Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells.
DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) 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 primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.
Brénot-Bosc
F
F
Institut de Biologie Structurale, Grenoble, France.
Gupta
S
S
Margolis
R L
RL
Fotedar
R
R
D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D002474:Cell-Free System; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism / Q000648:ultrastructure; D016213:Cyclins / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D004265:DNA Helicases / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism / Q000648:ultrastructure; D004261:DNA Replication; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D004587:Electrophoresis, Agar Gel; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D007399:Interphase / Q000502:physiology*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000648:ultrastructure; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000648:ultrastructure; D018741:Replication Origin / Q000502:physiology; D051716:Replication Protein A; D016196:S Phase / Q000502:physiology; D013539:Simian virus 40 / Q000235:genetics
D018797:Cell Cycle Proteins; D016213:Cyclins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D004257:DNA Polymerase II; D004265:DNA Helicases