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

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Abstract
Replication protein A (RPA) is a three subunit single-stranded DNA-binding protein required for DNA replication. In Xenopus, RPA assembles in nuclear foci that form before DNA synthesis, but their significance in the assembly of replication initiation complexes has been questioned. Here we show that the RPA34 regulatory subunit is dephosphorylated at the exit of mitosis and binds to chromatin at detergent-resistant replication foci that co-localize with the catalytic RPA70 subunit, at both the initiation and elongation stages of DNA replication. By contrast, the RPA34 phosphorylated form present at mitosis is not chromatin bound. We further demonstrate that RPA foci assemble on chromatin before initiation of DNA replication at sites functionally defined as initiation replication sites. Association of RPA with these sites does not require nuclear membrane formation, and is sensitive to the S-CDK inhibitor p21. We also provide evidence that RPA34 is present at initiation complexes formed in the absence of MCM3, but which contain MCM4. In such conditions, replication foci can form, and short RNA-primed nascent DNAs of discrete size are synthesized. These data show that in Xenopus, the hypophosphorylated form of RPA34 is a component of the pre-initiation complex.