Human Minichromosome Maintenance Proteins and Human Origin Recognition Complex 2 Protein on Chromatin

Abstract

Considerable progress has recently been made in describing the components of the machinery required for the initiation of eukaryotic genome replication. Much of the original work was performed studying yeast mutants that had lost the ability to perform defined steps in the progression of proliferating cells through the late G 1 phase or early S phase of the cell cycle. Genetic studies were complemented by cell biological and biochemical experiments leading to the identification of yeast DNA elements, known as ARS (autonomously replicating sequences), which serve as origins of replication, and of the sixmembered protein complex origin recognition complex (Orc) Mcm proteins occur in high copy numbers in nuclei of human cells. They appear to be either free in the nucleoplasm or bound to chromatin.Although not yet formally proven, it can be assumed that the binding of mammalian Mcm proteins to chromatin is also contingent upon the previous binding of Orc proteins and Cdc6 protein. The fraction of structure-bound mammalian Mcm proteins is highest at the beginning of S phase, but gradually decreases during progression of replication (21-23). Released nucleoplasmic Mcm proteins are found as large multiprotein complexes, composed of the six known members of the Mcm protein family. This complex has the tendency to desintegrate into more stable subcomplexes of which one is a dimer of the MCM3 protein (MCM3p) and the MCM5 protein (MCM5p), while the second is a trimer of proteins MCM4 (MCM4p), MCM6 (MCM6p), and MCM7 (MCM7p). The sixth member of the family, human protein MCM2 (MCM2p), is usually found to be loosely associated with these subcomplexes (24 -26).The present study was performed to investigate the mode of binding of Mcm proteins and ORC2p, a member of the origin recognition complex, on human chromatin. We prepared chromatin from proliferating human HeLa cells using low ionic strength conditions in the presence of the nonionic detergent Nonidet P-40. As first shown by Hancock (27), the nuclear envelope is removed by this procedure, while the general form and the ultrastructural characteristics of chromatin appear to remain largely unchanged. The maintenance of form in this structure allows biochemical manipulations such as pipetting and low speed centrifugations for a separation of structurebound from soluble nuclear material. We have used these prep-* This work was supported by Fonds der Chemischen Industrie and Deutsche Forschungs-gemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Present address:

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…(17) Although the replicator sequences in other eukarya are larger and less defined than in S. cerevisiae, ORC has been conserved during evolution and is required for initiation of DNA replication in different species. (16) In Xenopus, Drosophila melanogaster and mammalian cells, ORC associates with the chromatin, (18)(19)(20)(21)(22) but its binding sites are poorly defined. In flies, a subset of ORCbinding sites have been identified at loci that promote developmentally regulated DNA amplification through repeated rounds of replication.…”

Minichromosome maintenance (Mcm) proteins, cyclin B1 and D1, phosphohistone H3 and in situ DNA replication for functional analysis of vulval intraepithelial neoplasia

“…The Mcm proteins form an essential prereplicative complex by binding to DNA sites at which origin recognition complex and cdc6 proteins have sequentially bound. Mcm proteins are only present during the cell cycle and are lost in quiescent and differentiating cells (Ritzi et al, 1998). The use of an antibody directed against Mcm 5 has been shown to improve the specificity and sensitivity of cytological screening for CIN compared with the use of conventional cervical cytology alone (Freeman et al, 1999).…”

Minichromosome maintenance protein in myxofibrosarcoma

“…MCM2 is a component of the prereplicative complex, 7 that is an essential licensing factor for DNA replication and which limits replication to once per cell cycle. 8,11 MCM proteins are highly sensitive and specific markers of cells that are in cycle (regardless of the rate of cycling) and therefore indicate the cell cycle state within a tissue.…”

“…4 Here, we have tested the hypotheses that evaluation of the cell cycle state of myxofibrosarcoma tumors may facilitate the assessment of grade in needle biopsies and/or cytological specimens and may also be predictive of outcome. In order to obtain an accurate indication of cell cycle state in myxofibrosarcoma, we have examined the expression of minichromosome maintenance (MCM) protein 2, which is a member of the prereplicative complex [5][6][7] that serves as an essential licensing factor for DNA replication in eukaryotic cells. 8 MCM proteins are detectable throughout the cell cycle but are lost in quiescence and differentiation.…”