Abstract
Replication protein A (RPA) is the major single strand-specific DNA-binding protein in eukaryotic cells. We have investigated the distribution of RPA in nuclei of proliferating HeLa cells and found that only one-third of the detectable RPA appeared to be bound to DNA in chromatin, whereas the remainder was free in the nucleosol. This distribution did not significantly change when cells were released from a double thymidine block into the S phase of the cell cycle. Single strand-specific endonucleases failed to mobilize RPA bound to chromatin in G1 phase and S phase HeLa cells. In contrast, brief treatments with pancreatic DNase I or with micrococcal nuclease sufficed to release RPA from its chromatin-binding sites. Sucrose gradient analysis of soluble micrococcal nuclease digests showed that the released RPA sedimented free of mono- or oligonucleosomal chromatin fragments, possibly indicating that most of the detectable RPA may be associated with chromatin sites, which are more open to nuclease attack than bulk chromatin. The surprising conclusion is that the majority of the detectable RPA is, either directly or indirectly, associated with double-stranded DNA regions in chromatin from HeLa cells in G1 phase and in S phase.
Highlights
The major eukaryotic single strand-specific DNA-binding protein is known as replication protein A (RPA)1 because it was originally discovered as a factor essential for simian virus 40 DNA replication in vitro
Distribution of RPA in Nuclei—Using RPA-specific antibodies, we show by immunofluorescence that all of the detectable RPA resides in HeLa cell nuclei and that RPA was evenly distributed in pre-replicative nuclei, but partially appeared in a punctate form over a uniformly stained background in nuclei from S phase cells (Fig. 2A), as described in detail before by Murti et al [22]
A first result was that the majority of RPA is present in the soluble fraction even after gentle lysis of cells and that maximally one-third remains in the chromatin pellet
Summary
We have investigated the distribution of RPA in nuclei of proliferating HeLa cells and found that only one-third of the detectable RPA appeared to be bound to DNA in chromatin, whereas the remainder was free in the nucleosol. This distribution did not significantly change when cells were released from a double thymidine block into the S phase of the cell cycle. Immunofluorescence studies indicate that RPA may change its localization in the nucleus in a cell-cycle dependent manner This has been investigated in pseudonuclei that form in Xenopus egg extracts around added sperm chromatin or DNA. Released RPA sediments in sucrose gradients just like isolated free RPA
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