Journal of Histochemistry and Cytochemistry, Vol. 46, 1435-1442, December 1998, Copyright © 1998, The Histochemical Society, Inc.


ARTICLE

Ultrastructural Aspects of the DNA Polymerase {alpha} Distribution During the Cell Cycle

Giovanna Lattanzib, Angela Galanzia, Pietro Gobbia, Mirella Falconib, Alessandro Matteuccib, Lorenzo Breschia, Marco Vitalec, and Giovanni Mazzottia
a Istituto di Anatomia Umana Normale, Università di Bologna, Bologna, Italy
b Istituto di Citomorfologia CNR c/o Istituti Ortopedici Rizzoli, Bologna, Italy
c Dipartimento di Scienze Biologiche e Biotecnologie, Brescia, Italy

Correspondence to: Giovanni Mazzotti, Istituto di Anatomia Umana Normale, Università di Bologna, Via Irnerio 48, 40126 Bologna, Italy..


  Summary
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Materials and Methods
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Discussion
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We studied the nuclear topography of the replicating enzyme DNA polymerase {alpha} in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase {alpha} antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase {alpha} observed in G1- and S-phase cells was represented by circular structures 80–100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase {alpha} complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase {alpha}. The evidence of DNA polymerase {alpha} circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas. (J Histochem Cytochem 46:1435–1442, 1998)

Key Words: DNA polymerase {alpha}, cell cycle, DNA replication, chromatin structure, Transmission and field emission, scanning electron microscopy


  Introduction
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Introduction
Materials and Methods
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The morphological organization of the interphase nucleus reflects and influences important aspects of its function. In fact, molecular biology and biochemical evidence suggests that many cell functions, including DNA synthesis and transcription, require an ordered nuclear organization (Abney et al. 1997 ; Hickey and Malkas 1997; Spann et al. 1997 ). In previous studies we analyzed DNA fiber ultrastructure and organization (Rizzoli et al. 1994 ; Rizzi et al. 1995 ) by field emission in lens scanning electron microscopy (FEISEM) which allows ultra-high resolution detection of samples at low accelerating voltages and without any coating. Furthermore, we described by FEISEM the fine organization of isolated metaphase chromosomes after in situ hybridization and probe position was precisely detected over each DNA fiber (Rizzi et al. 1995 ). This finding, together with the detection of incorporated bromodeoxyuridine (BrdU) obtained on ultrathin sections of deresinated FLC cells (Mazzotti et al. 1998 ), allows us to define the fiber network here observed in situ in the nuclei as chromatin. We also demonstrated by transmission electron microscopy (TEM) the different labeling patterns of DNA synthesis in early, middle, and late S-phase of the cell cycle in exponentially growing cells (Mazzotti et al. 1990 ) and synchronized fibroblasts (Neri et al. 1992 ; Rizzoli et al. 1992 ) after administration of the thymidine analogue 5-BrdU. However, the spatial organization of the enzymes involved in DNA replication and their reciprocal interactions represent the other fundamental aspect for the functional understanding of the events that characterize cell progression through S-phase (Martelli et al. 1992 ; Coll et al. 1996 ). The fine localization of DNA polymerase {alpha} within the nucleus could therefore help to clarify the dynamics of DNA synthesis (Stokke et al. 1991 ), and the co-localization with newly synthesized DNA would define the morphology and the organization of the replication units, alternatively considered as fixed or transient structures by different authors (Fakan and Hancock 1974 ; Nakamura et al. 1986 ; Adachi and Laemmli 1992 ; Hozak et al. 1993 ; Hassan et al. 1994 ; Cook 1995 ).

To this aim, we analyzed at high resolution the BrdU incorporation pattern and DNA polymerase {alpha} organization and its relationship to the DNA replication sites in the cell cycle of HeLa cells. The immunogold technique enabled us to visualize the incorporated nucleotide and/or the polymerizing enzyme at the sites of their reciprocal interaction.

