150 s dna polymerase a-primase megacomplexes solubilized ... · (berezney and coffey, 1977; smith...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 12, Issue of April 25, pp. 5857-5865. 1987 Printed in U. S. A.

Identification of 100 and 150 S DNA Polymerase a-Primase Megacomplexes Solubilized from the Nuclear Matrix of Regenerating Rat Liver*

(Received for publication, October 20, 1986)

Ross A. TuboS and Ronald Berezneyg From the Department of Biological Sciences, State University of New York, Buffalo, New York 14260

The majority of DNA polymerase a and primase activities bound to the nuclear matrix of regenerating rat liver were released into an extract by a mild sonication procedure. During maximal in v ivo replication (22-h posthepatectomy) most of the solubilized a-polymerase and primase cosedimented at approximately 100 and 150 S as discrete megacomplexes with smaller amounts at 10 and 17 S. In contrast, high salt extracts obtained during nuclear matrix isolation as well as matrix extracts prepared just before the onset of in vivo replication (14-h posthepatectomy) were completely devoid of megacomplexes. In vitro incubation of the matrix extracts resulted in rapid dissolution of the megacomplexes to the 10 and 17 S forms. These relationships lead us to propose a dynamic assembly of the eucaryotic replisome which is initiated pre-replicatively as 10 and 17 S complexes and functionally expressed during in vivo replication as 100 and 150 S megacomplexes or “clustersomes.”

The nuclear matrix is a proteinaceous nuclear structure which remains after extraction of chromatin and soluble nuclear components from isolated nuclei (Berezney and Cof- fey, 1974; Berezney, 1979; Shaper et al., 1979; Agutter and Richardson, 1980; Bouteille et al., 1983; Hancock, 1983; Ber- ezney, 1984). It is composed predominantly of nonhistone proteins with smaller amounts of tightly bound DNA and RNA and retains many of the characteristic structural fea- tures of the intact nucleus including a surrounding nuclear lamina with nuclear pore complexes and an internal matrix composed of residual nucleoli and a nonchromatin fibrogran- ular network structure (Berezney and Coffey, 1974, 1977; Comings and Okada, 1976; Herlan and Wunderlich, 1976; Wunderlich and Herlan, 1977; Hodge et al., 1977; Berezney, 1979; Long et al., 1979; Agutter and Richardson, 1980; van Eekelen and van Venrooij, 1981; Brasch, 1982; Capco et al., 1982; Bouteille et al., 1983; Hancock, 1983; Berezney, 1984; Lewis et al., 1984). A similar nuclear matrix structure has been obtained in a wide range of cells and organisms from yeast to man (Berezney and Coffey, 1974, 1977; Herlan and Wunderlich, 1976; Hodge et al., 1977; Berezney et al., 1979; Mitchelson et al., 1979; Snead et al., 1979; Poznanovic and

* This work was supported by National Institutes of Health Grant GM-23922 (awarded to R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

School, 44 Binney St., Boston, MA 02115. $ Present address: Dana-Farber Cancer Institute, Harvard Medical

dressed. §To whom correspondence and reprint requests should be ad-

Sevaljevic, 1980; Maundrell et al., 1981; Bekers et al., 1981; Nakayasu and Ueda, 1981; Fisher et al., 1982; Lafond and Woodcock, 1983; Ierardi et al., 1983; Berezney, 1984; Staufen- biel and Deppert, 1984; Potashkin et al., 1984; Fey et al., 1986; Ghosh and Dey, 1986), and a growing body of evidence has demonstrated the association of a variety of functional properties with these subnuclear fractions (Agutter and Richard- son, 1980; Bouteille et al., 1983; Hancock, 1983; Berezney, 1984).

Perhaps the most studied and best characterized function associated with the isolated nuclear matrix is DNA replication (Berezney et al., 1982; Berezney, 1984, 1985). DNA in the eucaryotic cell nucleus is constrained within repeating supercoiled domains of approximately 50-200 kilobases (Cook and Brazell, 1975; Ide et al., 1975; Benyajati and Worcel, 1976; Pinon and Salts, 1977; Igo-Kemenes and Zachau, 1978; Har- twig, 1978; Nakane et al., 1978). The attachment of these supercoiled domains or loops to isolated interphase nuclear matrix in conjunction with the kinetics of association of in vivo replicating DNA and replication origins with the matrix fraction have formed the experimental basis for a new topographical model of eucaryotic DNA replication (Dingman, 1974; Berezney and Coffey, 1975; Wanka et al., 1977; Dijkwel et al., 1979; McCready et al., 1980; Pardoll et al., 1980; Vogel- stein et al., 1980; Berezney and Buchholtz, 1981a, 1981b; Buongiorno-Nardelli et al., 1982; Aelen et al., 1983; Valenzuela et at., 1983; Van der Valden et at., 1984; Smith et al., 1984; Carri et al., 1986; Dijkwel et al., 1986; Jackson and Cook, 1986) in which it is proposed that DNA is replicated by the reeling of DNA loops through matrix-bound replicational complexes or replisomes. Consistent with this matrix-bound replisome model, a number of studies have demonstrated a cell cycle and replicative dependent association of the eucaryotic replicative enzyme DNA polymerase a with the nuclear matrix (Smith and Berezney, 1980,1982,1983; Jones and Su, 1982; Foster and Collins, 1985).

