The control of eukaryotic DNA replication
One of the major limitations on research into the control of eukaryotic DNA replication has been the lack of any cell-free system that initiates DNA replication in vitro. The first part of the disseration describes the establishment of a eukaryotic system, derived from the activated eggs of the South African clawed toad, Xenopus laevis, that efficiently initiates and completes DNA replication in vitro. Using a variety of biochemical techniques I show that DNA added to the extract in the form of sperm nuclei is efficiently replicated over a period of 4 - 6 hours. Replication of nuclear DNA represents a single round of semiconservative, semidiscon-tinuous replication. The extract will also replicate naked DNA incubated in it, regardless of sequence, though less efficiently than nuclear templates. This is probably related to the unusual ability of the egg extract to assemble apparently normal interphase nuclei from any DNA molecule incubated in it Evidence is presented that initiation, rather than chain elongation, is the rate-limiting step for replication in vitro. In this and in other ways the cell-free system behaves as though it were an early embryo blocked in a single cell cycle. The second part of the dissertation describes experiments that examine the control of DNA replication in the extract The first set of experiments suggest that on replication, DNA is marked in some way so that it can no longer act as a substrate for further initiation. This provides a mechanism by which the template DNA is replicated precisely once per incubation in vitro (or per cell cycle in vivo). The second set of experiments investigate the relationship between nuclear assembly and the initiation of DNA replication in vitro. A novel method for quantifying DNA replication in intact nuclei using the nucleotide analogue biotin-11-dUTP is described. This technique reveals that although they are in the common cytoplasm of the egg extract, different nuclei start to replicate at different times. Entry into S-phase is characterised by a burst of many synchronous or near-synchronous initiations within individual nuclei. This means that nuclei act as independent and integrated units of replication in the cell-free system, and suggests a fundamental role for nuclear assembly in controlling DNA replication in vitro.