The minimal DNA sequence required for directing DNA replication initiation in S. cerevisiae has been determined by taking a segment of DNA that spans an origin of replication and testing smaller and smaller DNA fragments for their ability to function as origins. Most DNA sequences that can serve as an origin of replication are found to contain (1) a binding site for a large, multisubunit initiator protein called ORC, for origin recognition complex; (2) a stretch of DNA that is rich in As and Ts and therefore easy to melt; and (3) at least one binding site for proteins that facilitate ORC binding, probably by adjusting chromatin structure.
In bacteria, once the initiator protein is properly bound to the single origin of replication, the assembly of the replication forks seems to follow more or less automatically. In eukaryotes, the situation is significantly different because of a profound problem eukaryotes have in replicating chromosomes: with so many places to begin replication, how is the process regulated to ensure that all the DNA is copied once and only once?
The answer lies in the sequential manner in which the replicative helicase is first loaded onto origins and is then activated to initiate DNA replication. This matter is discussed in detail in Chapter 17, where we consider the machinery that underlies the cell-division cycle. In brief, during G1 phase, the replicative helicases are loaded onto DNA next to ORC to create a prereplicative complex. Then, upon passage from G1 phase to S phase, specialized protein kinases come into play to activate the helicases. The resulting opening of the double helix allows the loading of the remaining replication proteins, including the DNA polymerases.
origins of replication telomere centromere telomere
Figure 5–30 The origins of DNA replication on chromosome III of the yeast S. cerevisiae. This chromosome, one of the smallest eukaryotic chromosomes known, carries a total of 180 genes. As indicated, it contains 18 replication origins, although they are used with different frequencies. Those in red are typically used in less than 10% of cell divisions, while those in green are used about 90% of the time.
Figure 5–31 DNA replication initiation in eukaryotes. This mechanism ensures that each origin of replication is activated only once per cell cycle. An origin of replication can be used only if a prereplicative complex forms there in G1 phase. At the beginning of S phase, specialized kinases phosphorylate Mcm and ORC, activating the former and inactivating the latter. A new prereplicative complex cannot form at the origin until the cell progresses to the next G1 phase, when the bound ORC has been dephosphorylated. Note that the eukaryotic Mcm helicase moves along the leading-strand template, whereas the bacterial helicase moves along the lagging-strand template (see Figure 5–25). As the forks begin to move, ORC is displaced, and new ORCs rapidly bind to the newly replicated origins.
The protein kinases that trigger DNA replication simultaneously prevent assembly of new prereplicative complexes until the next M phase resets the entire cycle (for details, see pp. 974–975). They do this, in part, by phosphorylating ORC, rendering it unable to accept new helicases. This strategy provides a single window of opportunity for prereplicative complexes to form (G1 phase, when kinase activity is low) and a second window for them to be activated and subsequently disassembled (S phase, when kinase activity is high). Because these two phases of the cell cycle are mutually exclusive and occur in a prescribed order, each origin of replication can fire once and only once during each cell cycle.
Features of the Human Genome That Specify Origins of Replication Remain to Be Discovered