Journal of Cell Science 101, 25-34 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

25

The role of primer recognition proteins in DNA replication: association with

nuclear matrix in HeLa cells

JAMBOOR K. VISHWANATHA*, HITESH K. JINDALf and RANDALL G. DAVIS

Department of Biochemistry, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-4525, USA

•Author for correspondencetPresent address: Department of Biochemistry, University of British Columbia, Vancouver, BC, Canada V6T 1W5

Summary

Primer recognition proteins (PRP) enable DNA polym-erase a to utilize efficiently DNA substrates with lowprimer to template ratios. We have previously identifiedthe protein-tyrosine kinase substrate annexin n , and theglycolytic enzyme 3-phosphoglycerate kinase as com-ponents of PRP. As a step towards elucidation of the roleof PRP in the process of DNA replication, we haveinvestigated the subcellular distribution and specificassociation of these proteins with the nuclear matrix inHeLa cells. Nuclear extracts prepared from HeLa cellsin S phase contain the enzymatic activity of 3-phosphoglycerate kinase (PGK) and phospholipase A2

inhibitory activity of annexin II. Monomer annexin II isapproximately equally distributed between the nuclearand cytoplasmic fractions, while a majority of PGK is inthe cytoplasm. Immunoblot analyses reveal the presenceof these two proteins in nuclei, specifically associated

with the nuclear matrix. This is further confirmed byobservation of the presence of annexin II and PGK inisolated nuclear matrices by immunoelectron micro-scopy. The phospholipase A2 inhibitory activity ofannexin II colocalizes with the nuclear matrix-boundannexin II. A related protein, annexin I, is not detectablein the nuclear extracts and nuclear matrix. A slower-migrating (perhaps modified) form of annexin II is foundto be associated with the nuclear matrix. Attempts todissociate PGK and annexin II from the nuclear matrixwith octyl-/J-glucoside, high salt or metal ion chelatorswere unsuccessful, suggesting that the interaction is verystrong.

Key words: DNA replication, primer recognition proteins,nuclear matrix.

Introduction

DNA replication involves various protein-protein andprotein-DNA interactions (Thommes and Hubscher,1990). DNA polymerase a (Pol a) plays a major role inmammalian DNA replication (DePamphilis and Was-sarman, 1980; Thommes and Hubscher, 1990). Pol aexists in multiple forms; a multiprotein Pol a complexfrom HeLa cells (Vishwanatha et al., 1986) was shownto be associated with DNA primase, primer recognitionproteins, deoxyribonuclease and Ap4A binding pro-tein. Primer recognition proteins (PRP) enable Pol or toutilize efficiently DNA substrates with low primer totemplate ratios (Pritchard and DePamphilis, 1983;Vishwanatha et al., 1986), a situation found in lagging-strand DNA replication. PRP has been purified tohomogeneity from HeLa cells and human placenta, andis composed of two polypeptides of 36 and 41kilodaltons (Jindal and Vishwanatha, 1990a). By aminoacid sequence analysis of tryptic peptides derived fromthe two proteins, we have identified the 41 kilodaltonpolypeptide as the glycolytic enzyme, 3-phosphoglycer-

ate kinase (PGK) (Jindal and Vishwanatha, 1990b).The 36 kilodalton protein has been identified as theprotein-tyrosine kinase substrate, annexin II (Jindal etal., 1991). The complex of PGK and annexin IIfunctions as PRP; neither of them alone can act as PRP.

Annexins are major in vivo substrates of oncogeneand growth-factor receptor tyrosine kinases (reviewedby Klee, 1988). The annexin family is composed of atleast six different members including annexin II (alsocalled p36, lipocortin II or calpactin I; Pepinsky et al.,1988). The annexin family has been implicated inmitogenic signal transduction (Pepinsky et al., 1988)and regulation of inflammation (Flower et al., 1984;DiRosa, 1985). Annexin II exists in two forms: themonomer (representing 20-30% of the total cellularannexin II) is mainly cytosolic and the heteromer, inassociation with a 10 kilodalton annexin II light chain, ismainly cytoskeletal (Klee, 1988). The physiological roleof such a distribution is not clear and may indicateinvolvement of annexin II in more than one cellularfunction. The involvement of annexin II in DNAsynthesis is supported by the following observations: (a)

26 /. K. Vishwanatha and others

annexin II is part of the primer recognition proteincomplex (Jindal et al., 1991); (b) the growth-regulatedgene 1B6 is identical to annexin II (Keutzer andHirschhorn, 1990); (c) annexin II expression is basal innormal hepatocytes and significantly enhanced inhuman hepatocellular carcinoma (Frohlich et al., 1990);(d) enhanced expression of annexin II in hamsterpancreatic cancers is specifically associated with pro-liferating and metastatic areas (Kumble et al., 1992);and (e) immunodepletion of Xenopus egg extracts withanti-annexin II monoclonal antibodies results in loss ofreplicative ability of the extracts (S.Kumble and J.K.Vishwanatha, in preparation).

