In mammalian cells, the replication of DNA in the region between one replication origin and the next should normally require only about an hour to complete, given the rate at which a replication fork moves and the largest distances measured between replication origins. Yet S phase usually lasts for about 8 hours in a mammalian cell. This implies that the replication origins are not all activated simultaneously; indeed, replication origins are activated in clusters of about 50 adjacent replication origins, each of which is replicated during only a small part of the total S-phase interval.
Figure 5–28 Use of DNA microarrays to monitor the formation and progress of replication forks. For this experiment, a population of cells is synchronized so that they all begin replication at the same time. DNA is collected and hybridized to the microarray; DNA that has been replicated once gives a hybridization signal (dark green squares) twice as high as that of unreplicated DNA (light green squares). The spots on these microarrays represent consecutive sequences along a segment of a chromosome arranged left to right, top to bottom. Only 81 spots are shown here, but the actual arrays contain hundreds of thousands of sequences that span an entire genome. As can be seen, replication begins at an origin and proceeds bidirectionally. For simplicity, only one origin is shown here. In human cells, replication begins at 30,000–50,000 origins located throughout the genome. Using this approach it is possible to observe the formation and progress of every replication fork across a genome.
Figure 5–29 The four successive phases of a standard eukaryotic cell cycle.
During the G1, S, and G2 phases, the cell grows continuously. During M phase growth stops, the nucleus divides, and the cell divides in two. DNA replication is confined to the part of the cell cycle known as S phase. G1 is the gap between M phase and S phase; G2 is the gap between S phase and M phase.
It seems that the order in which replication origins are activated depends, in part, on the chromatin structure in which the origins reside. We saw in Chapter 4 that heterochromatin is a particularly condensed state of chromatin, while euchromatin, where most transcription occurs, has a less condensed conformation. Heterochromatin tends to be replicated very late in S phase, suggesting that the timing of replication is related to the packing of the DNA in chromatin.
Once initiated, however, replication forks seem to move at comparable rates throughout S phase, so the extent of chromosome condensation seems to influence the time at which replication forks are initiated, rather than their speed once formed.
A Large Multisubunit Complex Binds to Eukaryotic Origins of Replication
Having seen that a eukaryotic chromosome is replicated using many origins of replication, each of which “fires” at a characteristic time in S phase of the cell cycle, we turn to the nature of these origins of replication. We saw earlier in this chapter that replication origins have been precisely defined in bacteria as specific DNA sequences that attract initiator proteins, which then assemble the DNA replication machinery. We shall see that this is the case for the single-cell budding yeast S. cerevisiae, but it appears not to be strictly true for most other eukaryotes.
For budding yeast, the location of every origin of replication on each chromosome has been determined. The particular chromosome shown in Figure 5–30— chromosome III from S. cerevisiae—is one of the smallest chromosomes known, with a length less than 1/100 that of a typical human chromosome. Its major origins are spaced an average of 30,000 nucleotide pairs apart, but only a subset of these origins is used by a given cell. Nonetheless, this chromosome can be replicated in about 15 minutes.