Alterations in Purα levels and intracellular localization in the CV-1 cell cycle

Levels of the single-stranded DNA-binding protein Purα, previously implicated in control of both DNA replication and gene transcription, are altered during the CV-1 cell cycle. Just prior to the onset of S phase, Purα levels drop precipitously, after which they recover nearly 8-fold throughout S and G₂ to peak just after mitosis. As observed previously, Purα binds the hypophosphorylated form of the retinoblastoma protein, Rb. Coimmunoprecipitation of Purα and Rb reveals that the complex declines as cells enter S phase and does not reform as Purα levels recover in S and G₂. As Purα levels recover, the protein is localized to nuclear foci containing newly replicated DNA, as determined by immunoelectron microscopy using different sized gold beads and antibodies against Purα and bromodeoxyuridine-labeled DNA. These foci also contain cyclin A, and Purα coimmunoprecipitates with cyclin A from extracts of cells in S and G₂ phases. Purα remains with these foci throughout G₂, after the bulk of DNA synthesis has ceased. Changing levels of Purα may affect Purα functions at the onset of S phase and during progression to mitosis.