In addition, the decline in Purer levels preceding Sphase may itself play a regulatory role. The increase in Purerlevels occurs over a period when DNA replication is ceasing,in late S and G2 (Figs. 1C; Fig. 3, bottom; Tables i and 2).Although Purer exerts an inhibitory effect on initiation of JCVDNA replication (4), it is not known whether Purer is inhibitoryto replication at genomic PUR elements. If it is, the appear-ance of apparently new Purer molecules with cyclin A inreplication foci throughout S and G2 could play a role inpreventing rereplication of once-replicated DNA. This is con-sistent with recent evidence suggesting a role for cyclin A inpreventing rereplication (63). The decline in Purer levels at thebeginning of S phase could represent a positive signal for theonset of DNA replication. In human glial cells, Purer acts in cisas a suppressor of JCV DNA replication (4). This suppression

Cell Growth & Differentiation �_

may be due in part to an interaction with T antigen at com-

mon binding sites in the JCV origin (4, 5).

Materials and MethodsSynchronIzation of CV-1 Cells. Cells were grown to -33% confluencein 10-cm dishes with DMEM containing 10% fetal bovine serum. Cellswere synchronized by the addition of lovastatin (i 0 �i.t�i; generouslyprovided by A. W. Alberts of Merck Sharp and Dohme ResearchLaboratories)for 20 h (31). Afterthis period, cells wore washed with 150mM NaCI-lO mM NaPO4 (pH 7.4; PBS) and placed in fresh medium

containing mevalonic acid (1 mM; Sigma Chemical Co.) The G1 blockimposed (31) is reversible upon removal of lovastatin and addition ofmevalonic acid. For indicated experiments, cells were pulse-labeledwith is �LM BrdUrd for 10 mm at 37#{176}C.Phases of the CV-i cell cyclewere monitored by assaying [�H�thymidine incorporation, by counting

of mitotic figures after staining Unicryl sections with methylone blueand azure II, by visualizing incorporated BrdUrd, and by counting cellson plated grids. As synchronized here, 92% of CV-i cells showed highlevels of BrdUrd incorporation 6 h after release from lovastatin, mdi-cating that at least this percentage of cells reenter the cell cycle afterrelease from the block. Cell death due to the two drugs was insignif-

icant. A few cells (<3%) were either not blocked or blocked in G2

because they entered mitosis within the first 4 h.lmmunoprecipltation from Cell or Nuclear Lysates. Plates of cells,

synchronized as described, were washed twice with 0.15 M NaCI andplaced on ice. Lysis procedures were carried out quickly to avoid aggre-gation of proteins and to minimize proteolysis. For each time point,whole-cell extracts of i 0� cells were prepared by lysis at 0#{176}Cin 4.5 ml of

Lysis 250 buffer [50 mM Tris-HCI (pH 7.4), 250 m�.i NaCI, 5.0 m� EDTA,0.i % Nonidet P-40, 50 mM NaF, i .0 m� phenylmethylsulfonyl fluoride, 1�g/ml aprotinin, and 1 �.tg/ml loupeptin; Ref. 28]. After 2 mm in lysis buffer,extracts were clarified by centrifugation at 14,000 x g for 4 mm in anEppondortcentrifuge. Sait was adjusted to 150 m� NaCI (Lysis 150 buffer)

by addition of the above buffer with no NaCI. Nuclear extracts wereprepared from 1o� cells lysed by 20 strokes of a Dounce homogenizer in4.5 ml of 10 mM Tris-HCI buffer (pH 7.5) containing 10 n�i KCI, 1 m�phonylmethylsulfoxyl fluoride, 5 M MEDIA, 2 m� Na2MoO4, 0.25 M su-crose, 0.1 % Triton X-iOO, and 1 pg/mI leupeptmn. Aliquots (2 ml) of lysatewere layered over 4 ml of 0.5 M sucrose in the same buffer and subjectedto centrlfugation at 2000 x g for 10 mm. Nuclear pellets were resus-pended in 0.2 ml of the same buffer, with sucrose omitted and with theinclusion of 4.5 M NaCI, 25% glycerol, and 2.5 units of DNasel (Sigma; 2.5