A similar immunocytochemical procedure was utilized to evaluate the different labeling patterns of the DNA polymerase {alpha} in cryosectioned HeLa cells by FEISEM. This technique allowed ultra-high resolution of chromatin fibers without coating the samples and gave a three-dimensional view of the organization assumed by the enzyme and of the relationship with its chromatin substrate.

The immunocytochemical reaction using an anti-DNA polymerase gold-conjugated antibody demonstrated the same recurrent structural organization of DNA polymerase {alpha}, whether detected by scanning electron microscopy or by transmission electron microscopy (TEM).


  Materials and Methods
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Materials and Methods
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Cell Culture
HeLa cells were grown as a monolayer in Dulbecco's modified minimal essential medium supplemented with 10% fetal calf serum in a 5% CO2 atmosphere. To obtain synchronized cells, samples were blocked with 5 µM aphidicolin (Sigma; St Louis, MO) for 22 hr and were treated for 30 min with 100 µM BrdU (Sigma) at different times (0, 4, 6, 8, and 10 hr) after the release. Each sample was then collected and washed in PBS.

FACS Analysis
Fluorescence-activated cell sorter (FACS) analysis was performed to confirm the synchronization of the cells (Vitale et al. 1993 ). In brief, cell samples from each step of treatment were fixed in 70% ethanol, washed in PBS, and counterstained with propidium iodide. A FACStar Plus flow cytometer (Becton Dickinson; Palo Alto, CA) equipped with an argon ion laser tuned at 488-nm wavelength, 50 mW light output was used to analyze the cell cycle.

TEM Analysis
For TEM observation cell pellets were fixed in 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 1 hr at room temperature (RT), dehydrated in increasing concentrations of acetone, and embedded in Epon (Fluka; Buchs, Switzerland). Silver–gold ultrathin sections of about 100 nm were mounted on nickel grids (200 mesh square) without Formvar. Resin-embedded samples were labeled by multiple immunogold techniques (Bensch et al. 1982 ; Bendayan and Stephens 1984 ; Wang and Larsson 1985 ; Bastholm et al. 1987 ; Rizzoli et al. 1992 ) involving etching of the ultrathin sections with 10% H2O2 for 10 min at RT. BrdU-treated samples were incubated with 5% normal goat serum (NGS) for 30 min at RT and labeled with a mouse anti-DNA polymerase {alpha} monoclonal antibody (MBL; Nagoya, Japan) specific for human cells diluted 5 ng/ml in TBS buffer (0.05 M Tris-HCl, pH 7.6, containing 0.15 M NaCl and 0.1% BSA) overnight at 4C. The secondary antibody, a goat anti-mouse IgG conjugated with 15-nm colloidal gold particles (BioCell; Cardiff, UK) diluted 1:10 in 0.02 M Tris-HCl, pH 8.2, plus 0.1% BSA, was applied for 90 min at RT.

To obtain simultaneous evidence of both markers, a similar sequential procedure was randomly performed utilizing in some grids the same side of the sections and in others the opposite ones. BrdU immunodetection was always carried out after the DNA polymerase {alpha} localization. Before BrdU labeling on the same side of the grid, the sections were treated with 2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 30 min at 4C (modified from Wang and Larsson 1985 ; Bastholm et al. 1987 ), then incubated overnight with a monoclonal anti-BrdU antibody (Becton Dickinson), followed by a 30-nm (or 5-nm as control) gold-labeled anti-mouse IgG (BioCell).

After the immunocytochemical reaction, TEM grids were stained with uranyl acetate and lead citrate and observed with a Philips CM10 (Philips Electron Optics; Eindhoven, The Netherlands) TEM at 80 kV accelerating voltage.

FEISEM Analysis
Cell pellets were fixed with 1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, cryopreserved with 2.3 M sucrose in 0.1 M phosphate buffer, pH 7.2, frozen in liquid nitrogen, and cryosectioned in a Reichert JUNG FC 4/E (Leica; Wien, Austria) cryoultramicrotome. Sections of 100–120 nm were mounted on silicon chips which were utilized as a specimen support. Samples were incubated overnight with the anti-DNA polymerase {alpha} antibody (MBL), and then reacted with a secondary antibody (anti-mouse IgG) conjugated with 15-nm colloidal gold particles (BioCell). Anti-DNA polymerase {alpha}-treated cryosections were then processed for FEISEM observation. They were postfixed in 1% osmium tetroxide in phosphate buffer 0.15 M, pH 7.2, for 30 min, dehydrated in an increasing ethanol series, and critical point-dried (critical point dryer CPD 030; Bal-Tec, Lichtenstein).

