CHANGES IN THE SUBCELLULAR-LOCALIZATION OF REPLICATION INITIATION PROTEINS AND CELL-CYCLE PROTEINS DURING G1-PHASE TO S-PHASE TRANSITION INMAMMALIAN-CELLS

DNA replication in eukaryotic cells is restricted to the S-phase of th e cell cycle. In a cell-free replication model system, using SV40 orig in-containing DNA, extracts from G1 cells are inefficient in supportin g DNA replication. We have undertaken a detailed analysis of the subce llular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and theref ore available for DNA replication. Cyclin A and cdk2 have been implica ted in regulating DNA replication, and may be responsible for activati ng components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the re plication proteins RPA (the eukaryotic single-stranded DNA binding pro tein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primar ily cytoplasmic and there is no detectable cyclin A. An apparent chang e in the distribution of these proteins occurs as the cell enters S-ph ase. Cyclin A becomes abundant and both cyclin A and cdk2 become local ized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 sub units of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a di stinct punctate nuclear pattern. The initiation of DNA replication the refore most likely occurs by modification and activation of these repl ication initiation proteins rather than by their recruitment to the nu clear compartment.