Noc3p, a bHLH Protein, Plays an Integral Role in the Initiation of DNA Replication in Budding Yeast

Abstract

Initiation of eukaryotic DNA replication requires many proteins that interact with one another and with replicators. Using a yeast genetic screen, we have identified Noc3p (nucleolar complex-associated protein) as a novel replication-initiation protein. Noc3p interacts with MCM proteins and ORC and binds to chromatin and replicators throughout the cell cycle. It functions as a critical link between ORC and other initiation proteins to effect chromatin association of Cdc6p and MCM proteins for the establishment and maintenance of prereplication complexes. Noc3p is highly conserved in eukaryotes and is the first identified bHLH (basic helix-loop-helix) protein required for replication initiation. As Noc3p is also required for pre-rRNA processing, Noc3p is a multifunctional protein that plays essential roles in two vital cellular processes.

Characterization and Structure Determination of the Cdt1 Binding Domain of Human Minichromosome Maintenance (Mcm) 6

“…The 52 experimental RDCs (not used in the structure calculations) of the well resolved resonances of the amino acids in the secondary structural elements agreed nicely with the values back-calculated from the structure that was determined based on the NOEs and the dihedral angle constraints, with the quality factor equal to 0.32. The surface residues and hydrogen-bond network exhibited in the NMR structures were confirmed from a paramagnetic mapping experiment 5 and hydrogen exchange data obtained from solventexposed amides-HSQC experiment (45). Taken together, data from the backbone dynamics study, RDC, and amide hydrogen exchange measurements demonstrate that the first few turns of the helix ␣1 are not as tightly packed as the remaining secondary structural elements in CBD.…”

“…The physical and functional interactions between binding domains of Cdt1 and Mcm6 were also verified by co-immunoprecipitation and dominant negative assays in human cells. 5 As we observed that the Cdt1-(392-471) was unstable and unsuitable for structural study, several truncation mutations of this Cdt1 fragment fused with GB1 tag (40, 41) were constructed in an attempt to make them suitable for NMR analysis. We found that the region of Cdt1 spanning residues 410 -445 is the core MBD, which is sufficient to interact with CBD (Fig.…”

“…For pre-RC assembly, the sixsubunit origin recognition complex first binds replication origin on newly synthesized chromatin. The origin recognition complex serves as an origin marker and recruits the initiation factors Noc3p, Cdc6, and Cdt1 to origins for the chromatin loading of the heterohexameric Mcm2-7 complex (3)(4)(5). Once the Mcm complex is loaded onto chromatin and pre-RC is formed, the cell is licensed for DNA replication, awaiting additional signals for the activation of the licensed origins (6).…”

Cell Cycle Control of DNA Replication by Phosphorylation and Dephosphorylation of Replication-Initiation Proteins in Budding Yeast

“…Noc3p was isolated as a multicopy suppressor of the mcm5-1 ts mutant (Zhang et al, 2002). Noc3p mutants display classical ARS-number suppressible plasmid loss phenotype.…”

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…Noc3 protein associates with origins of replication and its elimination from the cell with an inducible degron element impaired the origin binding of Cdc6 and MCM proteins. (60) The precise biochemical function of Noc3 in DNA replication has not been established, but it also has a role in pre-rRNA processing, (61) suggesting a link between control of DNA replication and ribosome biogenesis. This link is reinforced by the recent identification of Yph1 as an ORC-interacting protein.…”