As a step toward identifying and characterizing these putative nuclear matrix-bound replisomes, we report the solubilization of DNA polymerase a and primase from regenerating rat liver nuclear matrix. We find that most of the matrix-released a-polymerase and primase are organized into discrete 100 and 150 S megacomplexes. The absolute replicative dependence of these megacomplexes leads us to propose a dynamic assembly model of the eucaryotic replisome. Briefly, it is suggested that 10 and/or 17 S a-polymerase- primase complexes are assembled pre-replicatively at poten- tial sites of eucaryotic DNA replication and are subsequently recruited into large 100 and 150 S megacomplexes during active DNA replication.

MATERIALS AND METHODS

Preparation of Samples and Sucrose Gradient Analysis-Normal and regenerating rat liver nuclei were isolated as described previously

5857

5858 Nuclear Matrix Polymerase-Primase Megacomplexes

(Berezney and Coffey, 1977; Smith and Berezney, 1982). Nuclear

endogenously digested for 45 min at 37 "C, centrifuged (3000 rpm for matrices and extracts were prepared as follows. Isolated nuclei were

15 rnin), resuspended in high salt buffer (2.0 M NaC1, 10 mM Tris, pH 7.4, 0.2 mM MgC12, 1 mM phenylmethylsulfonyl fluoride), and centrifuged (5000 for 15 min). The high salt extraction was repeated twice, and the combined extract was saved for polymerase assay. The pellet following high salt extraction was resuspended to one-third the original nuclear volume and divided into two equal aliquots. One aliquot was washed two additional times with low salt buffer (10 mM Tris, pH 7.4, 0.2 mM MgCl,, 1 mM phenylmethylsulfonyl fluoride), resuspended to the initial volume with low salt buffer, and made 50% in glycerol. Final nuclear matrix was stored at -20 'C and generally contained 2-5% of the total nuclear DNA. The other high salt matrix aliquot was sonicated with two 20-s pulses at the lowest setting possible (Branson Sonifier, model 145W). The sonicate was centrifuged (11,000 rpm for 10 min) in an Eppendorf microcentrifuge. The high salt sonicated supernatant (matrix extract) was carefully re- moved, made 50% in glycerol, and stored at -20 "C. The high salt sonicated pellet (matrix pellet) was washed twice with low salt buffer (3,000 rpm for 15 min), resuspended in low salt buffer to the original aliquot volume, made 50% in glycerol, and stored at -20 "C.

Nuclear matrix extracts (600 pl) in 2 M NaCl, 10 mM Tris, pH 7.4, 0.2 mM MgC4 were loaded onto the top of a (9.2-ml) 5-20% linear sucrose gradient containing 2 M NaCl, 10 mM Tris, pH 7.4, 0.2 mM MgCl,. Gradients were centrifuged at 35,000 rpm for 2 or 16 h in an SW-41 rotor (Beckman Instruments). Following centrifugation, gradients were fractionated from the top using an auto-densi-flow gradient fractionator (Buchler Instrument) and Minipulse I1 peristaltic pump (Gilson) into 15 equal fractions (22 drops/fraction). Fractions were immediately immersed in liquid Nz and stored at -70°C until assayed. Samples from the high salt extract were loaded, centrifuged, fractionated, and stored as described above. Sedimentation of the marker proteins bovine serum albumin (4.4 S), catalase (11.2 S), and thyroglobulin (19.2 S) indicated that the 16-h gradients were approximately isokinetic. For simplicity these marker protein values are depicted as 4, 11, and 20 S on the scales above the approximate sucrose gradient profiles. Sedimentation of 70 S Escherichia coli ribosomes and 30 and 50 S ribosomal subunits also indicated approximately isokinetic migration for at least the top half of the 2-h gradients.

Nuclear matrices before and after sonication were prepared for thin-section electron microscopy by standard methods (Smith et al., 1984) and examined with a Hitachi H-500 electron microscope oper- ating at 75 kV.

DNA Polymerase a-Total DNA polymerase a activity was assayed (Smith and Berezney, 1982) in a 50-p1 reaction mix containing the appropriate nuclear fraction, 50 mM Tris, pH 7.2,600 pg/ml DNase- free bovine serum albumin, 15% glycerol, 2 mM dithiothreitol, 2 mM EGTA,' 8 mM MgC12, 15 mM KC1, or 20 mM NaCI, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM ATP, 80 p~ dATP, dCTP, and dGTP, 40 p~ t3HJTTP (2.4 Ci/mmol, ICN), and 400 pg/ml activated calf thymus DNA. Assays were corrected for N-ethylmaleimide-resistant

after preincubation of sample with 10 mM N-ethylmaleimide at 0 "C incorporation (DNA polymerase p) measured in a parallel reaction

for 30 min. In some experiments endogenous DNA synthesis was measured by leaving out the exogenous DNA template. Reactions were run in triplicate at 37 "C for 30 min and terminated by addition of ice-cold trichloroacetic acid (5%) on ice. Acid-precipitable counts were trapped on No. 30 glass fiber filters (Schleicher and Schuell) and counted in Liquiscint (National Diagnostics) with a Delta 300 liquid scintillation system (Tracor Analytic). The total recovery of DNA polymerase (Y activity measured in the gradient fractions was typically 75-90% of the total activity applied to the gradients.

DNA Primase-DNA primase reactions were carried out as described by Conaway and Lehman (1982) with some modifications. The reaction mixture in a total volume of 50 pl contained the appropriate nuclear sample with 50 mM Tris, pH 7.4, 2 mM MgClz, 1 mM dithiothreitol, 100 pg/ml DNase-free bovine serum albumin, 1 unit of E. coli DNA polymerase I, 500 p~ ATP, and 40 p M [3H]dATP (3.4 Ci/mmol, E N ) . All reactions were carried out in the presence and absence of 1 pg of unprimed poly(dT) template. The endogenous incorporation (minus template reactions) were subtracted as back- graund from reactions containing template to give net primase activity on the exogenous template. The incorporation into DNA demon-

] The abbreviation used is: EGTA, [ethylenebis(oxyethyleneni- tri1o)ltetraacetic acid.

