ALTERATIONS IN PUR-ALPHA LEVELS AND INTRACELLULAR-LOCALIZATION IN THECV-1 CELL-CYCLE

Levels of the single-stranded DNA-binding protein Pur alpha, previousl y 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, Pur alpha levels drop precipitously, after which they recover nearly 8-fold throughout S and G(2) to peak just after mitosis, As obs erved previously, Pur alpha binds the hypophosphorylated form of the r etinoblastoma protein, Rb, Coimmunoprecipitation of Pura: and Rb revea ls that the complex declines as cells enter S phase and does not refor m as Pur alpha levels recover in S and G(2). As Pur alpha levels recov er, the protein is localized to nuclear foci containing newly replicat ed DNA, as determined by immunoelectron microscopy using different siz ed gold beads and antibodies against Pur alpha and bromodeoxyuridine-l abeled DNA. These foci also contain cyclin A, and Pur alpha coimmunopr ecipitates with cyclin A from extracts of cells in S and G(2) phases. Pur alpha remains with these foci throughout G(2), after the bulk of D NA synthesis has ceased, Changing levels of Pur alpha may affect Pur a lpha functions at the onset of S phase and during progression to mitos is.