Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1999-01-25
2000-08-22
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 15, 435 911, C12Q 168, C12P 1934
Patent
active
061070424
DESCRIPTION:
BRIEF SUMMARY
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 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.
The initiation of DNA replication 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 synchronised 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).
Genetic evidence in the yeast Saccharomyces cerevisiae provided independent evidence for an inducer of S phase (Hartwell, 1974). The protein kinase CDC28 is a key regulator of the yeast cell division cycle (Nasmyth, 1993). It associates with different regulatory CLN and CLB cyclin subunits throughout the progression of the cell cycle to execute cell cycle-specific functions in a temporally regulated way. Association with G1 and S phase specific cyclins, followed by the postranslational activation of the catalytic subunit is essential for the onset of S phase in yeast (Nasmyth, 1993). In higher eukaryotes, the situation is more complex in that different catalytic cyclin-dependent kinase (Cdk) subunits associate with different cyclins at different times to regulate the progression of the cell cycle [reviewed in (Heichman and Roberts, 1994; Nurse, 1994; Sherr, 1994)]. Experiments with antibodies, antisense RNA constructs or ectopic expression of cyclin genes indicate a role of cyclins D, E and A in inducing the G1-to-S phase transition in somatic mammalian cells in vivo (Girard et al., 1991; Pagano et al., 1992; Zindy et al., 1992; Ohtsubo and Roberts, 1993; Resnitzky et al., 1994; Wimmel et al., 1994; Ohtsubo et al., 1995; Resnitzky and Reed, 1995).
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. Existing cell-free DNA replication systems are inadequate for studying the initiation of chromosomal DNA replication for the following reasons:
First, viral cell-free systems such as that derived from Simian Virus 40 (SV40) and its somatic host cells depend on essential viral control elements in addition to mammalian cell extracts; namely cis-acting DNA viral sequences including the origin of bidirectional DNA replication and the trans-acting initiator protein T antigen (Li and Kelly, 1984; Challberg and Kelly, 1989; Stillman, 1989). Both control elements are virus-encoded and serve to overcome the cellular once-per-cell-cycle replication control allowing viral DNA runaway-replication to occur in the infected cell nucleus.
Second, a cell free system derived from activated eggs of the frog Xenopus laevis can r
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Jackman Mark Richard
Krude Torsten
Laskey Ronald Alfred
Pines Jonathan Noe Joseph
Cancer Research Campaign - Technology Limited
Horlick Kenneth R.
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