REGENERATING RAT LIVER

I I I I

ISOLATED NUCLEI

(45 MINUTES, 3 7 T )

ENDOGENOUSLY DGESTED NUCLEI

/------I HIGH SALT EXTRACTION (ZM NaCI)

NUCLEAR MATRIX l20-30% 01 alpha polymerase (70-80% of alpha polymerase

and primase) and primase)

HIGH SALT EXTRACT

I

/\ SONICATION (2M NaCl)

(-60% 01 alpha polymerase (-40% of alpha polymerase MATRIX EXTRACT MATRIX PELLET

and prlmase) and primase)

FIG. 1. Scheme for isolation and fractionation of nuclear matrix from regenerating rat liver. The major steps for nuclear matrix isolation and preparation of the matrix extract are outlined (see "Materials and Methods" for more details). The average recovery of DNA polymerase (Y and primase activities is presented in paren- theses below the approximate fraction.

strated an absolute dependence on ATP. A more detailed study of this DNA primase activity has been performed and will be reported elsewhere?

RESULTS

Solubilization of Nuclear Matrix-bound DNA Polymerase a and Primwe-Fig. 1 briefly outlines the procedures for nuclear matrix isolation from regenerating rat liver and subsequent fractionation into soluble matrix extract and residual matrix pellet. The standard "4-step" procedure for rat liver nuclear matrix isolation involving nuclease digestion and extractions with low salt, high salt, and Triton X-100 (Ber- ezney, 1979; Berezney and Buchholtz, 1981b; Smith and Berezney, 1982) was shortened in this study to include only endogenous nuclease digestion of isolated nuclei followed by high salt extraction (Fig. 1 and see "Materials and Methods"). This enabled very rapid preparation of the nuclear matrix and matrix extracts and avoided possible perturbation effects of detergent treatment. In support of this abbreviated method, the recoveries of DNA polymerase a and primase activities, the degree of solubilization of a-polymerase, and the sedimentation properties of the a-polymerase-primase complexes in the matrix extract were all virtually identical to results obtained with standard detergent-treated matrices (data not shown).

The recoveries of total nuclear protein and DNA in these matrix preparations were generally 12-15 and 2-5%, respectively. Most of the nuclear DNA (>go%) and protein (>75%) were recovered in the high salt extract. Similar to previously reported results (Smith and Berezney, 1982,1983), the recovery of total nuclear DNA polymerase a endogenous and exogenous template activities was generally 20-30% in nuclear matrix prepared from regenerating liver during maximal in vivo DNA replication (22-h posthepatectomy). A similar recovery was found for DNA primase activity. An average of 60.8 f 9.1% (S.E.; n = 6) of total nuclear matrix DNA polymerase a endogenous template activity, 62.3 -+ 13.8% (S.E.; n = 6 ) of exogenous template activity, and 64.7 f 9.6% (S.E.; n = 4) of DNA primase activity were released into the matrix extract following the brief sonication step (see Fig. 1).

In contrast to the release of the majority of DNA polymerase a and primase into the matrix extract, only a small percentage (520%) of total matrix protein was extracted. Monitoring of the matrix pellets following sonication revealed

Tubo, R. A., and Berezney, R. (1987) J. Bid. Chem. 262, in press.

Nuclear Matrix Polymerase-Primase Megacomplexes 5859

no visible damage or modification of the typical nuclear matrix structures (Fig. 2). We conclude that the sonication procedure results in preferential release of DNA polymerase a and primase with no obvious effect on the overall structural integrity of the nuclear matrix.

a-Polymerase-Prime Complexes in Matrix Extracts Pre- pared during Maximal in Vivo Replication-Fig. 3 shows the results of sucrose gradient centrifugation of the matrix extracts prepared during maximal in vivo replication in regenerating rat liver (22-h posthepatectomy). Gradients were run for 16 h to resolve components 520 S (Fig. 3A) and for 2 h to resolve components 5200 S (Fig. 3B). In this particular experiment the major peaks of DNA polymerase a activity sedimented at approximately 10,90, and 150 S. DNA primase co-sedimented with each of the a-polymerase peaks. In addition a small amount of primase activity sedimented ahead of the 10 S peak at about 6 S. This is similar to the S value reported for purified free primase from mammalian cells (Tseng and Ahlem, 1983). While the results shown in Fig. 3 are typical for the matrix extract, a small amount of DNA polymerase a and primase activities often sedimented at 17 S as well. The mean sedimentation values k S.E. of the a- polymerase-primase complexes from four different preparations of matrix extract were 10.4 f 0.8 S, 16.9 rt 0.8 S; 95 f 4.0 S, and 149 k 5.9 S. For simplicity we have designated these as the 10, 17,100, and 150 S complexes (Table I). The

10, 100, and 150 S forms were always detected as major components. The 17 S complex was generally found in relatively small amounts or not detected (see Fig. 3A). In addition, other larger forms at about 45, 65, or 75 S were occasionally detected as minor or major components (see e.g. Fig. 5).