The identification of annexin II and PGK as subunitsof PRP activity led us to examine the existence of theseproteins in nuclei and nuclear matrix. We havepreviously demonstrated, using immunoelectron mi-croscopy, that annexin II monomer and PGK arepresent in the nuclei of normal and transformed cells(Kumble and Vishwanatha, 1991). Nuclear matrix (alsocalled nuclear cage or nuclear scaffold) is a dynamicproteinaceous nuclear framework obtained by treat-ment of isolated nuclei with nuclease, salt and detergent(Berezney, 1984). Replication forks are associated withthe nuclear matrix (Vaughn et al., 1990), and associ-ation of replicative enzyme activities with the nuclearmatrix has been demonstrated in HeLa cells andregenerating rat liver (Smith and Berezney, 1983;Foster and Collins, 1985; Tubo and Berezney, 1987;Collins and Chu, 1987). Isolated nuclear matrices arecapable of in vitro DNA synthesis (Smith and Berezney,1983; Foster and Collins, 1985). Nuclear matrix is alsoimplicated in transcription (Jackson et al., 1984) andRNA processing (Ciejek et al., 1982). Data presentedhere show the association of PGK and annexin II withthe nuclear matrix, indicating that the complex of PGKand annexin II like PRP may have a role in DNAreplication.

Materials and methods

Cell fractionationHeLa S3 cells were maintained in suspension cultures inJoklik's modified Eagle's medium supplemented with 5%defined iron-supplemented calf serum (Hyclone Labora-tories, Utah, USA) at 37°C. Cells were synchronized by thedouble thymidine block technique (Collins and Chu, 1987)and harvested 5 hours after release from the second thymidineblock. The cell pellet was rinsed twice with Hank's balancedsalt solution and once with hypotonic buffer (10 mM Tris-HC1, pH 7.5, 10 mM NaCl and 1 mM MgCl2). Cells wereresuspended and held in hypotonic buffer for 2 hours at 4°C.The cells were disrupted with a Dounce homogenizer (30strokes with pestle B) and disruption was monitored by lightmicroscopy. No intact cells were observed by microscopy afterhomogenization. The homogenate was subjected to centrifu-gation in a Sorvall SS-34 rotor at 2,000 revs/min for 30 minutesat 4°C. The supernatant was designated as "cytoplasmicextract". The nuclear pellet was further rinsed three timeswith the hypotonic buffer and the supernatant from eachwashing was added to the cytoplasmic extract. The nucleiwere resuspended in 0.4 M potassium phosphate, pH 7.2,

1 mM EDTA, 1 mM DTT (dithiothreitol) and 10% glyceroland extracted for 1 hour at 4°C. Following centrifugation in aSorvall SS-34 rotor at 10,000 revs/min for 30 minutes at 4°C,the supernatant was collected as "nuclear extract". Thecytoplasmic and nuclear extracts were dialyzed against 50 mMpotassium phosphate, pH 7.2, 1 mM DTT, 1 mM EDTA and10% glycerol, and stored at -80°C until further use.

Antibodies against annexinsThe polyclonal rabbit anti-chicken annexin II antiserum(Cooper and Hunter, 1983) was generously provided by Dr.Tony Hunter, Salk Institute, San Diego, California, USA. Inimmunoblot analysis, we used a 1:200 dilution of thisantiserum. The polyclonal rabbit anti-human annexin IIantiserum (no. 774), the rabbit anti-human annexin HIantiserum (no. 646) (Pepinsky et al., 1988) and the mousemonoclonal anti-recombinant human annexin III (Lipo-103)(Pepinsky et al., 1990) were generously provided by Dr. BlakePepinsky, Biogen, Massachusetts, USA. Antisera 646 and 774were used at 1:2000 dilution and the lipo-103 antibody wasused at 20 ̂ g/ml in our immunoblot analysis.

Polyclonal antiserum against human PGKNew Zealand white rabbits were immunized by multipleinjections (intradermal and intraperitoneal) with 200 ng ofhuman placental PGK purified to homogeneity by publishedprocedures (Kuntz and Krietsch, 1982). The Ribi adjuvant forrabbits (Ribi Immunochem., Montana, USA) was used as anadjuvant in immunization. Immunization was repeated twoweeks later with 100 ng of purified PGK. Antisera wereobtained weekly for four weeks after the boost and antibodytiters were determined by enzyme-linked immunosorbentassay.

