Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells.

Abstract

A distinguishing feature of eukaryotic cells is the great variability they exhibit in both genome size and organization. For example, the budding yeast Saccharomyces cerevisiae contains little more DNA per cell than the prokaryote Escherichia coli, whereas cells from many plants such as the lily, Lilium longiflorum, contain nearly l0 s times as much DNA per cell. In addition to being large, eukaryotic genomes are also fragmented: DNA can be contained on just a few chromosomes or can be distributed on literally thousands of individual chromosomes. The evolution of a mechanism to rapidly replicate large amounts of DNA on multiple chromosomes was probably an important prerequisite to the expansion of genome size and consequently to the explosion of morphological and functional diversity seen among eukaryotes. One of the most important features of this mechanism is that bidirectional DNA replication initiates from multiple origins on eukaryotic chromosomes (Cairns 1966; Huberman and Riggs 1966, 1968). While budding yeast contains only about four times the amount of DNA per haploid cell as E. coli, it replicates its genomic DNA from 250-400 replication origins distributed on 16 chromosomes, whereas the E. coli genome, contained on a single chromosome, is replicated from a single replication origin (oriC). Thus, although eukaryotic genomes are generally larger than prokaryotic genomes, the amount of DNA replicated from an individual replication origin is generally considerably smaller. This can allow very rapid replication. Even under optimal growth conditions, the E. coli genome takes -40 min to replicate. Drosophila melanogaster cells contain almost 100 times as much DNA as E. coli, yet in cells of early Drosophila embryos, the DNA synthetic phase of the cell cycle (S phase) takes only 3-4 min because replication initiates synchronously from many origins spaced at short (roughly 8-kb) intervals (Blumenthal et al. 1973). Perhaps equally striking is that the number of origins used to replicate eukaryotic genomes can vary from cell type to cell type within an organism. This can allow a great deal of flexibility in the length of S phase. For example, although S phase in Drosophila embryos is 3-4 min, it takes nearly 10 hr for the same process to occur in cells from adult animals grown in tissue culture (Blumenthal et al. 1973). This increase in the length of S phase is partly attributable to a reduction in the number of origins used. In addition, origin firing in these tissue culture cells becomes highly asynchronous so that some parts of the genome replicate much earlier than others. The firing of multiple replication origins throughout S phase also presents a potential logistical nightmare; as S phase progresses, origins within unreplicated DNA must be located and fired against the background of an increasingly large pool of replicated DNA. Furthermore, within a single S phase, DNA replication must never reinitiate from an origin that has already fired. In this review I will summarize some of the important discoveries and concepts that have led to our current understanding of how replication origins are specified and how multiple initiation events are regulated to ensure efficient and precise duplication of the genome.