Ultrastructural aspects of the DNA polymerase distribution during the cell cycle

Abstract

We studied the nuclear topography of the replicating enzyme DNA polymerase α 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 α antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polyroerase α 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 α 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 α. The evidence of DNA polymerase α 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.