Preparation of the nuclear matrixNuclear matrices were prepared from freshly isolated nucleiof S phase HeLa cells. Briefly, nuclei prepared as described byCollins and Chu (1987) were digested with 80 ;Ug/ml DNase Ifor 30 minutes at 37°C. The cytosolic fraction was keptseparately and called "soluble fraction". Digested nuclei werewashed 3 times with PBS and 3 times with TMP buffer (10 mMTris-HCl, pH 7.5, 0.2 M MgCl2, 1 mM PMSF). Supernatantsfrom each of these washings were combined and this fractionwas designated as "low-salt nuclear extract". Low-saltextracted nuclei were resuspended in TMP buffer, and TMPbuffer containing 5 M NaCl was added slowly over a 1 hourperiod to a final concentration of 2 M NaCl. Followingcentrifugation, the nuclear pellet was washed 3 times withPBS, 4 times with low-salt phosphate buffer (20 mMpotassium phosphate, pH 7.5, 2 mM /S-mercaptoethanol),three times with high-salt phosphate buffer (0.5 M potassiumphosphate, pH 7.5, 2 mM /3-mercaptoethanol) and finally, 3times with the low-salt phosphate buffer. Supernatants fromthe above washes were combined and designated as "high-saltnuclear extract". The low-salt and high-salt nuclear extractswere concentrated with membrane concentrators (OmegaCell, Pharmacia LKB, California, USA), dialyzed against 50mM potassium phosphate, pH 7.2,1 mM DTT, 1 mM EDTA,10% glycerol, and stored at —80°C. The final nuclear matrixwas resuspended in the same buffer and stored in aliquots at-80°C.

Electron microscopy of cells, nuclei and nuclearmatrixHeLa cells, isolated nuclei and the nuclear matrix (preparedas described above) were fixed in 1.5% paraformaldehyde,1% glutaraldehyde, 0.13% picric acid, 0.1 M phosphate

Association of PRP with nuclear matrix 27

buffer, pH 7.4. The fixed cells, nuclei and matrix were thenwashed with 0.1 M phosphate buffer, pH 7.4, and dehydratedthrough a series of increasing ethyl alcohol concentrations.Following dealcoholization in the presence of propyleneoxide, cells, nuclei and matrix were embedded in Aralditeresin. Thin sections were cut using the Portex-Blum MT 2ultramicrotome and then mounted on nickel grids (200 mesh)for electron microscopy. The nickel grids were double stainedwith uranyl acetate and Reynold's lead citrate, and viewedthrough a Philips 410 LS electron microscope at 60-80 kV.

Immunoelectron microscopy of nuclear matrixUltrathin sections of nuclear matrices mounted on nickel gridswere prepared as described above and used for immunolabel-ing. The grids were initially fixed with 3% hydrogen peroxideand after rinsing in PBS and blocking buffer (PBS containing1% BSA) for 1 min each, incubated with the primary antibody(diluted in PBS) for 2 h. The grids were then washed withblocking buffer and incubated with the Protein A-goldconjugate (30 nm particle size) for 60 min at roomtemperature. At the end of this incubation, the grids were jetwashed successively with blocking buffer, PBS and distilledwater, and finally dried. The tissue sections were doublestained with uranyl acetate and Reynold's lead citrate. Thesections were viewed using a Phillips 410 LS electronmicroscope at 60-80 kV.

Solubilization of nuclear matrixExtensively washed nuclear matrix preparation was sonicated5 times for 15 seconds each at 20 second intervals and allowedto remain on ice for 2 hours. Following this, an aliquot of thehomogenate was incubated with 22 mM octyl-/3-glucoside and0.5 M KC1 for 1 hour at 4°C. Another aliquot of thehomogenate was adjusted to 5 mM final concentrationof ethyleneglycol-bis(jS-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and incubated for 1 hour at 4°C.Following centrifugation at 40,000 revs/min in a Beckmantype 65 rotor for 1 hour at 4°C, the supernatants were saved as"dissociated fraction". The pellet was resuspended in PBS,washed three times with PBS and the "residual matrix" wasstored in 50 mM potassium phosphate, pH 7.2,1 mM DTT, 1mM EDTA, 10% glycerol at -80°C. The "dissociatedfraction" was dialyzed against the same phosphate buffer andstored at -80°C.

Enzyme assays, SDS-PAGE and immunoblot analysisPreviously published procedures were used for assays of DNApolymerase or activity (Vishwanatha et al., 1986) and PGKactivity (Jindal and Vishwanatha, 1990b). Phospholipase A2inhibition assay (Haigler et al., 1987) was conducted withmodifications as previously described (Jindal et al., 1991).Proteins were resolved on 4% to 15% gradient polyacryl-amide gels containing SDS and immunoblotting was per-formed as previously described (Jindal et al., 1991).

