Mol Cell Biol 20(22): 8602-8612

Chromatin Association of Human Origin Recognition Complex, Cdc6, and Minichromosome Maintenance Proteins during the Cell Cycle: Assembly of Prereplication Complexes in Late Mitosis

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724

^{Corresponding author. Mailing address: Cold Spring Harbor Laboratory, P.O. Box 100, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Phone: (516) 367 8384. Fax: (516) 367 8879. E-mail:}gro.lhsc@namllits.

Received 2000 Jul 7; Revisions requested 2000 Aug 2; Accepted 2000 Aug 15.

Abstract

Evidence obtained from studies with yeast and Xenopus indicate that the initiation of DNA replication is a multistep process. The origin recognition complex (ORC), Cdc6p, and minichromosome maintenance (MCM) proteins are required for establishing prereplication complexes, upon which initiation is triggered by the activation of cyclin-dependent kinases and the Dbf4p-dependent kinase Cdc7p. The identification of human homologues of these replication proteins allows investigation of S-phase regulation in mammalian cells. Using centrifugal elutriation of several human cell lines, we demonstrate that whereas human Orc2 (hOrc2p) and hMcm proteins are present throughout the cell cycle, hCdc6p levels vary, being very low in early G₁ and accumulating until cells enter mitosis. hCdc6p can be polyubiquitinated in vivo, and it is stabilized by proteasome inhibitors. Similar to the case for hOrc2p, a significant fraction of hCdc6p is present on chromatin throughout the cell cycle, whereas hMcm proteins alternate between soluble and chromatin-bound forms. Loading of hMcm proteins onto chromatin occurs in late mitosis concomitant with the destruction of cyclin B, indicating that the mitotic kinase activity inhibits prereplication complex formation in human cells.

Abstract

The molecular mechanism that restricts firing of origins of replication to once per cell cycle invokes the ordered binding to and/or release of different replication proteins from specific DNA sequences (replicators) located in the vicinity of the actual origins of DNA replication. Following separation of sister chromatids at mitosis and during the subsequent G₁ phase, prereplication complexes (pre-RCs) are formed at origins of DNA replication. Initiation of DNA replication is triggered by the action of at least two sets of protein kinase activities, cyclin-dependent kinases (CDKs) and Dbf4p-Cdc7p. After initiation, the protein complex at each origin changes to a postreplication state (post-RC), thereby preventing further initiation events for the rest of the cell cycle (reviewed in references 15 and 57).

The origin recognition complex (ORC), a six-subunit initiator protein (2), is present in both pre- and post-RCs (10), and one of its functions is to mark the position of replication origins in the genome. The pre-RC is established by the regulated binding of additional factors, which include Cdc6p and the minichromosome maintenance (MCM) proteins. Yeast CDC6 displays a genetic interaction with the ORC and is a critical factor for establishing the competence of replication origins once per cell cycle (12, 33, 47, 48). Besides its function in DNA replication, it may also be involved in a mitotic checkpoint control, because Cdc6-deprived yeast cells that do not replicate DNA still undergo a reductional mitosis (4, 47, 63).

Cdc6p is a member of the large AAA^{superfamily of ATPases, which includes Orc1p, Orc4p, Orc5p, MCM, proteins and replication factor C (}42). Based on sequence similarities between Cdc6p, replication factor C, and other AAA^{family members and on the characterization of a dominant-negative}CDC6 mutant, it has been proposed that yeast Cdc6p might function as an ATP-dependent MCM protein loader (45, 63). Indeed, the association of MCM proteins with chromatin is dependent on Cdc6p (1, 12, 34, 59). Biochemical studies with Xenopus provided additional support for the idea of Xenopus Cdc6p (XCdc6p) being an essential factor for establishing pre-RCs. In Xenopus egg cell extracts, XCdc6 could bind to chromatin only in the presence of XOrc2, and it was absolutely required for the subsequent loading of XMcm3 (6).

Yeast Cdc6p is a highly unstable protein, and many factors seem to be involved in its degradation, including the CDC4-CDC34-CDC53-Skp1 pathway (13, 14, 54). However, ectopic expression of Cdc6p in G2 cells is not deleterious for the cell, and it has been shown that Cdc6p cannot induce MCM protein binding to chromatin at this point unless CDKs are inactivated (9, 59). Interestingly, a dominant gain-of-function allele of CDC6 causes persistent MCM protein binding to chromatin and overreplication of the genome in a single cell cycle (34).

Cdc18^{is the}Schizosaccharomyces pombe homologue of CDC6 and performs similar functions in regulating initiation of DNA replication and possibly entry into mitosis (28, 40, 43). Gross overexpression of Cdc18^{results in repeated rounds of DNA replication in the absence of mitosis (}23, 43). p65^{is also a very labile protein targeted for destruction by CDK phosphorylation after cells enter S phase (}24). At least in S. pombe and Xenopus, another protein, called Cdt1, participates in the assembly of pre-RCs (37, 44).

MCM proteins are essential for DNA replication (reviewed in reference 27). A subcomplex of human MCM proteins may function as a replicative helicase (22). This idea has been recently reinforced by the finding that yeast MCM proteins are required for replication fork progression (32) and by the characterization of a processive helicase activity in an archaeal MCM protein (5, 29).

The regulation of initiation of replication in mammalian cells is much less understood, mainly for two reasons: discrete replicator sequences have not been defined, and the information regarding mammalian proteins involved in this process has been limited. The recent identification of human homologues of ORC subunits (18, 49, 50, 62), Cdc6 (65), and MCM proteins (reference 27 and references therein) suggests that the overall mechanism for replication initiation is conserved between mammals and yeast.

We have therefore investigated the dynamics of chromatin association of endogenous human Orc2, Cdc6, and Mcm proteins (referred to as hOrc2p, hCdc6p, and hMcm proteins) throughout the cell cycle. We used centrifugal elutriation as a method to synchronize cells without interfering with their normal metabolism, and we raised new monoclonal anti-hCdc6p antibodies and polyclonal anti-hMcm antibodies. Our data show that a fraction of hCdc6p is targeted to chromatin during the entire cell cycle and help to define the point at which the hMcm proteins are loaded onto chromatin during late mitosis. We also report a previously unrevealed regulated destruction of hCdc6p and discuss the possible execution points for hCdc6p during the cell cycle.

ACKNOWLEDGMENTS

We thank W. Tansey for his help with the proteasome inhibition and in vivo ubiquitination experiments, R. S. Williams for providing purified recombinant hCdc6p, C. Bautista for her work at the Cold Spring Harbor Laboratory monoclonal antibody facility, K. Brown for her help with epitope mapping of the anti-hCdc6p antibodies, A. Koff for his help with the elutriation protocol, N. Hernandez for anti-TFIIB antibodies, D. Bohmann for plasmid pMT107, A. Verreault for his suggestions on the fractionation protocol, K. Cronin for excellent technical assistance, and A. Losada and J. Chong for useful comments on the manuscript.

This work was supported by the National Cancer Institute (grant CA13106). J.M. was the recipient of postdoctoral fellowships from Fundación Ramón Areces (Spain) and the Human Frontier Science Program Organization.

ACKNOWLEDGMENTS

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