Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

Background: Orc1 is the largest subunit of the origin recognition complex that promotes genome duplication. Results: We studied the dynamics of Orc1 during the cell division cycle. Conclusion: Orc1 binds to mitotic chromosomes, and during G(1) phase in the daughter cells it then forms spatial-temporal patterns in the nucleus. Significance: The large subunit of ORC orchestrates the earliest stages of chromosome inheritance. Replication of eukaryotic chromosomes occurs once every cell division cycle in normal cells and is a tightly controlled process that ensures complete genome duplication. The origin recognition complex (ORC) plays a key role during the initiation of DNA replication. In human cells, the level of Orc1, the largest subunit of ORC, is regulated during the cell division cycle, and thus ORC is a dynamic complex. Upon S phase entry, Orc1 is ubiquitinated and targeted for destruction, with subsequent dissociation of ORC from chromosomes. Time lapse and live cell images of human cells expressing fluorescently tagged Orc1 show that Orc1 re-localizes to condensing chromatin during early mitosis and then displays different nuclear localization patterns at different times during G(1) phase, remaining associated with late replicating regions of the genome in late G(1) phase. The initial binding of Orc1 to mitotic chromosomes requires C-terminal amino acid sequences that are similar to mitotic chromosome-binding sequences in the transcriptional pioneer protein FOXA1. Depletion of Orc1 causes concomitant loss of the mini-chromosome maintenance (Mcm2-7) helicase proteins on chromatin. The data suggest that Orc1 acts as a nucleating center for ORC assembly and then pre-replication complex assembly by binding to mitotic chromosomes, followed by gradual removal from chromatin during the G(1) phase.