Results

Presence of annexin H and PGK in nuclear extractsPGK in association with the protein-tyrosine kinasesubstrate, annexin II, forms the primer recognitionprotein complex that stimulates DNA polymerase aactivity on substrates with long single-stranded DNAtemplates. Annexin II is abundant in cells mainly inassociation with plasma membrane in a complex withcalpactin light chain. Monomer annexin II is cytosolic.PGK is abundant in the cytoplasm. In order for theseproteins to be involved in DNA replication, they mustbe present in the nucleus during S phase of the cellcycle. We investigated the presence of these proteins innuclear extracts of HeLa cells in S phase. Nuclei wereprepared by Dounce homogenization of cells incubatedin hypotonic buffer and extensively washed to removecytoplasmic contamination. Nuclear extracts from suchwashed nuclei contain significant amounts of PRP(Jindal and Vishwanatha, 1990a,b; Jindal et al., 1991).The activities of PGK, annexin II and Pol a in cytosoland nuclear extracts of HeLa cells are presented inTable 1. As expected, nuclear extracts have signifi-cantly higher activity of Pol a than is found in thecytosol. There is a tenfold higher activity of PGK incytosol compared to nuclear extracts. The monomerannexin II activity (as judged by the inhibition of PLA2activity) is present in both nuclear and cytosolic extractsto the same extent. We were unable to determine thestoichiometry of Pol a, PGK and annexin II in thenuclear extract and in the DNA Pol a2 complex, due tothe unavailability of antisera or monoclonal antibodiesto DNA Pol a that would react quantitatively onWestern blots. Fig. 1 shows the activities of annexin II(A) and PGK (B) as a function of protein concentrationin the reaction. At the various protein concentrationsused, annexin II activity in the cytosol and nuclearextract are nearly equal. No activity is seen with variousconcentrations of a non-specific protein gelatin. Immu-noblot analysis using polyclonal antibodies against PGKand annexin II demonstrates the presence of theseproteins in the nuclear extracts (Fig. 2). The immuno-blot from Fig. 2 was analyzed on a Betascope 603(Betagen, Inc.) radioanalytical imager and the radioac-tivity corresponding to the PGK and annexin II bandswas quantitated. A correlation of 0.996 is seen betweenimaging data obtained from the Betascope 603 andradioactivity determined with a gamma counter (R. G.MacDonald and J. K. Vishwanatha, unpublished). We

Table 1. Pol a, annexin II and PGK activities in cytosolic and nuclear extracts of HeLa cells

Fraction

CytosolNuclear extract

Volume(ml)

9059

Protein(mg)

324183

Pol a(units/mg)

13003230

PGK(units/^g)

31.03.4

Annexin II(% inhibition)

6867

The starting material was 20 g wet weight of HeLa cells collected in early S phase of cell cycle. The preparation of extracts and assaysfor Pol a with activated DNA template, PGK and annexin II are described under Materials and methods. One unit of Pol a activitycorresponds to 1 pmol of dNTP incorporated per hour at 35°C; one unit of PGK activity corresponds to oxidation of 1 /anol ofNADH/min, using a molar extinction coefficient for NADH of 3.4 x 10"1 cm"1. Annexin II activity is expressed as % inhibition ofphospholipase A2 activity at a protein concentration of 5 / l

28 /. K. Vishwanatha and others

==• 120co

n

- 0

ctiv

CO

<in.

100-

80-

60-

40-

20-

^s^=a^

1 10Protein concentration

100 5 10 20Protein concentration (^g/ml)

Fig. 1. Annexin II and PGK activities of cytosolic and nuclear extracts. HeLa cell cytosolic and nuclear extracts wereprepared and assayed for PGK and PLA2 inhibitory activities, as described under Materials and methods. (A) PLA2inhibitory activity profiles of cytosolic extract (filled squares), nuclear extract (open squares), crude lipocortin preparation(filled circles) and gelatin (open circles) at various indicated protein concentrations in the reaction; 100% activity representsPLA2 activity in the absence of added inhibitory or control protein. (B) PGK activity of nuclear extract (hatched bars) andcytosolic extract (filled bars) at various indicated protein concentrations.

