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

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 G 1 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.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 G 1 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

Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1

“…The 3xMBT region of L3MBTL1 is sufficient for its chromatin association in vivo and in vitro To test whether L3MBTL1 is chromatin-associated in vivo, we isolated nuclei from K562 erythroleukemia cells that express appreciable levels of L3MBTL1 (MacGrogan et al, 2004), and generated soluble (nucleoplasm) and insoluble (chromatin) fractions, as described (Mendez and Stillman, 2000). L3MBTL1 protein is found in the chromatin fraction (residual chromatin pellet; lane 1, Figure 1b).…”

Camptothecin-induced downregulation of MLL5 contributes to the activation of tumor suppressor p53

“…Cellular protein fractionation was performed as previously described (Mendez and Stillman, 2000). In brief, HCT116 cells were lysed (40 million cells/ml) for 5 min at 4 1C in buffer A (10 mM (4-(2-hydroxyethyl-1-piperazineethane sulfonic acid) pH7.9, 10 mM KCl, 1.5 mM MgCl 2 , 0.34 M sucrose, 10% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100) supplemented with protease and phosphatase inhibitors (2 mM phenylmethanesulfonylfluoride, 2 mg/ml leupeptin, 2 mg/ml aprotinin, 1 mg/ml pepstatin A, 1 mM Na 3 VO 4 and 5 mM NaF).…”

DNA replication licensing and cell cycle kinetics of oligodendroglial tumours

“…We have examined expression of the standard proliferation marker Ki67, the DNA replicationlicensing factor Mcm2 and the repressor of origin-licensing Geminin during the proliferative cell cycle, using a novel, nonchemical method for cell synchronisation (membrane elution; Thornton et al, 2002;Helmstetter et al, 2003; Figure 1C). We have employed membrane elution because use of chemical methods for cell synchronisation may account in part for reported discrepancies in temporal periodicities and subcellular localisation of replication proteins such as Orc1 (Pak et al, 1997;Kreitz et al, 2001;Okuno et al, 2001;Li and DePamphilis, 2002) and Cdc6 (Fujita et al, 1999;Jiang et al, 1999;Mendez and Stillman, 2000;Petersen et al, 2000).…”