The genome of E. coli is contained in a single circular DNA molecule of 4.6 × 106 nucleotide pairs. DNA replication begins at a single origin of replication, and the two replication forks assembled there proceed (at approximately 1000 nucleotides per second) in opposite directions until they meet up roughly halfway around the chromosome (Figure 5–24). The only point at which E. coli can control DNA replication is initiation: once the forks have been assembled at the origin, they synthesize DNA at relatively constant speed until replication is finished. Therefore, it is not surprising that the initiation of DNA replication is highly regulated. The process begins when initiator proteins (in their ATP-bound state) bind in multiple copies to specific DNA sites located at the replication origin, wrapping the DNA around the proteins to form a large protein–DNA complex that destabilizes the adjacent double helix. This complex then attracts two DNA helicases, each bound to a helicase loader, and these are placed around adjacent DNA single strands whose bases have been exposed by the assembly of the initiator protein–DNA complex. The helicase loader is analogous to the clamp loader we encountered above; it has the additional job of keeping the helicase in an inactive form until it is properly loaded onto a nascent replication fork. Once the helicases are loaded, the loaders dissociate and the helicases begin to unwind DNA, exposing enough single-strand DNA for DNA primase to synthesize the first RNA primers (Figure 5–25). This quickly leads to the assembly of remaining proteins to create two replication forks, with replication machines that move, with respect to the replication origin, in opposite directions. They continue to synthesize DNA until all of the DNA template downstream of each fork has been replicated.
In E. coli, the interaction of the initiator protein with the replication origin is carefully regulated, with initiation occurring only when sufficient nutrients are available for the bacterium to complete an entire round of replication. Initiation is also controlled to ensure that only one round of DNA replication occurs for each cell division. After replication is initiated, the initiator protein is inactivated by hydrolysis of its bound ATP molecule, and the origin of replication experiences a “refractory period.” The refractory period is caused by a delay in the methylation of newly incorporated A nucleotides in the origin (Figure 5–26). Initiation cannot occur again until the A’s are methylated and the initiator protein is restored to its ATP-bound state.
Eukaryotic Chromosomes Contain Multiple Origins of Replication
We have seen how two replication forks begin at a single replication origin in bacteria and proceed in opposite directions, moving away from the origin until all of the DNA in the single circular chromosome is replicated. The bacterial genome is sufficiently small for these two replication forks to duplicate the genome in about 30 minutes. Because of the much greater size of most eukaryotic chromosomes, a different strategy is required to allow their replication in a timely manner.
A method for determining the general pattern of eukaryotic chromosome replication was developed in the early 1960s. Human cells growing in culture are labeled for a short time with 3H-thymidine so that the DNA synthesized during this period becomes highly radioactive. The cells are then gently lysed, and the DNA is stretched on the surface of a glass slide coated with a photographic emulsion. Development of the emulsion reveals the pattern of labeled DNA through a technique known as autoradiography. The time allotted for radioactive labeling is chosen to allow each replication fork to move several micrometers along the DNA, so that the replicated DNA can be detected in the light microscope as lines of silver grains, even though the DNA molecule itself is too thin to be visible.