The initiation of DNA replication in eukaryotes requires the loading of the origin recognition complex (ORC), Cdc6, and minichromosome maintenance (MCM) proteins onto chromatin to form the preinitiation complex. In Xenopus egg extract, the proteins Orc1, Orc2, Cdc6, and Mcm4 are underphosphorylated in interphase and hyperphosphorylated in metaphase extract. We find that chromatin binding of ORC, Cdc6, and MCM proteins does not require cyclin-dependent kinase activities. High cyclin A-dependent kinase activity inhibits the binding and promotes the release of Xenopus ORC, Cdc6, and MCM from sperm chromatin, but has no effect on chromatin binding of control proteins. Cyclin A together with ORC, Cdc6 and MCM proteins is bound to sperm chromatin in DNA replicating pseudonuclei. In contrast, high cyclin E/cdk2 was not detected on chromatin, but was found soluble in the nucleoplasm. High cyclin E kinase activity allows the binding of Xenopus ORC and Cdc6, but not MCM, to sperm chromatin, even though the kinase does not phosphorylate MCM directly. We conclude that chromatin-bound cyclin A kinase controls DNA replication by protein phosphorylation and chromatin release of Cdc6 and MCM, whereas soluble cyclin E kinase prevents rereplication during the cell cycle by the inhibition of premature MCM chromatin association.

Replication protein A (RPA), the major eukaryotic single-strand specific DNA binding protein, consists of three subunits, RPA70, RPA32, and RPA14. The middle subunit, RPA32, is phosphorylated in a cell cycle-dependent manner. RPA occurs in two nuclear compartments, bound to chromatin or free in the nucleosol. We show here that the chromatin-associated fraction of RPA contains the phosphorylated forms of RPA32. Treatment of chromatin with 0.4 M NaCl releases bound RPA and causes a separation of the large and the phosphorylated middle RPA subunit. Unmodified RPA in the nucleosolic fraction remains perfectly stable under identical conditions. Phosphorylation is most likely an important determinant of RPA desintegration because dialysis from 0.4 to 0.1 NaCl causes the reformation of trimeric RPA only under dephosphorylating conditions. Biochemical studies with isolated Cyclin-dependent protein kinases showed that cyclin A/CDK1 and cyclin B/CDK1, but not cyclin E/CDK2, can phosphorylate human recombinant RPA in vitro. However, only a small fraction of in vitro phosphorylated RPA desintegrated, suggesting that phosphorylation may be one, but probably not the only, determinant affecting subunit interaction. We speculate that phosphorylation and changes in subunit interaction are required for the proposed role of RPA during the polymerase switch at replication forks. RPA1 (replication protein A) is the most abundant eukaryotic single strand-specific DNA-binding protein with copy numbers of up to 10 5 /mammalian cell nucleus (1). Mammalian RPA, like RPA in other eukaryotic organisms, is a heterotrimeric protein with subunits of apparent molecular masses of 70, 32, and 14 kDa, respectively known as RPA70, RPA32, and RPA14 (2). Within the three subunits, RPA may contain at least four potential binding sites for single-stranded DNA as recently suggested based on weak homologies to the bacterial single strand binding protein (3). Two of the sites in the central part of the RPA70 subunit are characterized by cocrystallization of the central RPA70 binding domain with an oligonucleotide ligand (4), and recent cross-linking experiments suggest that additional sites are localized in carboxyl-terminal regions of RPA70 and RPA32 (5).The high affinity for single-stranded DNA is one property of RPA, another is its ability to interact with a variety of proteins involved in DNA replication (6 -8), DNA repair (9, 10), recombination (11, 12), and, possibly, transcription (13). Thus, RPA binding to single-stranded DNA and its interaction with DNA polymerase ␣-primase and with other replication factors are required for the establishment and the propagation of replication forks (14). In recombination, RPA stimulates DNA strand exchange between Rad51-coated single-stranded DNA and the double-stranded DNA substrate (11); and in nucleotide excision repair, binding of RPA may help to stabilize the locally unwound DNA and to localize the repair endonucleases at damaged DNA sites (15). RPA32 is phosphorylated when cells transit from the G 1 phase...