DNA replication is a stringently regulated cellular process. In proliferating normal cells, replication initiation proteins (RIPs) are sequentially loaded on to origins of replication during the M-to-G1 transition to form the pre-replication complex (pre-RC), in a process known as replication licensing. Critically, RIPs ensure chromosomal DNA is replicated only once per cell cycle, following origin activation. RIPs (Noc3p, Ipi3p, Cdt1p, Cdc6p, Mcm2-7p) are recruited by the origin recognition complex (ORC), which binds and marks replication origins throughout the cell cycle to form the pre-RC. The detailed mechanism(s) and regulation of the pre-RC and its architecture still remain unclear.

In this study, 146 combinations of pairwise protein-protein interactions among 16 hu...[ Read more ]

DNA replication is a stringently regulated cellular process. In proliferating normal cells, replication initiation proteins (RIPs) are sequentially loaded on to origins of replication during the M-to-G1 transition to form the pre-replication complex (pre-RC), in a process known as replication licensing. Critically, RIPs ensure chromosomal DNA is replicated only once per cell cycle, following origin activation. RIPs (Noc3p, Ipi3p, Cdt1p, Cdc6p, Mcm2-7p) are recruited by the origin recognition complex (ORC), which binds and marks replication origins throughout the cell cycle to form the pre-RC. The detailed mechanism(s) and regulation of the pre-RC and its architecture still remain unclear.

In this study, 146 combinations of pairwise protein-protein interactions among 16 human pre-RC proteins were systematically and comprehensively examined for the first time. The yeast two-hybrid assay identified 72 positive interactions, of which 41 were previously unknown. hNOC3 and hIPI3 proteins had interactions with several RIPs, suggesting a role in DNA replication, similar to that found in the budding yeast homologs. The self-interactions of hORC2, -4, -5 and hNOC3 proteins were also of particular interest. Critically this study may provide a foundation for understanding the architecture(s) and function(s) of the human pre-RC.

ORC self-interaction in the budding yeast was also examined to verify the Orc1p, -2p, -5p and -6p self-interactions. Subsequent experiments further supported the ORC dimerization mechanism. According to this model, ORC components self-interact to form dimer complexes (double-hexamers), at replication origins, prior to pre-RC formation. Following replication initiation, at origins in S phase, ORC double-hexamers dissociate into single-hexamers, which bind to the duplicated origins until late M phase, prior to replication-licensing. During the M-to-G1 transition, the double-hexamers, completing a semi-conservative, cell cycle dependent ORC 'dimerization cycle'. Depletion of non-chromatin bound ORC from the nucleus during the M-to-G1 transition abrogates ORC self-interactions, pre-RC formation and DNA replication.

Three different yeast NOC3 temperature sensitive mutants were also examined in relation to DNA replication (ORC dimerization, pre-RC formation and maintenance, and cell cycle progression) and ribosome biogenesis in budding yeast to conclude that the functions of Noc3p in DNA replication and ribosome biogenesis are separable. Inactivation of Noc3p results in the failure of ORC dimerization, and MCM loading at the M-to-G1 transition and maintenance in G1 phase. Noc3p is the only non-ORC/MCM RIP that possesses self-interaction and potential dimerization. Noc3p and its self-interaction may be a prerequisite for ORC dimerization and eventual MCM double-hexamer loading.

The 'ORC dimerization' model provides a symmetric platform to load the symmetric pre-RCs and guards against origin re-licensing within the same cell cycle by marking and protecting the nascent sister replication origins until the next licensing event. To further understand the 'ORC dimerization cycle', it would be necessary to investigate the mechanisms and other factors involved in its regulation in all eukaryotes, including normal and cancerous human cells.