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 eukar yotic cells, Previous studies of the subcellular distribution and stability of the RPA heterotrimer during the mammalian cell cycle have produced conf licting results, Here, we present evidence that these inconsistencies can b e accounted for by the presence of an extractable pool of soluble RPA withi n the nucleus. Indirect immunofluorescence experiments in both CHO and HeLa cells showed that all three RPA subunits associated specifically with site s of ongoing DNA synthesis, similar to the replication fork protein prolife rating cell nuclear antigen. Furthermore, we found no evidence for disassem bly of the chromatin-bound heterotrimeric RPA complex in vivo. Our results are consistent with a role for RPA in the initiation and elongation steps o f replication, as previously defined in the viral in vitro replication syst ems. (C) 2000 Academic Press.