The analysis was performed on uncoated samples with an FEISEM Jeol JSM 890 (Jeol; Tokyo, Japan) at 7 kV accelerating voltage and 1 x 10-11 A probe current.

Controls
To check the amount of gold labeling and to avoid low marker efficiency due to steric hindrance of the gold particles, a 5-nm gold-linked antibody was employed to detect BrdU in the double labeled samples. In the specimens where the double labeling was performed on the same side of the grid, controls for specificity of the labeling consisted of omission of the fixative agent between the first and the second immunocytochemical procedure and omitting the primary antibody of the second reaction. The irreversible hindrance of free anti-IgG binding sites by the fixative agent was checked by glutaraldehyde treatment between the first monoclonal antibody (anti-DNA polymerase {alpha} or anti-BrdU) and the second gold-linked one and verifying the absence of relative labeling. In the case of BrdU incorporation analysis, control samples also consisted of cells not exposed to the nucleotide or not incubated with the primary antibody. For DNA polymerase {alpha} detection, controls were performed by omitting incubation with the primary antibody and also by means of unsynchronized HeLa cells. Moreover, the enzyme was tested in each experiment in a murine cell line (Friend erythroleukemia cells) to check the human specificity of the anti-DNA polymerase {alpha} antibody. Cryosections of synchronized and unsynchronized HeLa cells for the FEISEM analysis were also observed before any immunocytochemical treatment.


  Results
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DNA Polymerase {alpha} Shows Two Distinct Distribution Patterns During the Cell Cycle
Cells obtained after aphidicoline blockage were >80% in G1-phase as shown by FACS analysis. A similar percentage of synchronization was found in samples obtained at 4, 6, 8, and 10 hr after release of the block. TEM analysis of DNA polymerase {alpha} showed two different nuclear labeling patterns throughout the cell cycle. G2- and M-phase cells exclusively showed a weak, diffuse labeling (Figure 1A and Figure 1B). The FEISEM analysis provided three-dimensional images after immunocytochemical reactions. Dispersed chromatin regions appeared as a regular network of 10-nm fibers with nodal points of about 30 nm where fibers crossed and overlapped each other, thus delimiting spherical empty areas of about 100 nm in diameter. The analysis of DNA polymerase {alpha} labeling by FEISEM, also confirmed the same arrangement of the enzyme in the different phases of the cell cycle in cryosectioned samples. Gold particles appeared scattered throughout the cell nucleus in G2-phase of the cell cycle (Figure 1C). During G1- and S-phases, at the TEM level the enzyme was organized in nuclear clusters (Figure 2A). These clusters assumed a circular shape in most cases and they surrounded or covered electron-dense chromatin areas 80–100 nm in diameter (Figure 2B). Analysis of cryosectioned HeLa cells by FEISEM showed the enzyme markers clustered in a rosette-like shape in G1- and S-phase cells (Figure 2C) surrounding or covering electron-dense globular areas about 100 nm in diameter. A precise localization of single enzyme-linked gold particles over the chromatin fibers was obtained by secondary electron images (Figure 2D), whereas analysis of the same sample in the backscattered mode (Figure 2D, inset) distinguished gold particles localized in different focal planes, thus enabling us to determine that DNA polymerase {alpha}-linked gold particles were arranged at the periphery of spherical structures. On average, the number of these complete circular structures per section present in G1-phase nuclei was a maximum of two, whereas a higher number (approximately five to ten) was observed during S-phase. However, some single DNA polymerase {alpha} ring-like clusters were also detectable in sections from S-phase nuclei. A small amount of isolated DNA polymerase {alpha}-linked gold particles was also scattered all over the nucleus, whereas negative controls for DNA polymerase {alpha} were consistently unlabeled.



