The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis
ABSTRACT 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.
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ABSTRACT: Normal DNA replication starts following the stepwise recruitment of replication initiators to assemble Mini-chromosome Maintenance (MCM) 2-7 protein complexes at an adequate amount of DNA replication origins. Under normal conditions, the monoubiquitination of Fanconi Anemia (FA) group D2 protein (FANCD2) occurs in each S-phase of cell cycle, which is the basal level of FANCD2 monoubiquitination. However, little is known regarding the roles of this basal level of monoubiquitinated FANCD2. Here we show that monoubiquitinated FANCD2 in each S-phase of normal cell cycle is essential for replication origins to fire at a normal rate. We found that the basal level of the monoubiquitinated FANCD2 can interact with replication origins as well as mini-chromosome maintenance protein 3 (MCM3) in an S-phase specific manner to secure an enough number of the licensed-origins to fire. Non-monoubiquitinated FANCD2 or mutant MCM3 lacking AA 477-480 responsible for interacting with FANCD2 can lead to an insufficient amount of licensed origins to fire and, thereby, enlarged intervals between the fired origins. Our results demonstrate that the monoubiquitinated FANCD2 in each S-phase of normal cell cycle is required to maintain an enough number of licensed origins to initiate the normal DNA replication. This finding is the first to provide insights into how FANCD2 functions under normal condition of cell cycle to maintain genome stability, as well as resulting implications in the strategic improvement for the fight against human cancer.Oncotarget 03/2014; · 6.63 Impact Factor