a-Polymerase-Prime Complexes in the High Salt Extract and in Pre-replicative Nuclear Matrix Extracts-The high salt extract contained approximately 70-80% of total nuclear DNA polymerase a, with 20-30% remaining bound to the nuclear matrix (Fig. 1). It was, therefore, of interest to determine the size of DNA polymerase a complexes in this fraction, since it represents the major portion of DNA polymerase activity in the isolated cell nucleus. Fig. 4A shows that greater than 80% of the polymerase was of the 10 S form, with approximately 20% at 17 S. There were no high salt-extract- able DNA polymerase a complexes > 20 S (Fig. 4, A and C ) . While significant amounts of primase activity cosedimented with the 10 and 17 S polymerase peaks, free primase activity at about 6.5 S was the most prominent component (Fig. 4A). These results, along with previous findings that a-polymerase-primase complexes purified from the cytosol or salt extracts of whole cells generally sediment in the range of 7-10 S (Yagura et al., 1983; Chang et al., 1984; Wahl et al., 1984), suggest that the large 100 and 150 S megacomplexes are unique to the nuclear matrix fraction.

The proposed role of the nuclear matrix as the intranuclear

FIG. 2. Electron microscopic thin sections of the nuclear matrix fraction from 22-h regenerating rat liver before and after the sonication step in matrix extract preparation. A, before sonication; B, after sonication. Typical nuclear matrix structures are observed with no detectable differences following the sonication step (see Fig. 1 and “Materials and Methods” for more details). Burs represent 5 pm.


Replicotive Motrix Extract

4s F”T 100

g 75 > 5 50 a

a -J 25 E a x 0 I LL

O c I 30F?-Ys

50

25

OOL JOP FRACTION NUMBER BOTTOM

FIG. 3. Separation of a-polymerase-primase complexes in 22-h regenerating rat liver nuclear matrix extracts. Nuclear matrix extracts prepared during maximal in vivo replication (22-h posthepatectomy) were centrifuged on linear 5-20% sucrose gradients containing 2 M NaCl at 35,000 rpm for either 16 h (A) or 2 h (B) . Gradients were fractionated from the top and assayed for DNA polymerase a activity (0) with activated calf thymus DNA as template and DNA primase activity (0) with single-stranded poly(dT) as template (see “Materials and Methods”). Maximum peaks of incorporation per gradient fraction were 7.54 and 1.50 pmol of incorporated radioactive nucleotide for A and 8.23 and 1.53 pmol for B for DNA polymerase CY and primase, respectively.

TABLE I Discrete sizes of nuclear matrix-solubilized a-polymerase-primae

Complexes Sedimentation on sucrose

gradients Designation

Mean k S.E. ( n = 4) Range

9-12 S 10.4 f 0.4 S 10 S complex 16-18 S 16.9 * 0.4 S 17 S complex 85-110 S 95 * 2.0 s 100 S complex

140-155 S 149 f 1.9 S 150 S complex

site of eucaryotic DNA replication (see the Introduction) has prompted us to determine whether the 100 and 150 S megacomplexes are related to the replicative state of the cell. We previously demonstrated that the association of DNA polymerase a with the nuclear matrix is replicative dependent and is initiated pre-replicatively in regenerating liver by 12 h after partial hepatectomy (Smith and Berezney, 1983). We, therefore, prepared nuclear matrix extracts from regenerating liver at a time period (14-h posthepatectomy) just preceding the onset of in vivo replication which was initiated 16-h posthepatectomy (data not shown; see Berezney and Buchholtz, 1981b; Smith and Berezney, 1982).

The results of this experiment are shown in Fig. 4, B and D. Virtually all of the pre-replicative DNA polymerase a solubilized from matrix sedimented a t 10 S (80%) with a smaller amount at 17 S (20%). The very trace levels of polymerase a detected at higher sedimentation values (Fig.

A. High Salt Extract C. High Salt Extract

25 a

Pre-replicative Matrix I Extract

n 4s 30S50S 705

TOP 15 0 5 io 15

BOTTOM BOTTOM TOP

FRACTION NUMBER FIG. 4. Separation of a-polymerase-primase complexes in

the high salt and pre-replicative matrix extracts. High salt extracts of 22-h regenerating rat liver nuclei (A and C ) and nuclear matrix extracts prepared from pre-replicative regenerating rat liver at 14 h ( B and D ) were centrifuged on linear 5-20% sucrose gradients containing 2 M NaCl at 35,000 rpm for 16 (A and B ) or 2 h (C and D ) . Gradients were fractionated from the top and assayed for DNA polymerase a activity (0) with calf thymus-activated DNA and DNA primase activity (0) with single-stranded poly(dT) (see “Materials and Methods”). Maximal peaks of incorporation per gradient fraction for A were 10.08 and 2.43 pmol of incorporated radioactive nucleotide for polymerase and primase, respectively, 2.02 and 0.28 pmol, respectively, for B, 14.2 and 7.4 pmol, respectively, for C , and 2.64 and 0.78 pmol, respectively, for D.

40) were not significant or reproducible. DNA primase cosedimented with the 10 and 17 S peaks along with a distinct free primase peak a t approximately 6 S (Fig. 4B). The virtual absence of a-polymerase-primase complexes greater than 20 S in the pre-replicative state (Fig. 4, B and D ) as compared to the replicative state (Fig. 3) is particularly striking. These results are consistent with the possibility that the 10 and 17 S polymerase-primase complexes begin to associate pre-replicatively with the nuclear matrix as a first step toward the assembly of much larger megacomplexes which function during active DNA replication.

