Several replication-initiation proteins are assembled stepwise onto replicators 
to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA 
replication. We performed a yeast functional proteomic screen and identified the 
Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical 
determinants of replication licensing and replication-initiation frequency. 
Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS 
sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is 
required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during 
the M-to-G₁ transition and for pre-RC maintenance in G₁ phase-independent of its 
role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome 
biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and 
maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin 
association and function in pre-RC formation. These results establish a new 
framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex 
provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication 
licensing.