Orc6 is required for dynamic recruitment of Cdt1 during repeated Mcm2–7 loading

Abstract

The origin recognition complex (ORC) nucleates DNA replication initiation in eukaryotic cells. This six-protein complex binds replication origin DNA, recruits other initiation factors, and facilitates loading of the DNA helicase. Studying the function of individual ORC subunits during pre-RC formation has been hampered by the requirement of most subunits for DNA binding. In this study, we investigate the function of the Saccharomyces cerevisiae Orc6, the only ORC subunit not required for DNA binding. In vivo, depletion of Orc6 inhibits prereplicative complex (pre-RC) assembly and maintenance. In vitro, ORC lacking Orc6 fails to interact with Cdt1 and to load the Mcm2-7 helicase onto origin DNA. We demonstrate that two regions of Orc6 bind Cdt1 directly, and that the extreme C terminus of Orc6 (Orc6-CTD) interacts tightly with the remaining five ORC subunits. Replacing Orc6 with a fusion protein linking Cdt1 to the Orc6-CTD results in an ORC complex that loads Mcm2-7 onto DNA. Interestingly, this complex can only perform a single round of Mcm2-7 loading, suggesting that a dynamic association of Cdt1 with ORC is required for multiple rounds of Mcm2-7 loading.

Assembly of the Human Origin Recognition Complex Occurs through Independent Nuclear Localization of Its Components

“…Orc6 is required for binding ORC to DNA in flies (19) but not in yeast (20,21), frogs (6), or humans (Hs) (13,14). On the other hand, Orc6 is essential for initiation of DNA replication in yeast and flies (19,(22)(23)(24)(25) although not in frogs or humans (6,13,14). A role for Orc6 in mitosis and cytokinesis also has been reported in flies and humans (19, 26 -28), although it is not essential for these events in budding yeast (22,23).…”

“…On the other hand, Orc6 is essential for initiation of DNA replication in yeast and flies (19,(22)(23)(24)(25) although not in frogs or humans (6,13,14). A role for Orc6 in mitosis and cytokinesis also has been reported in flies and humans (19, 26 -28), although it is not essential for these events in budding yeast (22,23). In addition, various ORC subunits appear to play a role in heterochromatin assembly, ribosomal biogenesis, centrosome and kinetochore function, sister chromatin cohesion, and neural dendritic branching (5, 29 -31).…”

A spontaneous Cdt1 mutation in 129 mouse strains reveals a regulatory domain restraining replication licensing

“…Identification of the Cdt1 D 6 PEST allele. During a recent analysis of DNA replication in mouse pluripotent cells 22,23 , we characterized the expression of various replication factors.…”

“…While analysing DNA replication in mouse embryonic stem (ES) cells, we discovered a novel, spontaneous mutation of the Cdt1 gene that consists of a six-amino acid deletion within the previously uncharacterized PEST-like domain in the aminoterminal part of Cdt1 (D 6 PEST allele). Here we show that this PEST domain is important for repressing Cdt1 activity, independently of the Geminin and E3 ligase pathways.…”

“…Cdc6 and Cdt1 are two essential cofactors for pre-RC establishment, a process known as licensing 4,5 . Differently from other pre-RC components, Cdt1 does not display enzymatic activity and is believed to bridge together the ORC and MCM2/7 complexes during licensing 6,7 .…”