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, Noc3p, Cdc6p, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). The architecture and the detailed mechanism of regulation of pre-RC remain unknown. To study the pre-RC in a comprehensive manner, we systematically tested pairwise protein-protein interactions among the budding yeast replication-initiation proteins by using the yeast two-hybrid assay, and identified 121 pairs of interactions. Three novel interactions were identified, i.e.. the self-interactions of Noc3p and of some ORC subunits, and the interaction between Cdt1p and...[ Read more ]

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, Noc3p, Cdc6p, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). The architecture and the detailed mechanism of regulation of pre-RC remain unknown. To study the pre-RC in a comprehensive manner, we systematically tested pairwise protein-protein interactions among the budding yeast replication-initiation proteins by using the yeast two-hybrid assay, and identified 121 pairs of interactions. Three novel interactions were identified, i.e.. the self-interactions of Noc3p and of some ORC subunits, and the interaction between Cdt1p and Mcm6p. This thesis describes three follow-up studies on these novel interactions.

I investigated the biological significance of the self-interaction of ORC. ORC apparently exist as dimers, before the pre-RC formation in vivo. The dimerized ORC separates into two monomers upon the initiation of DNA replication but redimerizes at the M-to-G1 transition. Supporting this notion, preventing the dimerization of ORC by depleting non-chromatin bound Orc2p in G1 phase abolishes pre-RC formation and DNA replication in the next cell cycle. Our findings suggest a novel 'dimerization cycle' of ORC that regulates the initiation of DNA replication.

Noc3p was dissected to map the self-interacting domain. I identified a coiled-coils motif in Noc3p that is essential for the self-interaction, S phase entry and cell viability. Additional mutants of Noc3p were constructed. These Noc3p mutants showed varying degrees of defects in DNA replication and ribosome biogenesis. My results suggest that the functions of Noc3p in these two biological processes are separable.

In an effort to better understand the physiological roles of Cdt1p in pre-RC formation and the assembly mechanism of the MCM complex, I provide the first evidence that Cdt1p, through its interaction with Mcm6p via the C-terminal regions of the two proteins, is critical for the formation of the MCM complex in both the cytoplasm and nucleus. I show that disrupting 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. These findings suggest besides the role of loading the Mcm2-7 complex onto chromatin, Cdt1p promotes the assembly and maintains the integrity of the MCM complex by interacting with Mcm6p.