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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..

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


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