Effect of NaCl Concentrations on Matrix-released a-Polym- erase-Primase Complexes-Since the matrix extract is prepared in 2 M NaCl and separated on sucrose gradients containing 2 M NaCl (see “Materials and Methods”), it was essential to determine what effect this salt concentration had on the size of the released a-polymerase-primase complexes. More specifically, we asked whether the 100 and 150 S megacomplexes were the result of massive aggregation induced by the high salt conditions. This seemed unlikely since the matrix-released complexes from pre-replicative regenerating rat liver were completely devoid of the 100 and 150 S megacomplexes. Moreover, high salt solutions are commonly used to prevent aggregation of DNA polymerase a during purification (Matsukage et al., 1976; Fichot et al., 1979; Chang et al., 1984). We could not, however, rule out the possibility that certain components unique to the complexes or associated matrix during active DNA replication are susceptible to aggregation or induce this hypothetical salt precipitation.

To test this possibility we prepared matrix extracts from

Nuclear Matrix Polymerase-Primae Megacomplexes 586 1

22-h regenerating rat liver by sonication in the absence of NaCl, with 0.6 M NaC1, and with the typical 2.0 M NaC1. The amount of total matrix-bound DNA polymerase a activity which was released into the extract in this experiment in- creased from 55% with no salt to 80% with 0.6 M NaCl and 95% with 2.0 M NaCl. The results of sucrose gradient separation of these matrix extracts are shown in Fig. 5. In all cases, complexes containing DNA polymerase a were detected near the top of the sucrose gradient (<20 S ) as well as large complexes at approximately 65, 90, and 150 S. The apparent decreasing amount of 65 S complex with increasing salt concentration does not appear to be significant since the detec- tion of this component is extremely variable (e.g. a repeat experiment showed no 65 S under any of the salt conditions).

In conclusion, the virtually identical DNA polymerase a sedimentation profiles independent of the salt concentration suggest that the 100 and 150 S megacomplexes are not a result of salt-induced aggregation. High salt concentrations, however, enabled maximal solubilization of polymerase-primase complexes.

Time-dependent Dissociation of a-Polymerase-Primase Me- gucomplexes-In an attempt to determine the relationship of the various DNA polymerase a-primase complexes to each other, that is whether smaller forms interact to create larger forms, the following experiment was performed. Nuclear matrices from 22-h regenerating rat livers were sonicated in the presence of 2.0 M NaC1. The solubilized extract was then either loaded immediately onto a sucrose gradient or incu- bated for up to 16 h on ice before sedimentation. Fig. 6A shows that, when matrix extract was loaded immediately to the gradient, approximately 75% of the released a-polymerase was detected in megacomplexes with the remaining activity divided between the 10 and 17 S forms. Following 6 h of preincubation on ice, all the DNA polymerase a activity sedimented at 10 and 17 S. Finally, after 16 h of preincubation on ice (Fig. 6C) only the 10 S form of DNA polymerase a remained.

Over 95% of the total DNA polymerase activity of the gradient run immediately after preparation of the matrix extract was recovered in the gradient run after 6 h of prein-

i

TOP 5 10 15 FRACTION NUMBER BOT TOM

FIG. 5. Effect of NaCl concentration on large forms of DNA polymerase a released from matrix. Nuclear matrix extract was prepared from 22-h regenerating rat liver using low salt (O), 0.6 M NaCl (A), and 2.0 M NaCl(0) for sonication and subsequent sucrose gradient analysis. 5-20% linear sucrose gradients with indicated salt were run at 35,000 rpm for 2 h. Gradients were fractionated from the top and assayed for DNA polymerase a activity with activated calf thymus DNA. Maximum peaks of incorporation per gradient fraction for low salt, 0.6, and 2.0 M NaCl were 7.96, 4.10, and 4.04 pmol of incorporation of dTMP, respectively.

1 A. 0 Hours

4s i l S 20s 100 1 " -

f 401 I

b '""t A

5 10 15 FRACTION NUMBER

FIG. 6. Dissociation of a-polymerase-primase complexes. Nuclear matrix extracts were prepared from 22-h regenerating rat liver as described under "Materials and Methods" and centrifuged on 5-20% sucrose gradients for 16 h at 35,000 rpm immediately (A) or following 6 h (€3) and 16 h (C) on ice. Gradients were fractionated from the top and assayed for DNA polymerase a activity with activated calf thymus DNA. Maximum peaks of incorporation for gradients loaded immediately or following 6 or 16 h of incubation on ice were 9.11, 3.71, and 5.54 pmol of dTMP incorporated per gradient fraction.

cubation. We conclude, therefore, that the absence of the 100 and 150 S megacomplexes after 6 h is not a result of differential inactivation of the 100 and 150 S activities but is rather a consequence of a time-dependent in uitro conversion of the megacomplexes to the 10 and 17 S forms. Only 47% of total DNA polymerase a activity is recovered following complete conversion to 10 S after 16 h of preincubation (Fig. 6C). While the 17 S form may be subsequently converted to the 10 S form, we cannot presently rule out differential inactivation of 17 S polymerase activity as an explanation.

Since it required a minimum of 20-30 min from the time of sonication to actually begin sucrose gradient centrifugation, the relatively small amounts of 10 and 17 S complexes detected in the gradients run immediately after matrix extract preparation (Fig. 6A) may also be a result of dissociation from megacomplexes. This suggests that virtually all of the matrix- bound a-polymerase-primase is organized into 100 and 150 S megacomplexes during active DNA replication.

