Cell-free replication system and uses thereof

Details Cell-free replication system and uses thereof Cell-free replication system and uses thereof

2000-03-09

2003-02-18

Jones, W. Gary (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S243000, C435S244000, C435S420000

Reexamination Certificate

active

06521405

ABSTRACT:

The present invention relates to in vitro procedures for DNA replication, and more particularly provides a system for initiating semi-conservative DNA replication under somatic cell cycle control. It also relates to induction of premature competence to replicate. It also relates to the use of such a system, for example in identifying agents that modulate DNA replication, in particular inhibit or stimulate it, thereby providing for example agents having utility based on therapeutic potential.
BACKGROUND OF THE INVENTION
The mechanisms which regulate the eukaryotic cell cycle are complex and the subject of much research. There is a continuing need for the development of good model systems which allow investigation of the mechanisms of action of particular components of the cell cycle under controlled conditions to provide insights into the control of DNA replication and its coupling to the cell cycle in eukaryotic, particularly human, cells. In addition, such systems will allow a range of uses in deriving products of practical benefit, such as screening and identifying therapeutic agents which inhibit DNA replication, and which could thus be used as anti-cancer drugs, and also agents which stimulate DNA replication, which could be used for tissue repair therapeutics.
An important aspect of a good model system is one in which a population of cells, or cellular nuclei, are synchronized with respect to the cell cycle. In proliferating cells, the cell cycle can be divided into four main stages. Following the production of a new cell by mitotic division, there is a period of time, G1, prior to the start of DNA synthesis in the S phase. During the S phase the genome of the cell is replicated and this is followed by an interval, G2, prior to mitosis (the M phase). Following mitosis, the cells reenter the G1 phase. Non-replicating cells generally exit the cell cycle during G1 into the G0 phase.
The initiation of DNA replication, i.e. the transition from G1 to S, is a key step in the regulation of the cell division cycle. A plethora of intra-and extracellular signals is integrated during G1 phase of the cell cycle into a decision to withdraw from the division cycle, or to initiate S phase and hence to continue proliferation (Heichman and Roberts, 1994). Once S phase is initiated, control mechanisms ensure that all chromosomal DNA is replicated before chromosomes are segregated into the two daughter cells at mitosis (Nurse, 1994).
Cell fusion and nuclear transplantation experiments provided compelling evidence that quiescent cell nuclei are induced to initiate DNA replication when introduced into S phase cells (Graham et al., 1966; Harris et al., 1966; de Terra, 1967; Johnson and Harris, 1969). When synchronized cells were fused, S phase cells induced DNA replication only in G1 nuclei, but not in G2 nuclei (Harris et al., 1966; de Terra, 1967; Guttes and Guttes, 1968; Ord, 1969; Rao and Johnson, 1970). These results indicated that S phase cells contain dominant specific factors that trigger DNA replication and are evolutionarily conserved. Unreplicated G1 nuclei are the physiological substrates for the initiation of DNA replication, whilst re-replication in G2 nuclei is prevented until they have undergone mitosis (Romanowski and Madine, 1996).
The transition from G1 to S is regulated by a number of proteins within the cell, in particular the cyclins A, D and E, and their associated cyclin-dependent kinases (Cdks), particularly Cdk2.
One of the major mechanisms by which a replication-competent state during the G1 phase of the cell cycle is achieved involves the regulated assembly of pre-replicative complexes (pre-RCs) or “replication licences” at origins of replication during G1 (Diffley et al., 1994, reviewed by Donovan and Diffley, 1996).
