||In Saccharomyces cerevisiae, initiation of DNA replication (IDR) requires the replicators ARS (autonomously replicating sequence) elements and replication-initiation proteins. Identification and characterization of replication-initiation proteins and their regulators have significantly advanced our understanding of eukaryotic DNA replication. However, the detailed mechanism of replication initiation is still elusive, and some initiation proteins may not have been discovered. By screening a collection of td (temperature-sensitive degron) mutants (Kanemaki et al., Nature 423:720-724, 2003) with a pair of tester plasmids p1ARS and p8ARSs (Zhang et al., Cell 109:849-860, 2002), we have identified several novel IDR genes, one of which is IPI3 (Involved in Processing ITS2). Plasmid loss assays and FACS analysis indicate that Ipi3p is required for the initiation of DNA replication, but not for replication elongation or mitosis. Co-IP and co-pull down experiments show that Ipi3p interacts with ORC and MCM proteins. ChlP assays reveal that Ipi3p specifically associates with ARS sequences in G1 cells. Chromatin binding assays indicate that Ipi3p binds chromatin in a cell cycle-dependent manner. Furthermore, in vivo and in vitro pre-RC assembly assays demonstrate that Ipi3p is required for and directly involved in loading Cdc6p, Cdt1p and MCM proteins onto chromatin during M-to-G1 transition and for pre-RC maintenance in G1 cells, without affecting the levels of the pre-RC components. Importantly, purified recombined Ipi3p can rescue the defect in pre-RC assembly of the G1 extracts from ipi3-td cells combined with G2/M chromatin from ipi3-td cells or with ARS1-plasmid in the presence of protein and RNA synthesis inhibitors. On the other hand, ORC and Noc3p are required for the chromatin association of Ipi3p. Our data show that Ipi3p is an essential DNA replication-initiation protein and plays direct roles in the assembly and maintenance of pre-RC in addition to its function in ribosome biogenesis. From these and other data, the interdependence of replication- initiation proteins in the following order for pre-RC formation can be established: ORC→Noc3p→Ipi3p→Cdc6p/Cdt1p→MCM proteins.