Rentian Wu

Structural insights into the Cdt1-mediated MCM2–7 chromatin loading

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2–7 (MCM2–7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410–440)/MCM6(708–821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2–7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2–7, our results suggest that the MCM2–7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2–7 chromatin loading.

Ctf4p facilitates Mcm10p to promote DNA replication in budding yeast.

Ctf4p (chromosome transmission fidelity) has been reported to function in DNA metabolism and sister chromatid cohesion in Saccharomyces cerevisiae. In this study, a ctf4(S143F) mutant was isolated from a yeast genetic screen to identify replication-initiation proteins. The ctf4(S143F) mutant exhibits plasmid maintenance defects which can be suppressed by the addition of multiple origins to the plasmid, like other known replication-initiation mutants. We show that both ctf4(S143F) and ctf4Delta strains have defects in S phase entry and S phase progression at the restrictive temperature of 38 degrees C. Ctf4p localizes in the nucleus throughout the cell cycle but only starts to bind chromatin at the G1/S transition and then disassociates from chromatin after DNA replication. Furthermore, Ctf4p interacts with Mcm10p physically and genetically, and the chromatin association of Ctf4p depends on Mcm10p. Finally, deletion of CTF4 destabilizes Mcm10p and Pol alpha in both mcm10-1 and MCM10 cells. These data indicate that Ctf4p facilitates Mcm10p to promote the DNA replication.

Origin Recognition Complex (ORC) Mediates Histone 3 Lysine 4 Methylation through Cooperation with Spp1 in Saccharomyces cerevisiae

The heterohexameric origin recognition complex (ORC) has been implicated in many cellular activities, including DNA replication, transcriptional control, heterochromatin assembly, centromere and telomere function, and so on. Here, we report a new function for ORC in mediating histone methylation. Using the yeast two-hybrid system, we identify a physical interaction between Orc2p and Spp1p, a member of the Set1 complex, and we demonstrate the interaction between the endogenous ORC and Spp1p by co-immunoprecipitation from yeast extracts. Furthermore, we find that Orc2p physically interacts with trimethylated histone 3 lysine 4 (H3K4) on chromatin by co-immunoprecipitation. Finally, we show that the trimethylation of H3K4 is decreased in orc2-1 cells and abolished in orc2-1, spp1Δ double mutants. Our data reveal a novel facet of ORC in mediating histone methylation in collaboration with Spp1p and demonstrate a connection between ORC and chromatin structure via the Set1 complex.

Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex

Regulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity by interacting with Mcm6p.

Far3p domains involved in the interactions of Far proteins and pheromone-induced cell cycle arrest in budding yeast.

Far3p (factor arrest), a protein that interacts with Far7-11p, is required for the pheromone-mediated cell cycle arrest in G1 phase. We used a combination of computational and experimental strategies to identify the Far3p self-association, to map the Far3p domains that interact with Far3p itself and with other Far proteins, and to reveal the importance of the two coiled-coil motifs of Far3p in the integrity and function of the Far complex. We show that Far3p self-associates through its central region and its C-terminal coiled-coil domain, that the amino acid 61-100 region of Far3p interacts with Far7p, and that the Far3p N-terminal coiled-coil domain interacts with Far9p and Far10p. Mutation of the N-terminal coiled coil disrupts the interactions of Far3p with Far9p and Far10p, and mutation of the C-terminal domain weakens the Far3p self-interaction. Although the N- and C-terminal coiled-coil mutants reserve some of the interactions with itself and some other Far proteins, both mutants are defective in the pheromone-mediated G1 arrest, indicating that both coiled-coil motifs of Far3p are essential for the integrity and the function of the Far complex.

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis

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.