Assembly of the Human Origin Recognition Complex

Abstract

The six-subunit origin recognition complex (ORC) was originally identified in the yeast Saccharomyces cerevisiae. Yeast ORC binds specifically to origins of replication and serves as a platform for the assembly of additional initiation factors, such as Cdc6 and the Mcm proteins. Human homologues of all six ORC subunits have been identified by sequence similarity to their yeast counterparts, but little is known about the biochemical characteristics of human ORC (HsORC). We have extracted HsORC from HeLa cell chromatin and probed its subunit composition using specific antibodies. The endogenous HsORC, identified in these experiments, contained homologues of Orc1-Orc5 but lacked a putative homologue of Orc6. By expressing HsORC subunits in insect cells using the baculovirus system, we were able to identify a complex containing all six subunits. To explore the subunitsubunit interactions that are required for the assembly of HsORC, we carried out extensive co-immunoprecipitation experiments with recombinant ORC subunits expressed in different combinations. These studies revealed the following binary interactions: HsOrc2-HsOrc3, HsOrc2-HsOrc4, HsOrc3-HsOrc4, HsOrc2-HsOrc6, and HsOrc3-HsOrc6. HsOrc5 did not form stable binary complexes with any other HsORC subunit but interacted with sub-complexes containing any two of subunits HsOrc2, HsOrc3, or HsOrc4. Complex formation by HsOrc1 required the presence of HsOrc2, HsOrc3, HsOrc4, and HsOrc5 subunits. These results suggest that the subunits HsOrc2, HsOrc3, and HsOrc4 form a core upon which the ordered assembly of HsOrc5 and HsOrc1 takes place. The characterization of HsORC should facilitate the identification of human origins of DNA replication.The initiation of DNA replication in lower eukaryotes is similar to that observed in bacteria in that it occurs at well defined origins of DNA replication that are recognized by specific initiator proteins (1-5). Saccharomyces cerevisiae origins of DNA replication are short, ϳ100-base pair (bp) 1 segments that consist of two essential regions: the A domain, which contains a conserved 11-bp consensus sequence (ACS) that is required for origin function, and the B domain, which contains several stimulatory elements (6, 7). The ACS is recognized specifically by the S. cerevisiae initiator protein, the origin recognition complex (ORC) (8, 9). S. cerevisiae ORC (ScORC) is comprised of six proteins (Orc1-6), each of which is essential for viability in S. cerevisiae (10 -13). A number of lines of evidence indicate that ScORC is required for initiation of DNA replication in vivo and functions in part to recruit other initiation factors, such as Cdc6 and the minichromosome maintenance proteins, to replication origins (13-15).Homologues of ORC subunits have been identified in a variety of other eukaryotic species (2, 16), and ORC has been purified from the fission yeast Schizosaccharomyces pombe and the metazoans Drosophila melanogaster and Xenopus laevis (17)(18)(19). Genetic and biochemical data indicate that specific ORC subunits from t...

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

“…3), consistent with earlier studies showing that Orc1 is required both for binding ORC(2-5) to DNA in vitro and for initiation of ORC-dependent DNA replication in Xenopus egg extract (13,14). In vitro, Orc1 first associates with ORC(2-5), and then ORC(1-5) binds to DNA in a reaction that requires binding of ATP to Orc1 (10,11,13).…”

“…Human ORC assembled from baculovirus-expressed proteins either lacks Orc6 or it is present only in trace amounts (11)(12)(13)(14). Nevertheless, interactions between mammalian Orc6 and other ORC subunits have been detected by yeast two-hybrid screens (15), by association of ORC subunits in vitro (10,11), and by immunoprecipitation of ORC subunits from cell extracts (11). Thus, the interaction of mammalian Orc6 with other ORC subunits appears tenuous.…”

“…ORC isolated from frog eggs lacks the Orc6 subunit, which is not essential for DNA replication (6). ORC isolated from human cells also lacks the Orc6 subunit (7)(8)(9)(10), and only the human ORC (1)(2)(3)(4)(5) complex is required to initiate DNA replication in an ORC-depleted frog egg extract (9,10). Human ORC (2)(3)(4)(5) binds to DNA and initiates DNA replication only when associated with Orc1 (9,10,26).…”

Nonreplicative functions of the origin recognition complex

“…Moreover, in mammals the BAH (bromo-adjacent homology) domain of ORC1 recognizes the H4K20me2 modification [11], which is associated with replication. Besides that, human ORC2, 3, 4, and 5 form the core of the complex and are associated with each other throughout the entire cell cycle, while the smallest subunit, ORC6, interacts with the complex in a transient manner [12,13]. Taken together, multicellular organisms have more complex regulation of ORC that reflects the evolutionary emergence of new additional functions of the complex.…”

A probabilistic view of gene function

“…Consider the example of the protein ORC2, which binds the origin of DNA replication to initiate the process of DNA replication [13][14][15] . The specific function of ORC2 is to bind DNA and, along with the proteins ORC1-ORC6, it forms the origin recognition complex [16][17][18] . The origin recognition complex is a single component of the prereplicative complex, which in turn is one component required for initiation of DNA replication, and so on up the hierarchy of functions, from specific to general, detailed to global.…”