Selective instability of Orc1 protein accounts for the absence of functional origin recognition complexes during the M-G(1) transition in mammals

To investigate the events leading to initiation of DNA replication in mamma lian chromosomes, the time when hamster origin recognition complexes (ORCs) became functional was related to the time when Orc1, Orc2 and Mcm3 protein s became stably bound to hamster chromatin, Functional ORCs, defined as tho se able to initiate DNA replication, were absent during mitosis and early G (1) phase, and reappeared as cells progressed through G(1) phase. Immunoblo tting analysis revealed that hamster Orc1 and Orc2 proteins were present in nuclei at equivalent concentrations throughout the cell cycle, but only Or c2 was stably bound to chromatin, Orc1 and McmS were easily eluted from chr omatin during mitosis and early G(1) phase, but became stably bound during mid-G(1) phase, concomitant with the appearance of a functional pre-replica tion complex at a hamster replication origin. Since hamster Ore proteins ar e closely related to their human and mouse homologs, the unexpected behavio r of hamster Orc1 provides a novel mechanism in mammals for delaying assemb ly of prereplication complexes until mitosis is complete and a nuclear stru cture has formed.