SDS-PAGE

200

92

anti-p36 anti-p41

6 9

4 6

302114

N M C N M C NFig. 2. Annexin II and PGK are present in HeLa cell nuclear extracts. HeLa cells collected in early S phase werefractionated into cytoplasmic and nuclear extracts as described under Materials and methods. Samples (10 jig) of proteinfrom cytoplasmic extract (lane C) and nuclear extract (lane N) from S phase HeLa cells were subjected to electrophoresison a 4% to 15% gradient polyacrylamide gel containing SDS. Proteins were stained with silver reagent (A), or transferredto Immobilon-P membrane, which was then incubated either with polyclonal rabbit anti-annexin II antiserum or polyclonalrabbit anti-human PGK antiserum (B). Reactivity was then assessed using [1Z5I]protein A followed by autoradiography.The positions of 14C-labeled marker proteins run on the same gel (lane M) are indicated (in kilodaltons). The blots wereexposed for 8 h at — 70°C using Kodak X-Omat AR film and a Cronex-plus intensifying screen.

firid that PGK is predominantly cytosolic (94%) andonly a small amount exists in the nucleus (6%).Annexin II, on the other hand, is distributed betweennucleus (40%) and cytosol (60%). The nuclear annexin

II represents that part of the monomer annexin II that isnot associated with the light chain. Monomer annexin IIhas been variously estimated to represent 20-30% of thetotal annexin II in cells. Thus, the amount of annexin II

Association of PRP with nuclear matrix 29

in the HeLa nucleus represents 8-12% of the totalannexin II in cells. These results are in agreement withthe activities shown in Table 1. The presence of annexinII and PGK in the nuclear extracts is further supportedby the demonstration of the presence of these proteinsin the nucleus by immunoelectron microscopy usingcolloidal gold-conjugated secondary antibody (Kumbleand Vishwanatha, 1991). Previously, we have shownthat the cytoplasmic protein, annexin I (also called p35,calpactin II or lipocortin I) is not present in nuclearextracts prepared by this procedure (Jindal et al., 1991).

Preparation of the HeLa cell nuclear matrixA number of proteins and enzymes implicated in DNAreplication are found associated with the nuclear matrix(Smith and Berezney, 1983; Foster and Collins, 1985;Tubo and Berezney, 1987; Collins and Chu, 1987) andrecently the association of replication forks with thenuclear matrix was demonstrated (Vaughn et al., 1990).We investigated the association of PRP with the nuclearmatrix in HeLa cells. We compared different pro-cedures for isolation of the nuclear matrix, sincedifferences in these procedures may influence thecomposition of the matrix. The final procedure that weroutinely used in all of the following experiments isessentially that of Collins and Chu (1987) and isdescribed under Materials and methods. Electronmicroscopic pictures of the prepared nuclear matrices,isolated nuclei and the original cells are shown in Fig. 3.The nuclear membrane maintains its integrity through-out the matrix preparation procedures (Fig. 3B). Thematrices exhibit well-preserved internal structure, withthe perichromatin granules and interchromatin in thematrix (Fig. 3A and B) being very similar to the isolatednuclei or the intact nucleus of the whole cell (Fig. 3C).Some debris present on the outside of the matrix (Fig.3A, B) possibly represents lysis of some nuclei, sincethe isolated nuclei are free of any cytoplasmic debris(Fig. 3D). The nuclear matrices that we prepared arevery similar in appearance to the nuclear matrices fromchicken erythroblast cells (Farache et al., 1990) and theyeast nuclear scaffolds (Cardenas et al., 1990). Nodifferences were seen in the quality of matricesobtained when nuclei were extracted with or withoutreducing agents in the extraction buffer.

Annexin II activity in nuclear matrixThe soluble fraction, low-salt and high-salt extracts ofnuclei and the nuclear matrix were prepared asdescribed under Materials and methods. The phospho-lipase A2 inhibitory activities of these fractions areshown in Fig. 4. Significant amounts of the nuclearannexin II activity are released by low-salt washing ofnuclei (bar 3). Further washing of nuclei with high-saltbuffers (bar 4) does not release any PLA2 inhibitoryactivity. Nuclear matrices prepared after these saltwashes contain significant levels of PLA2 inhibitoryactivity (bars 5,6,7). Increasing PLA2 inhibitory activityas a function of increasing protein concentration isobserved with the nuclear matrix preparation (bars 5,6,

c /

'4

A

-,...-' D

Fig. 3. Morphology of the isolated nuclear matrix and theoriginal cell. Ultrathin sections (100 nm) through nuclearmatrices (A and B), the original HeLa cells (C) andisolated nuclei (D) were viewed through an electronmicroscope following staining with uranyl acetate andReynold's lead citrate as described under Materials andmethods. Part of the matrix is shown in B to illustrate thenuclear membrane in greater detail.

and 7). Although PGK is present in the nuclear matrix(see below), measurable activities are not observed.

