Replication fork density increases during DNA synthesis in X-laevis egg extracts

Duplication of the eukaryotic genome depends on the temporal and spatial or ganization of DNA replication during the cell cycle. To investigate the gen omic organization of DNA replication in a higher eukaryote, multiple origin s of replication must be simultaneously analyzed over large regions of the genome as DNA synthesis progresses through S phase of the cell cycle. We ha ve employed a novel technique that allows for the quantitative analysis of DNA replication on a genome wide basis. The technique involves stretching a nd aligning individual DNA molecules on a glass surface. As a model system, Xenopus laevis egg extract was used to differentially label sperm chromati n at successive time points after the start of DNA synthesis. The different ially labeled DNA allows earlier and later replicating sequences to be dist inguished, and hence the sites of DNA synthesis at any given time can be di rectly visualized. Genomic DNA was extracted, and measurements made on the linearized molecules provided a comprehensive analysis of the spatial and t emporal organization of DNA replication in the X. laevis in vitro replicati on system. It was found that: (i) DNA synthesis initiates asynchronously at irregular intervals but continuously as DNA replication advances; and (ii) that the frequency of initiation (the number of activated origins per kilo base) increases as DNA synthesis nears completion. The implications of thes e findings for the regulation of DNA replication in early embryos is discus sed. (C) 2000 Academic Press.