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Figure 1. Electron micrographs of a labeled HeLa cell. (A) G2-phase cell shows at TEM analysis a spread nuclear distribution of gold markers for DNA polymerase {alpha} (arrows); rare randomly arranged clumps of few marker particles are also evident (asterisks). Bar = 0.7 µm. (B) TEM image of a metaphase cell. During mitosis, chromosomes (asterisks) are labeled by scattered gold particles (arrows). Bar = 0.5 µm. (C) Cryosectioned G2-phase cell at FEISEM analysis. The scattered distribution of the enzyme markers in the cell nucleus is evident (arrows). Bar = 0.2 µm.



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Figure 2. Electron micrographs of HeLa cells in G1- and early S-phases. (A) TEM observation of an S-phase cell. The gold markers for DNA polymerase {alpha} are arranged in organized circular clusters (asterisks). Bar = 0.3 µm. (B) TEM high magnification of a G1 cell. The detail shows an electron-dense area (asterisk) of about 100 nm inside the circular arrangement of the enzyme markers. Bar = 50 nm. (C) FEISEM observation of a cryosectioned G1 cell. A globular cluster of gold particles is detectable (asterisk) inside the cell nucleus. A few isolated or clumped gold particles are also evident (arrows). NM, nuclear membrane. Bar = 0.3 µm. (D) Detail of a globular cluster of DNA polymerase {alpha}-linked gold particles at FEISEM. The localization of the single gold particles over the chromatin fibers is evident (arrows). The marker appears to limit a dense chromatin area of about 100 x 70 nm (asterisk), and the neighboring chromatin network appears to be composed of small (4–10 nm in diameter) and large (30–40 nm in diameter) fibers. Bar = 75 nm. (Inset) The FEISEM backscattered image of the same sample shows the gold particles' arrangement around a spherical structure. Bar = 50 nm.

DNA Polymerase {alpha} Rosette-like Structures and BrdU-positive DNA Co-localize
BrdU labeling of newly synthesized DNA showed a typical early, middle, and late S-phase distribution that was in agreement with cell cycle FACS analysis as previously described (Rizzoli et al. 1992 ). Independently of the S-phase labeling pattern, DNA polymerase {alpha} formed circular structures as described above, with which BrdU labeling was always associated. Moreover, we also found that the association between polymerase {alpha} and incorporated BrdU depended on the immunocytochemical procedure followed. In fact, when the double labeling was performed on the same side of the sections, virtually all the ring-like polymerase {alpha} structures were associated with BrdU (Figure 3A), mostly localized on the polymerase {alpha} ring. When polymerase {alpha} labeling was performed on the opposite side of the section compared to the BrdU immunodetection, only about 60% of the polymerase {alpha} rings appeared associated with BrdU labeling (Figure 3B). When both immunocytochemical approaches were performed on G1 cells, only in a few ring-like structures was BrdU co-localization evident (Figure 3C). Negative controls were consistently unlabeled.



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Figure 3. TEM micrographs of double labeled HeLa cells in the G1- and S-cycle phases. (A) Early S-phase cell labeled on the same side of the section. DNA polymerase {alpha} is detectable as clustered gold particles (15 nm) mostly assuming a circular shape (asterisks). BrdU-linked gold markers (30 nm) (arrows) are localized in association with the rosette-like clusters. NM, nuclear membrane. Bar = 150 nm. (B) Middle S-phase cell in which the double labeling was carried out on the opposite side of the section. BrdU gold markers (30 nm) (arrows) co-localize in some of the ring-like enzyme structures (15-nm gold particles) (asterisks). Bar = 150 nm. (C) G1-phase cell labeled on the same side of the section. In one ring-like enzyme cluster (asterisk) (15-nm gold markers), BrdU (30-nm gold particles) is detectable (arrowhead) and some isolated BrdU markers are also present (arrows). Bar = 0.3 µm.


  Discussion
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Materials and Methods
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DNA polymerase {alpha} shows a specific pattern of labeling throughout the cell cycle. The immunolocalization of the enzyme described here was performed at both the TEM and the FEISEM level to compare and contact two different approaches to the morphological analysis of labeling in the cell nucleus. Moreover, the simultaneous localization of DNA polymerase {alpha} and incorporated BrdU was performed at the TEM level to show sites of active DNA replication.