Endogenous DNA Synthesis on Matrix-released a-Polym- erase-Primase Complexes-As previously stated, an approximately equivalent percentage (-60%) of total matrix-bound DNA polymerase a endogenous and exogenous template activities is released into the matrix extract. It was, therefore, of interest to determine whether one or more of the a- polymerase-primase complexes resolved on the sucrose gra-


dients also displayed endogenous template activity. Fig. 7 shows typical gradient profiles for DNA polymerase a measured in the absence of added DNA template. Similar results were obtained in three repeat experiments. We conclude that all the major forms of matrix-released a-polymerase-primase complexes (10,17,100, and 150 S) are also capable of synthesis using DNA associated with the complexes. Further studies on the nature of this putative DNA associated with the megacomplexes are in progress.

DISCUSSION

As a step toward the molecular elucidation of the putative replicational machinery associated with the nuclear matrix (Smith and Berezney, 1980, 1982, 1983; Jones and Su, 1982; Berezney, 1984, 1985; Nishizawa et al., 1984; Foster and Collins, 1985; Wood and Collins, 1986) we have developed a method for solubilization of matrix-bound replicational complexes containing DNA polymerase LY and primase activities. Following gentle sonication of nuclear matrix isolated from regenerating rat liver during maximal in vivo DNA synthesis (22-h posthepatectomy), the majority of DNA polymerase a and primase activities were released into a matrix extract (Fig. 1). The relatively mild conditions used for release of these replicative components is indicated by the low percentage of total nuclear matrix protein released (520%), the virtually complete recovery of total enzyme activities, and the absence of any detectable changes in the overall intactness and ultrastructure of the nuclear matrices following the sonication step (Fig. 2). Sedimentation analysis of the matrix extract revealed three major a-polymerase-primase complexes at 10, 100, and 150 S (Fig. 3, A and B) . Polymerase-primase complexes sedimenting at about 17,45, 65, and 75 S were also occasionally detected but usually as minor components. Sim- ilar to the matrix-bound activity, DNA polymerase LY in the

I

FRACTION NUMBER FIG. 7. Analysis of DNA polymerase (II endogenous template

activity in nuclear matrix-released polymerase-primase complexes. Nuclear matrix extracts from 22-h regenerating rat liver were centrifuged on linear 5-20% sucrose gradients containing 2 M NaCl at 35,000 rpm for either 16 h (A) or 2 h (B) . Gradients were fractionated from the top and assayed for DNA polymerase a activity in the absence of added DNA template. Maximum peaks of incorporation were 3.79 and 1.62 pmol of incorporation of dTMP per gradient fraction for A and B, respectively.

released complexes was capable of DNA synthesis in the absence of added DNA (endogenous DNA synthesis, see Fig. 7) as well as effectively accepting exogenous DNA templates.

The large 100 and 150 S megacomplexes were unique to the subset of total nuclear DNA polymerase a and primase associated with the nuclear matrix fraction (20-30%, Fig. 1) and were absolutely replicative dependent. The bulk of the total nuclear DNA polymerase a and primase activities (70-80%) were extracted by high salt (Fig. 1) and sedimented predominantly as 10 S complexes with smaller amounts at 17 S (Fig. 4A). No larger complexes were detected (Fig. 4, A and C). The replicative dependence of the 100 and 150 S megacomplexes was demonstrated by examining nuclear matrix extracts from pre-replicative regenerating rat liver. In matrix extracts prepared from 14-h regenerating liver (DNA synthesis begins at 16 h) only 10 and 17 S a-polymerase-primase complexes were detected (Fig. 4, B and D).

Although we cannot presently rule out the possibility that the released megacomplexes do not reflect the organization of DNA polymerase a and primase in the nuclear matrix-bound state, several of our findings argue against artifactual forma- tion of these large complexes following release into the matrix extract. For example, the virtually complete absence of 100 and 150 S forms in matrix extracts from pre-replicative liver (Fig. 4, B and D ) indicates that the megacomplexes reflect some aspect of the replicative state of the cell and are not spuriously created due to the conditions of sonication or extractions. Further supporting this conclusion are the findings of similar sized complexes independent of the salt conditions used during the sonication (Fig. 5). Finally, we find that the megacomplexes are actually unstable in the matrix extract and rapidly dissociate into 10 and 17 S complexes (Fig. 6). This behavior is the opposite of what would be expected for an aggregation phenomenon. Moreover, sucrose or glycerol gradients are typically run in the presence of high salt as a preventative measure against aggregation of DNA polymerase a complexes (Matsukage et al., 1976; Fichot et al., 1979; Chang et al., 1984).

It is known that each long molecule of eucaryotic chromosomal DNA is divided into hundreds to thousands of independent subunits of replication termed replicons (Huberman and Riggs, 1968; Hand, 1978). Replication proceeds bidirectionally in each replicon subunit. Individual replicons are further organized into families or clusters of tandemly repeated subunits which replicate as a unit at particular times in S phase (Lau and Arrighi, 1981). Up to 100 or more replicons may be organized into each replicon cluster with an estimated average size of approximately 25 (Hori and Lark, 1974; Hand and Tamm, 1974; Hand, 1975; Painter and Young, 1976; Hand, 1978; Lau and Arrighi, 1981). The numerous reports that specific DNA sequences are duplicated at very precise times within the S phase of eucaryotic cells (Goldman et al., 1984; Pierron et al., 1984; Jalouzot et al., 1985; Gilbert, 1986) further support the conclusion that replicon cluster synthesis is temporally and spatially regulated along the chromosomal DNA (Hand, 1978).

