A hypophosphorylated form of RPA34 is a specific component of pre-replication centers

Abstract

IntroductionReplication protein A (RPA) is a stable complex of three different subunits (p70, p34 and p11) that participates in different cellular processes: DNA replication, recombination and repair (Wold, 1997). The RPA70 subunit has a high affinity for single-stranded DNA, but a DNA-binding activity that is associated with the RPA34 and RPA11 subunits (Bochkareva et al., 1998). Cell-cycle-regulated phosphorylation of RPA34 at the G1-S transition has been described (Din et al., 1990;Fang and Newport, 1993;Pan et al., 1994). However, it is not yet clear whether phosphorylation of RPA is involved in the onset of DNA replication (Pan et al., 1995;Philipova et al., 1996), as DNA replication efficiency is not affected by DNAdependent protein kinase (Brush et al., 1994;Lee and Kim, 1995; Pan et al., 1995) or Cdc2 (Henricksen andWold, 1994), two kinases involved in RPA modification.The RPA70 large subunit alone is not sufficient for DNA replication, and the RPA complex cannot be replaced by E. coli single-stranded DNA-binding protein SSB (Dornreiter et al., 1992;Walter and Newport, 2000), suggesting that the RPA34 and RPA11 subunits are necessary for the function of RPA in DNA replication. RPA participates in the synthesis and processing of Okazaki fragments during DNA replication in viruses and in yeast (Mass et al., 1998;Bae et al., 2001). In multicellular organisms, however, its participation in the preinitiation complex is currently unclear. Notably, sites of DNA synthesis are detected as replication foci that co-localize with RPA and the DNA polymerase δ processivity factor PCNA (Adachi and Laemmli, 1992;Dimitrova et al., 1999), but none of the proteins forming the pre-replication complex (e.g. ORC, cdc6, cdt1, MCMs) exhibits such clear localization. The significance of RPA foci is therefore debated.In Xenopus in vitro systems, RPA is present on chromatin before initiation of DNA replication. It first localizes at distinct foci that might be pre-replication centers, but appears to be relocalized evenly throughout the nucleus after initiation of DNA replication (Adachi and Laemmli, 1992). In mammalian cells, RPA localizes to replication foci at the onset of S phase (Brenot-Bosc et al., 1995;Murti et al., 1996), but RPA foci were not observed in early G1 (Dimitrova et al., 1999;Dimitrova and Gilbert, 2000). These observations have led to the proposal that RPA foci in Xenopus may be unrelated to initiation of DNA synthesis and may represent a nonspecific storage of RPA bound to chromatin (Dimitrova et al., 1999).We have investigated the association of the regulatory subunit RPA34 with chromatin during the cell cycle and its participation in pre-replication complexes. We show that the regulatory RPA34 subunit is rapidly dephosphorylated upon mitosis exit, and then associates with chromatin prior to the initiation of DNA replication. RPA34 is detected in its hypophosphorylated form during the whole of S phase and assembles into detergent-resistant foci. Mitosis results in phosphorylation of RPA34 and its loss of chro...

Multiple human single-stranded DNA binding proteins function in genome maintenance: structural, biochemical and functional analysis

“…However this differs from mammalian cells where no RPA foci are observed in normal G1 cells (Dimitrova et al, 1999;Dimitrova and Gilbert, 2000a;2000b), but mainly in S-phase cells (Dimitrova et al, 1999). It has been speculated that the RPA foci observed in Xenopus are unrelated to DNA replication and represent non-specific storage of RPA prior to its requirement in replication (Francon et al, 2004).…”

“…The switch from hyper to hypo-phosphorylated RPA is likely to be catalysed by the 1A/2A phosphatases, as treatment with okadaic acid inhibits the dephosphorylation of RPA34 (Francon et al, 2004). This is further supported by the inhibition of DNA replication in Xenopus egg extracts immuno-depleted of phosphatase 2A (Lin et al, 1998).…”

Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

“…RPA, replication factor C (RFC), PCNA, minichromosome maintenance proteins, and DNA polymerase ␦ have been previously shown to be required for AAV replication in vitro and in cells (54,55,58). Interestingly, RPA phosphorylation excludes it from cellular sites of replication (25,82) and may alter its DNA affinity in order to promote repair (23). We have also previously shown that Rep can bind RPA (76) although we do not know how this interaction may be affected by phosphorylation.…”

Bocavirus Infection Induces a DNA Damage Response That Facilitates Viral DNA Replication and Mediates Cell Death

“…However, in our study, we found that inhibition of RPA32 phosphorylation by silencing ATR did not affect MVC DNA replication. In fact, it has been reported that RPA32 phosphorylation appears to occur outside the cellular replication sites (33,84). CGK733 inhibits both ATM and ATR activation (1,10,25,35,89,92) and was the most effective inhibitor of DDR-mediated cell death and cell cycle arrest in MVC-infected cells.…”