HeLa cells were positive for DNA polymerase {alpha} labeling throughout the cell cycle. The immunocytochemical method employed here enabled us to detect the enzyme during G1- and S-phase and even in G2 and mitotic cells. This finding is consistent with biochemical evidence showing that the enzyme is constitutively expressed throughout the cell cycle in exponentially growing cells, whereas it is absent during the G0-phase (Nakamura et al. 1984 ; Jackson and Cook 1986 ; Wang 1991 ; Lopez-Girona et al. 1995 ). Two different labeling patterns were detected in cell nuclei. A spread distribution of the enzyme was always observed, whereas DNA polymerase {alpha} was mostly organized in circular clusters during G1- and S-phase. The distribution of BrdU-linked gold particles among the different periods of the S-phase corresponded exactly to that observed in our previous studies on exponentially growing cells or synchronized fibroblasts (Mazzotti et al. 1990 ; Neri et al. 1992 ; Rizzoli et al. 1992 ) showing that different areas of the nucleus are in turn involved in DNA replication.

In a previous study (Mazzotti et al. 1998 ), utilizing cells from different cell lines treated with BrdU and by using different techniques, we showed that BrdU labeling assumes the same circular arrangement observed in this study for DNA polymerase {alpha}, strongly suggesting an ordered and repetitive organization of structures involved in DNA replication.

We have also demonstrated here that, during simultaneous labeling, BrdU co-localizes within the circular DNA polymerase {alpha} structures, thereby indicating the morphological relationship between the enzyme and its own substrate. Our observations suggest that the rosette-like labeling of both TEM and cryosectioned samples represents the active form of the enzyme. This is in agreement with previously reported data showing that DNA polymerase {alpha} assembles into replication complexes at the end of the G1-phase and persists throughout the S-phase (Nakamura et al. 1984 ; Adachi and Laemmli 1992 ), giving rise to foci of DNA replication (Kumble and Vishwanatha 1991 ; Hozak et al. 1993 ; D'Urso et al. 1995 ; Krude 1995 ). DNA polymerase {alpha} not actively involved in DNA synthesis (Wang 1991 ; D'Urso et al. 1995 ) is likely to be represented by the spread pattern distribution of the enzyme observed in the other phases of the cell cycle. This is supported by evidence that the BrdU labeling is rarely associated with diffuse DNA polymerase {alpha}-linked gold particles in S-phase cells. The BrdU labeling not associated with DNA polymerase {alpha} clusters observed in S-phase cells could represent DNA synthesized early during the 30-min administration of the precursor or nucleotides not yet assembled. The two immunocytochemical approaches for polymerase {alpha}/BrdU double labeling utilized here (same or opposite sides of the section) provided complementary information about the activity of the replication machinery. In fact, labeling on the same side of the section points out the relationship between polymerase {alpha} and its substrate at a given time point, independent of the length of BrdU administration. Under these conditions, BrdU co-localizes with virtually all the circular enzyme complexes. In contrast, when BrdU labeling is performed on the opposite side of the section, it is probable that we detect the progression product of DNA synthesis elongated through the section thickness (Nakamura et al. 1986 ; Fox et al. 1991 ; Neri et al. 1992 ). The topological superimposition of the two markers therefore occurs when DNA synthesis proceeds along a main axis perpendicular to the section surface.

The three-dimensional information obtained by FEISEM reinforced the functional interpretation of the circular arrangement of the enzyme molecules suggested by the TEM observation. The diameter of the labeled structures (80–100 nm) was comparable in TEM and FEISEM samples. Furthermore, the distribution of the enzyme in three-dimensional rosette-like structures observed by FEISEM matched the underlying chromatin structure.