units/pg). Following incubation for 30 mm at 4#{176}C,supematants wereretained after centrifugatlon at 20,000 x g for 15 mm, diluted with thesame buffer with NaCI omitted to an NaCI concentration of 0.15 PA, and

stored at -70#{176}C.For immunoprecipitation, monoclonal antibodies were added to 1 .0-mI

aliquots of either whole-cell or nuclear extract. Aliquots of each of variousantibodies were added as described for each figure. After incubation at0#{176}Cfor 30 mm, magnetic beads coupled to sheep antimouse or antirabbitantibody (Dynal; 500 �g of beads coupled to -5 �g of antibody) wereadded to each i-mI aliquot. After 2 h at 4#{176}Cwith gentle shaking, beadswore collected by magnetism and washed 5 times with 1 ml of 0.15 M

NaCI-0.0i5 M Na-citrate (pH 7.0) containing 1 mu EDTA. As an aitemativeto magnetic beads, after addition of antibodies to the lysate, proteinA-agaroso beads (25 mg beads per ml ofextract; Sigma) were added, andimmunoextraction was carried out by 1 0 successive centrifugation(14,000 x g for 5 mm) and washing (1.0 ml of Lysis 150 buffer, withaprotinin and leupeptin omitted, per Eppondorf tube) steps, all at 4#{176}C.Proteins attached either to magnetic beads or to protein A-agaroso beadswere eluted in 40 �sI of SOS sample buffer.

Electrophoresis and Immunoblot Detection of ProteIns. Proteins

either oluted from immunoprecipitates or obtained directly from cell ornuclear extracts wore subjected to SOS-PAGE in 10% polyacrylamidegels as described previously (28). Separated proteins were blotted tolmmobilon-P membranes (Millipore) and probed with various antibodies

as described (28). Detection was the Pierce SuperSignal chemilumines-cence system.

lmmunoelectron Microscopy. Cells from a single plate, synchro-nizod as described wore fixed by addition directly to the culture dish of

3 ml of 3.7% paraformaldehydo in PBS and incubation for 1 h at room

temperature. Fixed cells wore scraped from the dish and polleted at1500 x g in 3 Eppendorf centrifuge tubes. Pelleted cells were embed-ded in Unicryl and 1 -�m sections were cut and stained with methyleneblue azure II to identify representative areas for ultrathin sectioning and

immunoolectron microscopic staining. Ultrathin sections were cut andplaced on formvar-coated nickel grids and treated sequentially asfollows: first, with antibody to one of various nuclear proteins; second,with goat antirabbit or antimouse antibody coupled to 1 0-nm goldbeads; third, with mouse monoclonal anti-Pura antibody 9Ci 2; andfourth, with protein A coupled to 30-nm gold beads. The antibodieswore used at the following titers: rabbit polyclonal anti-cyclmn A C-22

(Santa Cruz Biotechnology), 1 :100; rabbit polyclonal anti-Rb 0.47 (gen-erous gift of Ors. Pheng-Lang Chen and Wen-Hwa Lee), 1 :200; mousemonoclonal and anti-BrdUrd (Bocton Dickinson), 1 :200; and mouse

monoclonal anti-Pura 9C12, 1:100. The goat antirabbit and goat anti-

mouse coupled to gold (both from Amorsham) were used at 1 :i 00, andthe protein A-gold (Amersham) was used at 1 :40. Blocking steps pre-

ceded and washing stops succeeded each antibody. After the proteinA-gold antibody washing steps, the sections were treated with 3%glutaraldehyde in PBS to fix the antibodies in place. Sections were thenwashed with water, stained with uranyl acetate and load citrate, andvisualized with a Jeol JEM100CX electron microscope. For each pair ofprimary and secondary antibodies, the antibodies wore tested alone

and in both forward and reverse orders to determine whether there wasa difference in labeling pattern and efficiency of labeling. Under theconditions described, no such differences were noted. An array ofadditional controls, performed to assess specificity and cross-roactiv-ity of the antibodies, using the two M-phase cell cycle checkpoints asexamples, is presented in Table 3.

AcknowledgmentsWe thank Lawrence Y. Wang for excellent technical assistance. We aregratefulto Drs. Phang-Lang Chen and Wen-Hwa Lee for anti-Rb antibod-ies 1107 and 0.47. Dr. Thomas M. Fasy generously provided purifiedhistone Hi.

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