While the existence of replicon subunits and their bidirectional replication and organization into functional replicon clusters is well documented, the mechanistic and molecular basis for these fundamental properties of eucaryotic DNA replication remains elusive. In this regard, numerous studies of replicating DNA and DNA polymerase a associated with nuclear matrix systems (Berezney and Coffey, 1975; Wanka et al., 1977; Dijkwel et al., 1979; McCready et al., 1980; Pardoll et al., 1980; Smith and Berezney, 1980,1982, 1983; Vogelstein et al., 1980; Berezney and Buchholtz, 1981b) have led to


models which incorporate all of these macroscopic properties of replication (Fig. 8). It is envisioned that nuclear matrix- bound replicating DNA loops correspond to individual replicon subunits and that replication proceeds bidirectionally by the reeling of DNA through matrix-bound replisomes (Fig. 8). Topographical organization of the replicating DNA loops and the associated replisomes into functional clusters or ciuster- somes may then provide the basis for replicon clustering. For the sake of simplicity certain details of these models such as the very important question of replicon origin sites (Aelen et al., 1983; Van der Valden et al., 1984; Carri et al., 1986; Dijkwel et al., 1986) are not presented in Fig. 8.

The results of this study are consistent with the possibility that the assembly of the putative matrix-bound replisome cluster or clustersome illustrated in Fig. 8 is initated pre- replicatively with the association of 10 and possibly 17 S a- polymerase-primase complexes (Fig. 9). In this regard we previously suggested that the nuclear matrix replisomes are

A. REPLICON CLUSTER ON LINEAR DNA B\uBBLE

REPLICONS

B. REPLICON CLUSTER ON DNA LOOPS

NUCLEAR MATRIX

REPLISOMES (Clustersome)

FIG. 8. The clustersome model of nuclear matrix-associated DNA replication. A, replicon cluster on linear DNA. Bidirectional replication along three tandomly arranged replicons in a hypothetical replicon cluster is illustrated along a linear DNA molecule. In ac- tuality the number of replicons in a functional cluster may average 25 or more (Hand, 1978). the arrows show the directions of the growing replicational bubbles. Unduplicated DNA is shown in white; duplicated DNA (replicational bubbles) in block. 8, replicon cluster on nuclear matrix-attached DNA loops. The growing evidence that DNA replication occurs as a series of DNA loops attached to the nuclear matrix has led to a general model of eucaryotic DNA replication which attempts to integrate the known phenomena of bidirectional replicon synthesis in regulated clusters with the topographical organization of DNA loops on the putative nuclear matrix-bound replisomes. This particular version is a modification of the model presented by DeDuve (1984) and emphasizes the clustering of the individual replisomes for the tandomly arranged DNA loops (replicons) into a higher order replicational apparatus termed the clustersome. The replication of the individual DNA loops of the replicon cluster is postulated to be regulated by the underlying clustersome apparatus. The arrows show the direction of movement of the DNA being reeled through the matrix-bound replisomes to form the bidirectional growing replicational bubbles. Unduplicated DNA is shown in white; duplicated DNA (replicational bubbles) in black.

- ELL lCLE -

Go

-ATE

G1

-

S

I DNA LOOP MODELS OF

REPLICON CLUSTER FUNCTION

NO RELEASE

MATRIX

FUNCTIONAL CLUSTERSOME 1 RELEASE OF UNITS WITH DNA LOOPS io0 + 150s

COMPLEXES

0 4 CONVERSION TO 10s COMPLEXES ....e

...e. Model RELEASE OF

COMPCEXES t o 0 + 150s

@ 4

CONVERSION TO 10s COMPLEXES

i. AND PSSOCIATED FACTORS s<;;

I ..." FIG. 9. Properties of nuclear matrix-released a-polymer-

ase-primase complexes and their possible role in DNA replication. This scheme summarizes the properties of the matrix-released a-polymerase-primase complexes found in this study and attempts to integrate these findings with appropriate DNA loop-nuclear matrix models of replicon cluster function. Go represents normal rat liver; late GI represents pre-replicative regenerating rat liver (14-h posthepatectomy); and S represents replicating regenerating rat liver (22-h posthepatectomy). The nuclear matrix of normal liver is virtually devoid of DNA polymerase a and primase activity (Smith and Berezney, 1980, 1982; R. Tub0 and R. Berezney, manuscript in preparation). Therefore, no a-polymerase-primase activity is detected in the matrix extract of normal rat liver (R. Tub0 and R. Berezney, unpublished experiments), and the nuclear matrix attachment sites for the DNA loops are devoid of these replicative activities (hatched black circles). By late GI there is significant assembly of a-polymerase and primase on the nuclear matrix which are released as predominantly 10 S complexes and depicted on the DNA loop attachment sites by the solid black circles. During active replication predominantly 100 and 150 S megacomplexes are released and the DNA loops are presumed to be organized into functional replicon clusters. Two possible interpretations of the megacomplexes in relationship to this clustersome model (see Fig. 8) are presented. In model A, assembly of a replicon cluster is mediated by the corresponding clustering of individual 10 S and/or 17 S complexes into a clustersome (see Fig. 8 for more details of multiple replicon synthesis along the clustersome). Supporting this model is the complete in vitro dissociation of the 100 and 150 S megacomplexes into 10 and 17 S complexes. Moreover, the number of 10 S complexes which might compose a megacomplex (10- 30) is in the same range as the estimated average number of replicons in a replicon cluster (Painter and Young, 1976). In model B, the pre- replicative 10 and/or 17 S complexes represent the core structures (black centers of stippled white circles) of much larger individual replisomes (the 100 and 150 S megacomplexes) which are assembled during active DNA replication. The putative clustersome then cor- responds to many of these rnegareplisomes.


dynamically assembled during replication and that this assembly process is initiated pre-replicatively (Smith and Ber- ezney, 1980,1982, 1983).