The pre-RC includes two heteromeric protein complexes, the minichromosome maintenance complex (MCM) and the origin recognition complex (ORC), together with the monomeric protein Cdc6 (reviewed by Dutta and Bell, 1997,; Newlon, 1997; Romanowski and Madine, 1997).
The six-subunit origin-recognition complex (ORC) binds specifically to
S. cerevisiae
autonomously replicating sequences (ARS) throughout the cell cycle (Bell and Stillman, 1992; Diffley and Cocker 1992; Aparicio et al.,1997; Liang and Stillman, 1997; Tanaka et al., 1997). Although origins of replication have been difficult to define in higher eukaryotes, homologues of the yeast ORC proteins have a similar function in that they are required for initiation of replication (Gavin et al., 1995; Carpenter et al., 1996; Coleman et al., 1996; Romanowski et al., 1996a; Rowles et al., 1996).
In yeast, it has been shown that the monomeric Cdc6 protein is essential for the initiation of DNA replication and is required for the assembly and maintenance of the pre-Rc (Kelly et al., 1993; Liang et al., 1995; Nishitani and Nurse, 1995; Piatti et al., 1995; Cocker et al., 1996; Muzi-Falconi et al., 1996, Detweiler and Li, 1997, 1998) but its role in mammalian cells is less well characterised (Yan et al., 1998).
The six members of the MCM protein family (MCM2-7) are also components of the pre-RC and association of these proteins with chromatin is required for initiation of DNA replication (Chong et is al., 1995; Dalton and Whitbread, 1995; Kubota et al., 1995; Madine et al., 1995a). During replication the MCM proteins become phosphorylated and displaced from chromatin (Kimura et al., 1994;Chong et al., 1995; Kubota et al., 1995; Madine et al., 1995a, 1995b; Todorov et al., 1995; Coué et al., 1996; Hendrickson et al., 1996; Krude et al., 1996). Cells arrested in vitro by serum starvation or contact inhibition lose chromatin-bound MCMs (after a few days). Although the total level of MCMs in the cells does not decrease greatly within 14 days, after 14 days it falls sharply. Cells which undergo differentiation in vitro (e.g. HL-60 cells induced to differentiate with DMSO or TPA) down-regulate MCM3 but not Orc2 (Musahl, Aussois Meeting on DNA Replication, Aussois, France, June 1997). Differentiated cells from tissues ex vivo do not express MCM proteins such as MCM2 and MCM5. In co-pending patent application GB 9722217.8, it is shown that MCM5 is absent from differentiated cells of the uterine cervix and breast.
The six MCM proteins MCM2-MCM7 form a multiprotein complex, which splits into two subcomplexes: MCM3 and MCM5 dimer; MCM2-4-6-7 tetramer. MCM3 and MCM5 may be displaced from chromatin during S phase more slowly than MCM2-4-6-7 (Kubota et al., 1997, EMBO J. 16, 3320-3331). MCMs are chromatin-bound in G1, displaced during S phase, and nuclear, although not bound to chromatin, in G2.
In yeast and Xenopus assembly of the pre-RC is sequential with ORC recruiting Cdc6, which results in recruitment of MCM proteins (Coleman et al, 1996).
Human Cdc6 amino acid sequence is disclosed in Williams et al., 1997,
PNAS
USA 94: 142-147, GenBank Acc. No. U77949 and in WO 97/41153.
Human MCM2 sequence is disclosed in Todorov et al., 1994,
J. Cell Sci.,
107, 253-265, GenBank Acc. No. X67334.
Human MCM3 sequence is disclosed in Thommes et al., 1992,
Nucl. Acid Res.,
20, 1069-1074, GenBank Acc. No. P25205.
Human MCM4 sequence is disclosed in Ishimi et al., 1996,
J. Biol. Chem.,
271, 24115-24122, GenBank Acc. No. X74794.
Human MCM5 sequence is disclosed in Hu et al., 1993,
Nucleic Acids Res.,
21, 5289-5293, GenBank Acc. No. X74795.
Human MCM6 sequence is disclosed in Holthoff et al., 1996,
Genomics,
37, 131-134, GenBank Acc. No. X67334.
Human MCM7 sequence is disclosed in Hu et al., 1993,
Nucleic Acids Res.,
21, 5289-5293.
ATPase enzymatic activity has been reported for Cdc6 and for MCM proteins. (Zwerschke et al., 1994; Ishimi et al., 1997). These proteins may have other enzymatic activities, for instance helicase activity as reported by Ishimi et al., 1997.
Direct biochemical analysis of replication initiation in eukaryotic somatic cells has been impeded by the lack of an efficient mammalian cell-free DNA replication system to complement these cellular and genetic approaches.
We have previously developed a cell free

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