Stability and nuclear distribution of mammalian replication protein A heterotrimeric complex.
Replication protein A (RPA), a stable complex of three polypeptides, is the single-stranded DNA-binding protein essential for DNA replication in eukaryotic cells. Previous studies of the subcellular distribution and stability of the RPA heterotrimer during the mammalian cell cycle have produced conflicting results. Here, we present evidence that these inconsistencies can be accounted for by the presence of an extractable pool of soluble RPA within the nucleus. Indirect immunofluorescence experiments in both CHO and HeLa cells showed that all three RPA subunits associated specifically with sites of ongoing DNA synthesis, similar to the replication fork protein proliferating cell nuclear antigen. Furthermore, we found no evidence for disassembly of the chromatin-bound heterotrimeric RPA complex in vivo. Our results are consistent with a role for RPA in the initiation and elongation steps of replication, as previously defined in the viral in vitro replication systems.
Dimitrova
D S
DS
Department of Biochemistry and Molecular Biology, State University of New York Health Sciences Center, 750 East Adams Street, Syracuse, New York 13210, USA. dimitrod@hscsyr.edu
Gilbert
D M
DM
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002843:Chromatin / Q000502:physiology / Q000648:ultrastructure; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D008322:Mammals; D051716:Replication Protein A
D002843:Chromatin; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C497167:RPA1 protein, human; D051716:Replication Protein A