It is further suggested that during active replication, the a- polymerase and primase are recruited into large 100 and 150 S megacomplexes. We envision two possible relationships of the megacomplexes to the putative functional clustersome illustrated in Fig. 8. In model A (Fig. 9), the megacomplexes correspond to the functional clusters of individual replisomes or clustersomes. Each clustersome consists of a number of repeating 10 and/or 17 S complexes which is determined by the number of replicons in the cluster. Once released from its nuclear matrix attachment, the clustersome is rapidly dissociated into the individual 10 and 17 S a-polymerase-primase complexes.

In Model B (Fig. 9), it is suggested that each 100 and 150 S megacomplex might correspond to a single matrix-bound replisome (see also Fig. 8). Once released from the nuclear matrix these “megareplisomes” are rapidly dissociated into 10/17 S a-polymerase-primase complexes and other associated factors. The clustersome in this model (i.e. the cluster of replisomes) would presumably have a sedimentation coeffi- cient which is approximately a magnitude or more greater than the 100 and 150 S complexes detected in this study.

Our present results do not enable a distinction between these two models as well as the other possibilities. This will require, among other things, an estimation of the number of molecules of DNA polymerase a and primase in each megacomplex. With regard to Model A, however, it is intriguing to note that the 100 and 150 S complexes are potentially large enough to be composed of at least 10-30 smaller 10 S complexes. This is in the range of the estimated average size of replicon clusters in mammalian cells (Painter and Young, 1976). Both models also predict that the 10 and/or 17 S complex is the core component of the eucaryotic replisome. This is of significance in light of the numerous reports of approximately 7-10 S DNA polymerase-primase complexes isolated from a wide variety of eucaryotic cells (Kaguni et al., 1983; Yagura et al., 1983; Chang et al., 1984; Gronostajski et al., 1984; Plevani et al., 1984; Wahl et al., 1984).

A major feature of this topographical model of replication is the prediction that large clusters of replicons are duplicated synchronously at discrete sites or clustersomes in the cell nucleus. Consistent with this possibility, recent immunocytochemical findings indicate that DNA polymerase a is arranged in the cell nucleus in discrete granular clusters (Ya- mamoto et al., 1984; Nakamura et al., 1984). Whether or not these represent specific function “replication centers” or clustersomes in the nucleus will require further investigation. I t is encouraging to note, however, that the granular structures observed in intact cells were maintained following extractions used for obtaining nuclear matrix structure (Yamamoto et al., 1984). Moreover, while this manuscript was in preparation, Nakamura et al. (1986) used an immunocytochemical ap- proach to demonstrate that the in situ sites of DNA replication within the interphase nucleus are organized into discrete ringlike structures which they believe correspond to structural domains of active replicon clusters. We are struck by the similarities of these in situ identified “replicational ring structures” to the clustersome models depicted in Figs. 8 and 9. Our results are also consistent with earlier studies demon- strating large replicative-dependent macromolecular complexes of DNA polymerase and other replicative enzymes in mammalian cells (Reddy and Pardee, 1980; Noguchi et al., 1983; Ottinger and Hubscher, 1984; Vishwanatha et al., 1986) and yeast (Jazwinski et al., 1983; Jazwinski and Edelman,

1984). Consistent with these earlier reports, we also find other replicative enzymes associated with the matrix-released complexes (Tubo and Berezney, 1987). The studies of Reddy and Pardee (1980) and Noguchi et al., (1983) are particularly relevant since they employed a similar sonication procedure of mammalian cell nuclei and sucrose density gradient separation technique.

Unfortunately, our attempts to isolate DNA polymerase a- primase complexes by applying the sonication procedure to isolated nuclei did not lead to reproducible release of com- ~ l e x e s . ~ Problems with endogenous proteases, nucleases, or aggregation with chromatin or other nuclear substructures during sonication of intact nuclei may have occurred and need to be clarified in future experiments. Moreover, the purification of large (220 S) multienzyme replicative complexes from salt extracts of mammalian cells (50.15 M KCl; Ottinger and Hubscher, 1984; Vishwanatha et al., 1986) also suggests that these putative multienzyme replicative complexes may only be transiently associated with the nuclear matrix. Whether these soluble forms of multienzyme complexes represent early stages in the assembly or disassembly of the proposed clustersomes on the matrix is a potentially important area of research for future investigation.

Two other communications dealing with the solubilization of DNA polymerase a from isolated nuclear matrix have recently appeared (Nishizawa et al., 1984; Wood and Collins, 1986). In one study both DNA polymerase a and primase were effectively solubilized, but no information was presented on the size of the putative complexes (Wood and Collins, 1986). In the other report only 7.5 S complexes were detected (Nishizawa et al., 1984). This data, however, is not necessarily in conflict with our results since we report a very rapid and complete dissociation of the 100 and 150 S megacomplexes to 10 S complexes following release from the matrix (Fig. 6). Indeed, this rapid dissociation could potentially explain the discrepancy between the very large complexes of our study and others (Noguchi et al., 1983; Jazwinski and Edelman, 1984; Ottinger and Hubscher, 1984; Vishwanatha et al., 1986) with the 7-10 S polymerase primase complexes typically found following extensive purification protocols (Kaguni et al., 1983; Yagura et al., 1983; Chang et al., 1984; Gronostajski et al., 1984; Plevani et al., 1984; Wahl et al., 1984).

Acknowledgments-We thank Alan J. Siege1 for performing the electron microscopy, Jim Stamos for the illustrations, and Dawn Styres for typing this manuscript.

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