Immunoblot and immunoelectron microscopicanalyses of annexin II and PGK in nuclear matrixThe presence of annexin II and PGK in the nuclearmatrix was demonstrated by immunoblot and immuno-electron microscopic analyses using various antibodies

30 /. K. Vishwanatha and others

120-1

1 2 3 4 5 6 7Fig. 4. Phospholipase A2 inhibitory activity of nuclearextracts and nuclear matrix. A sample of each fractionduring nuclear matrix preparation was assayed forinhibition of porcine pancreatic phospholipase A2 activityas described under Materials and methods. The results arepresented as percentage inhibition of phospholipase A2activity, with 100% activity being 1100 cts/min releasedfrom labeled E. coli cells by PLA2 in the absence of anyadded inhibitor protein. The different fractions used were 6Hg protein of a crude lipocortin preparation from placentalmembranes (bar 1), 20 jig protein of HeLa soluble extract(bar 2), 5 ng of low-salt nuclear extract (bar 3), 2 jig ofhigh-salt nuclear extract (bar 4), and nuclear matrix at 3.0Hg protein (bar 5), 6.0 ng protein (bar 6) and 9.0 jigprotein (bar 7).

that recognize the two proteins. Analyses of thepolypeptide composition of various fractions duringnuclear matrix preparation are presented in Fig. 5 (A).The complex nature of nuclear matrix seen (Fig. 5A) issimilar to that reported earlier for nuclear matrix andchromosome scaffolds (Berezney and Coffey, 1974;Earnshaw et al., 1985). We examined different fractionsobtained during nuclear matrix preparation for theirreactivity against a polyclonal rabbit antiserum raisedagainst chicken annexin II (Cooper and Hunter, 1983),and the data are presented in Fig. 5B. The antiserumrecognizes a 36 kilodalton polypeptide in all of thefractions analyzed. In addition, the antiserum reactswith a protein of 39 kilodaltons in the nuclear matrix.We have not seen a cross-reaction between a 39kilodalton protein and the anti-annexin II antiserum inany other fraction tested so far. The 39 kilodaltonprotein appears to be a modified form of annexin II,which leads to the intriguing hypothesis that thismodification is specific for a subset of annexin IImolecules bound to the nuclear matrix. Our prelimi-nary results indicate that phosphorylated annexin II isspecifically associated with the nuclei (K. D. Kumbleand J. K. Vishwanatha, unpublished). However, we donot know the precise nature of the modification in the39 kilodalton polypeptide recognized by the anti-annexin II antibody in the nuclear extracts. Althoughthere is no detectable enzymatic activity of PGK innuclear matrices (discussed above), immunoblot analy-

sis using polyclonal rabbit antiserum against humanPGK demonstrates the presence of PGK in nuclearmatrices (Fig. 5C). The anti-PGK antisera reacts withthe authentic PGK in the soluble fraction (lane 1).Cross-reactivity is also seen with a protein of greaterthan 200 kilodaltons. Strong cross-reactivity of theantisera is observed with the nuclear matrix (lane 4) butnot with salt extracts of nuclei (lanes 2 and 3). Incontrast to the nuclear matrix-associated annexin II, amodified form of PGK was not found. Specificassociation of annexin II and PGK with the nuclearmatrix was further confirmed by immunoelectronmicroscopic analysis of isolated nuclear matrices (Fig.6). Incubation of thin sections of nuclear matrices withanti-annexin II (A and B) or anti-PGK (C and D)antisera followed by incubation with Protein A-colloidal gold particles results in specific staining withinthe nuclear matrices for both proteins. The Protein A-gold particles are seen associated with the nuclearmatrix structures and we do not see significantassociation of these proteins with the nuclear mem-brane.

We have previously shown that a rabbit antiserumraised against recombinant human annexin I (no. 646)(Pepinsky et al., 1988) cross-reacts with annexin II(Jindal et al., 1991). Annexins I and II share a 50%amino acid sequence identity (Pepinsky et al., 1988).The 36 kilodalton protein that is recognized by anti-annexin II antiserum in the nuclear matrix (Fig. 5B) isalso recognized by the anti-annexin I antiserum asshown in Fig. 7A). However, the anti-annexin Iantiserum does not recognize the 39 kilodalton proteinthat cross-reacts with the anti-annexin II antiserum. Inthe soluble fraction (Lane 1), the anti-annexin Iantiserum reacts with a 32 kilodalton protein. The 32kilodalton protein is a proteolytic product of annexin I.The autoradiogram was intentionally overexposed inorder to observe the weak interaction of the antiserumwith the nuclear matrix. This resulted in overexposureof the lipocortin lane (lane 5). Annexin I is a cytosolicprotein (Klee, 1988) and mouse monoclonal anti-human annexin I (Pepinsky et al., 1990) does not cross-react with annexin II. Fig. 7B shows an immunoblot ofthe cross-reactivity of various nuclear fractions with themouse monoclonal anti-human annexin I antibody.While the annexin I in the soluble fraction is recognizedby this antibody (lane 1), no cross-reactivity is observedwith the proteins in the salt extracts of nuclei (lanes 2and 3) or the nuclear matrix (lane 4). Prolongedoverexposure of the blot did not result in appearance ofany other bands. Thus, annexin II is specificallyassociated with the nuclear matrix, while annexin I isnot present in either nuclear extracts or the nuclearmatrix.

