9.2 DNA Replication

1996

The replication of the genetic material during cell doubling is regulated at three apparently distinct levels. In the eukaryote nucleus, between 103 and 105 DNA replication initiation events occur each cell cycle (1). Though these events can occur asynchronously and can occur over an extended period of many hours, only a single initiation event is allowed to happen at any one site. Thus, on a long DNA fiber, multiple replication "eyes" that reveal start sites can be observed, but reinitiation within one eye is not detected (2). This once-and-only-once regulation of replication initiation helps maintain the relative copy numbers of genes. Furthermore, as first revealed by autoradiographic methods, the order of initiation events is determined, with certain regions replicating before others. This pattern can change during development, leading to the suggestion that such changes in the timing of initiation may result in a reprogramming of the gene expression program (3). At an apparently higher level of regulation, the entire synthesis phase (S) takes place within a discrete period, surrounded in time by two gap (G) periods, and resetting the clock to undergo a new round of DNA synthesis is dependent on passage through the mitosis (M) phase of the cell cycle. A classic demonstration of this interdependency is provided by cell fusion experiments in which heterokaryons containing combinations of S phase and Gi or G2 nuclei were observed; the DNA complement of the Gi nucleus was observed to enter S phase prematurely, while the G2 nucleus did not pass through another round of DNA replication without prior passage through mitosis (4).

The discovery of the DNA double helix structure half a century ago immediately suggested a mechanism for its duplication by semi-conservative copying of the nucleotide sequence into two DNA daughter strands. Shortly after, a second fundamental step toward the elucidation of the mechanism of DNA replication was taken with the isolation of the first enzyme able to polymerize DNA from a template. In the subsequent years, the basic mechanism of DNA replication and its enzymatic machinery components were elucidated, mostly through genetic approaches and in vitro biochemistry. Most recently, the spatial and temporal organization of the DNA replication process in vivo within the context of chromatin and inside the intact cell are finally beginning to be elucidated. On the one hand, recent advances in genome-wide high throughput techniques are providing a new wave of information on the progression of genome replication at high spatial resolution. On the other hand, novel super-resolution microscopy techniques are just starting to give us the first glimpses of how DNA replication is organized within the context of single intact cells with high spatial resolution. The integration of these data with time lapse microscopy analysis will give us the ability to film and dissect the replication of the genome in situ and in real time.

Current Opinion in Genetics & Development, 2002

1981

Arabinosylcytosine at a 1 • 1(Γ 4 molar concentration inhibited thymidine incorporation into DNA by more than 95% In sucrose gradients the labelled dThd was predominantly found in short DNA chains Labelled arabinosylcytosince (aC) was incorporated into DNA, as was labelled dThd, indicating that it causes a preferential inhibition of the chain polymerization rather than termi nation of nascent chains This was confirmed by the observation of the con version of short chains to normal size DNA molecules in chase experiments. In the absence of NaCl and at neutral pH a release of more than 50% of the nascent label as single strands was repeatedly observed upon sucrose gradient centnfugation This release could be significantly reduced by 1 M NaCl, indicat ing that in the presence of aC the in vivo structure was preserved This was further confirmed by the fact that aC did not cause detachment of DNA from a rapidly sedimenting nuclear structure Based on these results, a replication model slightly different from the one suggested by Okazaki, is proposed, in which initiations of new nascent chains can occur at certain distances ahead of the replication fork.

DNA replication is the process by which the genetic material is copied prior to distrubution into daughter cells  The original DNA strands are used as templates for the synthesis of new strands  It occurs very quickly, very accurately and at the appropriate time in the life cycle of the cell DNA replication relies on the complementarity of DNA strands  The AT/GC rule or Chargaff"s rule  The process can be summarized as follows:

1992

In common with other eukaryotes, plant DNA is organised for replication as multiple replicons. A key event is the recognition of origins by the origin-recognition complex (ORC). Despite earlier indications, it is now likely that as in mammals, plant replication origins are not defined strictly by sequence; it is thus important to ascertain what features of origins are recognised by the ORC. Activation of origins occurs by stepwise loading of the pre-replication complex, all components of which have been identified in plants. This step is important in restricting DNA replication to once per cell cycle, although in plants, this restriction is relatively easily overcome, thus permitting DNA endoreduplication. It is also important to note the flexibility of origin use in relation to aspects of plant development. Is this flexibility mediated by ORC binding or at the pre-replication step? Following origin activation, the origin is prepared for initiation, again by the stepwise loading of ...