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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Cycle / genetics
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Cell Cycle / physiology
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Cell Cycle Proteins / metabolism
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Chromatin / metabolism
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DNA Helicases / genetics
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DNA Helicases / metabolism
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DNA Replication*
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DNA-Binding Proteins / metabolism
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G1 Phase / physiology
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Minichromosome Maintenance 1 Protein / metabolism
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Nuclear Proteins / metabolism
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Nucleocytoplasmic Transport Proteins / metabolism
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Origin Recognition Complex / genetics
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Origin Recognition Complex / metabolism
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Proteome
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Replication Origin
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Ribosomes / metabolism*
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Trans-Activators / genetics
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Trans-Activators / metabolism
Substances
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CDC6 protein, S cerevisiae
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Cell Cycle Proteins
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Chromatin
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DNA-Binding Proteins
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Minichromosome Maintenance 1 Protein
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NOC3 protein, S cerevisiae
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Nuclear Proteins
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Nucleocytoplasmic Transport Proteins
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ORC1 protein, S cerevisiae
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Origin Recognition Complex
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Proteome
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Rix1 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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TAH11 protein, S cerevisiae
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Trans-Activators
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replication initiator protein
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DNA Helicases