Strong association of annexin II and PGK withnuclear matrixDNA replication proteins tightly associated with thenuclear matrix are dissociated by treatment of nuclearmatrices with the detergent octyl /3-D-glucoside underhigh-salt conditions (Collins and Chu, 1987). We

Association of PRP with nuclear matrix 31

A

200

B 200

92

69

46

302114

M

92-

69-

46

30-

21-14-

M

c200

Fig. 5. Immunoblot analysis of annexin II and PGK innuclear extracts and nuclear matrix. The soluble fraction(lane 1), low-salt nuclear extract (lane 2), high-salt nuclearextract (lane 3) and nuclear matrix (lane 4) from S phaseHeLa cells, and crude lipocortin from human placenta(lane 5), were electrophoresed on 4% to 15% gradientSDS-PAGE gels. Proteins were stained with silver reagent(A), or transferred to Immobilon-P membranes andincubated with polyclonal rabbit anti-annexin II antiserum(B) or polyclonal anti-human PGK antiserum (C).Reactivity was assessed using f^Ijprotein A andautoradiography. Positions of C-labeled marker proteins(lane M) run on the same gels are indicated with theirmolecular masses in kilodaltons.

92-

69

46

30-

21-14

M 1 2 3 4 5

attempted to dissociate the nuclear matrix-bound PGKand annexin II under a variety of conditions. The resultsare shown in Fig. 8. Treatment of nuclear matrices with22 mM octyl-y3-D-glucoside in the presence of 0.5 MKC1 or incubation of the matrix with 5 mM EGTA failsto dissociate either the annexin II (Fig. 8A) or PGK(Fig. 8B) proteins. Interestingly, the 39 kilodaltonprotein, presumably modified annexin II, is also tightlybound and is not dissociated. However, these proteinsare dissociated from the matrix by strong denaturingagents such as SDS.

Discussion

The complex of annexin II with the glycolytic enzyme,PGK, functions as PRP in stimulating Pol a-activity ontemplates with low primer to template ratios (Jindaland Vishwanatha, 1990a,b; Jindal et al., 1991). Themajority of annexin II in cells exists as a heteromer withcalpactin light chain (plO). However, the monomericannexin II is cytosolic (Klee, 1988). The physiologicalrole of annexins or the significance of two pools ofannexin II in cells is not yet understood. Presumably,

32 /. K. Vishwanatha and others

Fig. 6. Immunoelectronmicroscopy of ultrathinsections of nuclear matricesusing protein A-colloidal goldparticles. Ultrathin sections ofHeLa cell nuclear matriceswere prepared on nickel gridsas described in Materials andmethods. Grids were incubatedwith anti-annexin II polyclonalantiserum (A and B) at 1:150dilution or with anti-PGKpolyclonal antiserum (C andD) at 1:50 dilution. Afterwashing with the blockingbuffer, the grids wereincubated with Protein A-colloidal gold conjugate (30 nmparticle size). The grids werethen double stained withuranyl acetate and Reynold'slead citrate, and viewed usinga Philips 410 LS electronmicroscope. Locations of a fewof the gold particles areindicated with arrowheads.

annexin II is involved in different cellular processes.The identity of growth-regulated gene, 1B6, as annexinII (Keutzer and Hirschhom, 1990) and enhancedexpression of annexin II in human hepatocellularcarcinoma (Frohlich et al., 1990) and in proliferatingand metastatic areas of pancreatic cancer (Kumble etal., 1992) further support our findings that annexin II isinvolved in DNA synthesis (Jindal et al., 1991). Wehave previously demonstrated that the complex of PGKand annexin II is functional as PRP and that inhibitionof one of the components reduces the ability of thecomplex to function as PRP (Jindal and Vishwanatha,1990b; Jindal et al., 1991). We do not know thephysiological role of PRP in the DNA replicationprocess. It has been proposed that they interact with Pola in lagging-strand DNA replication (Vishwanatha etal., 1986).