The organization of chromatin as detected by FEISEM showed 30-nm and mostly 10-nm fibers. The 10-nm fibers were arranged in a three-dimensional network and crossed and overlapped each other, giving rise to nodal points about 30 nm in diameter. In previous ultrastructural studies we have described by FEISEM the fine organization of isolated metaphase chromosomes after in situ hybridization, and the DNA probe position was precisely detected over the 10-nm fibers (Rizzi et al. 1995 ). This finding, together with the detection of incorporated BrdU obtained on deresinated ultrathin sections of resin-embedded FLC cells (Mazzotti et al. 1998 ), enabled us to define the nuclear fiber network observed here as chromatin. The 10-nm fibers almost certainly represent the substrate of DNA polymerase {alpha}, as shown by the fact that the enzyme clusters are tightly joined to the foldings formed by these fibers. These data obtained on cryosectioned samples and comparable to those obtained with resin-embedded samples confirm the suitability of the use of cryosections in the study of the nuclear structure, as previously shown by other authors (Raska et al. 1989 , Raska et al. 1991 ; Woodcock and Horowitz 1995 , Woodcock and Horowitz 1997 ). The role of morphological studies in chromatin function research has been pointed out by Woodcock and Horowitz 1995 , Woodcock and Horowitz 1997 , who also noted that techniques now available, such as cryoultramicrotomy, allow high-resolution analysis of chromatin structure and strongly reduce artifacts. In fact, the possibility of observing samples without any coating at that FEISEM level enabled us to simultaneously visualize both the fibers and the gold particles at a high-resolution level.

The high resolution power of the techniques here employed enabled us to detect DNA polymerase {alpha} in the nucleus and sometimes in the nucleolus. The rosette-like clusters were also clearly detectable in the nucleolus, in a repetitive pattern almost certainly representing the replication sites of the nucleolus-associated chromatin (Wang 1991 ).

Although a well-defined spatial distribution (Nakamura et al. 1986 ; Nakayasu and Berezney 1989 ; Tomlin et al. 1995 ; Newport and Hong 1996 ) and a three-dimensional arrangement of functionally defined chromatin replication areas have already been proposed in previous reports (Woodcock and Horowitz 1995 ), a high-resolution morphological analysis of these areas was still lacking. Moreover, the nuclear areas described here could be related to the nuclear replication factories described by Cook and co-workers using a different experimental model (Hozak et al. 1993 , Hozak et al. 1994 ; Cook 1995 ), which showed similar dimensions and nuclear distribution to our rosette-like structures.

In conclusion, our study shows the presence of morphologically defined areas of DNA replication in which both the DNA polymerase {alpha} and its substrate, represented by the 10-nm chromatin fiber, are localized. The presence of the electron-dense areas within the circular labeled structures observed by TEM and by FEISEM suggests that other replicating enzymes could converge at these sites of the nucleus (Rogge and Wang 1992 ; Hozak et al. 1993 , Hozak et al. 1994 ; Cook 1995 ), and further supports the hypothesis that these circular areas include the active form of DNA polymerase {alpha}. Above all, by combining the results obtained with the two ultrastructural methods we achieved more information on the organization of these DNA replication structures. Analysis of both BrdU and DNA polymerase {alpha}, when performed only at the TEM level, suggested that the replication complexes might be spherical structures of 100 nm in diameter with the enzyme operating at the periphery. However, when the same structures were observed by FEISEM, it became evident that the DNA polymerase {alpha} arrangement structurally matched the underlying chromatin folding. On the basis of our results, we suggest that chromatin folding may represent the fundamental DNA structure that dictates functional enzyme organization. As a consequence, the rosette-like structure can be considered to represent a nuclear ultrastructural marker for cell proliferation.

The availability of a new experimental model for immunocytochemical study of the three-dimensional architecture of chromatin at high resolution would allow a better definition of the reciprocal interactions of the molecules involved in DNA synthesis to understand the organization of the DNA replication machinery.


  Acknowledgments

Supported by grants from Fondo 60% Ministero Università e Ricerca Scientifica, C.N.R. progetto finalizzato ACRO 96, and Fondi ISS 1% Ministero Sanità.

We are grateful to Marcello Maselli and Aurelio Valmori for the photographic work.

Received for publication February 9, 1998; accepted July 21, 1998.


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Summary
Introduction
Materials and Methods
Results
Discussion
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