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

Abstract

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.

ORC4 Surrounds Extruded Chromatin in Female Meiosis

“…It is not clear what role these four ORC proteins play in these structures, but it is intriguing that all four localize at both. A recent study has suggested that ORC2-5 assemble in the cytoplasm before being translocated to the nucleus [Ghosh et al, 2011]. The co-localization of ORC1-3 and 5 at the mitotic spindle and between the separating chromosomes at anaphase II suggest that these are storage sites for the initial ORC complexes in close proximity to the newly forming nucleus.…”

“…Previous models for human ORC proposed that in actively dividing somatic mammalian cells, ORC2-5 remain associated with DNA replication origins throughout the cell cycle while ORC1 is recruited to the ORC complex only during G1 [DePamphilis, 2005; Dhar et al, 2001; Thomae et al, 2008; Vashee et al, 2001]. However, more recent work has suggested that it is possible all the ORC proteins are recruited to the origins during each cycle [Ghosh et al, 2011]. Moreover, the DNA binding patterns of ORC to DNA vary during differentiation [Nordman and Orr-Weaver, 2012].…”

Genome Duplication

“…Nuclear DNA replication in the metazoa begins during the anaphase to G1 phase transition of the mitotic cell cycle with the assembly of prereplication complexes onto the DNA in chromatin (Baldinger & Gossen, 2009; Noguchi, Vassilev, Ghosh, Yates, & DePamphilis, 2006). In mammals, this event, termed “origin licensing,” begins by assembly of the six subunit “origin recognition complex” [ORC(1–6)] through independent nuclear localization of Orc1, ORC(2–5), and Orc6 (Ghosh, Vassilev, Zhang, Zhao, & DePamphilis, 2011). The BAH domain in human Orc1 facilitates the ability of Orc1 to activate replication origins in vivo and triggers assembly of ORC(1–6) (Kara, Hossain, Prasanth, & Stillman, 2015; Noguchi et al, 2006).…”

“…As cells exit mitosis, geminin and cyclin A are ubiquitinated by the anaphase promoting complex [originally termed cyclosome (APC/C)], an activity that is inhibited during S to early M phase by the APC/C-specific inhibitor Emi1. Reassembly of the helicase loader occurs by reassembly of Orc1, ORC(2-5), Orc6, and Cdc6 onto chromatin (Ghosh et al, 2011; Kara et al, 2015; Sonneville et al, 2012), an event that appears to be triggered by the Orc1 subunit (Noguchi et al, 2006). Geminin binding to Cdt1 and CDK-dependent phosphorylation of Cdc6 prevents targeting of Cdt1 and Cdc6 for degradation by the APC/C, thereby allowing them to participate in origin licensing (Ballabeni et al, 2004; Mailand & Diffley, 2005).…”

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

“…For immunoprecipitation experiments, equivalent amounts of protein samples were incubated at 4°C with HA-coupled beads for 4 h. After extensive washing with Triton lysis buffer, bound proteins were eluted in boiling Laemmli buffer. For Cdt1/MCM coimmunoprecipitation, TK100 lysis buffer (20 mM Tris-HCl pH 7.9; 100 mM KCl, 5 mM MgCl 2 , 10% glycerol; 0.5% NP-40) was used 57 . Purification of GST fusion proteins was done as described previously 53 .…”