For PRP to be involved in DNA replication, itscomponents must be present in the cell nucleus duringactive DNA synthesis. Published reports on the intra-cellular localization of annexin II indicate that it iscytoskeletal (reviewed by Klee, 1988). However, it isknown that the monomer (approximately 20-30% oftotal annexin II in cells) is cytosolic. Further, there areno reports of intracellular localization of annexin IIduring different phases of the cell cycle. In our attemptsto elucidate the physiological role of annexin II andPGK in DNA replication, we examined the presence ofannexin II and PGK in nuclei and nuclear matrix in cellscollected at early S phase during active DNA synthesis.

Nuclear extracts prepared from HeLa cells in S phasecontain PGK and annexin II. The presence of theseproteins in nuclear extracts is not due to cytoplasmiccontamination as evidenced by specific association ofthese proteins with nuclear matrix, immunoelectronmicroscopy and the differential distribution of PGK(only 6% in nuclear extracts) and annexin II (40%) inthe nuclear and cytosolic extracts. Further, a mono-clonal antibody to annexin I, a cytosolic protein, doesnot detect its antigen in the nuclear extracts. Immuno-electron microscopic studies have demonstratedspecific association of PGK and annexin II with nucleiin normal and transformed cells (Kumble and Vishwa-natha, 1991). Thus, our observations of PGK andannexin II being present in nuclei during S phasesupport a possible role of these proteins in DNAreplication.

A number of recent reports suggest the involvementof nuclear matrix in DNA replication in vivo (Berezney,1984; Cam et al., 1986; Tubo and Berezney, 1987).Replication forks are associated with the nuclearmatrices prepared either by the high-salt method or byhypotonic or isotonic LIS extraction (Vaughn et al.,1990). Replication-dependent association of Pol a,DNA primase, ribonuclease H, DNA methylase andother replicative enzymes with the nuclear matrix(Smith and Berezney, 1982, 1983; Foster and Collins,1985; Tubo and Berezney, 1987) further suggests theinvolvement of nuclear matrix in DNA replication.

Strong association of PGK and annexin II with the

Association of PRP with nuclear matrix 33

200A200

M 3 4

M 1 2 3 4Fig. 7. Reactivity of nuclear matrix with antisera andmonoclonal anti-human annexin I antibodies. Proteinfractions were subjected to SDS-PAGE and transferred toImmobilon-P as described in the legend to Fig. 5. Themembrane was incubated with either polyclonal anti-humanannexin I antiserum (no. 646) (A) or Lipo-103 monoclonalantibody (B). The blots were subsequently incubated with[125I]protein A, followed by autoradiography, as in legendto Fig. 5.

nuclear matrices prepared from S phase cells has beenshown in our studies. This is the first identification ofthese two activities being associated with the nuclearmatrix. The nuclear matrix-associated annexin II existsin two forms as distinguished by their apparentmolecular masses. Calpactin I is known to be phos-phorylated (Powell and Glenney, 1987) and myristy-lated (Soric and Gordon, 1985). The biochemicalconsequences or physiological role of such modifi-cations are not known. Preliminary studies indicate thatphosphorylated annexin II is specifically associated withthe nuclei (K. D. Kumble and J. K. Vishwanatha,unpublished). The mechanisms involved in the translo-cation of PGK and annexin II to the nuclei and theirassociation with the matrix are not known.

92

69-

46-

30-

21-

B200

M 1 2 3 4 5

9 2 - *

6 9 - *

46—• - • > • • »r

30—* — ,

2 1 - * —'

M 1 2 3 4 5

Fig. 8. Strong association of PGK and annexin II withnuclear matrix. HeLa cell nuclear matrix (lane 1) wasincubated with 22 mM octyl-^S-D-glucoside and 0.5 M KC1as described under Materials and methods. Followingincubation, the matrix was subjected to centrifugation. Thesupernatant (lane 2) and resuspended pellet (lane 3) weresubjected to electrophoresis. In another experiment, thematrix was incubated with 5 mM EGTA and, followingcentrifugation, the supernatant (lane 4) and resuspendedpellet (lane 5) were subjected to electrophoresis.Electrophoresis, transfer and Western blotting using 125I-labeled Protein A were performed as described underMaterials and methods. (A) Results using anti-chickenannexin II antiserum; (B) an immunoblot using anti-humanPGK antiserum. Lane M denotes 14C-labeled markerproteins run on the same gel.

34 /. K. Vishwanatha and others

The authors thank Richard MacDonald and Angie Rizzinofor critically reviewing the manuscript; Christopher Andersonand Demi Johnson for excellent technical help; Rick Vaughnfor help with Electron Microscopy and Pamela Walter forword processing.

This research was supported by grants from the LeukemiaResearch Foundation, Inc., Nebraska Cancer and SmokingDisease Research Program and the University of NebraskaMedical Center (Seed Grant).

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(Accepted 11 June 1991 - Accepted, in revised form,30 September 1991)