We have seen how a set of replication proteins rapidly and accurately generates two daughter DNA double helices behind a replication fork. But how is this replication machinery assembled in the first place, and how are replication forks created on an intact, double-strand DNA molecule? In this section, we discuss how cells initiate DNA replication and how they carefully regulate this process to ensure that it takes place not only at the proper positions on the chromosome but also at the appropriate time in the life of the cell. We also discuss a few of the special problems that the replication machinery in eukaryotic cells must overcome. These include the need to replicate the enormously long DNA molecules found in eukaryotic chromosomes, as well as the difficulty of copying DNA molecules that are tightly complexed with histones in nucleosomes.
As discussed previously, the DNA double helix is normally very stable: the two DNA strands are locked together firmly by many hydrogen bonds formed between the bases on each strand. To begin DNA replication, the double helix must first be opened up and the two strands separated to expose unpaired bases. As we shall see, the process of DNA replication is begun by special initiator proteins that bind to double-strand DNA and pry the two strands apart, breaking the hydrogen bonds between the bases.
leading strand of fork 1 of fork 2 leading strand lagging strand of fork 1 of fork 2
Figure 5–23 A replication bubble formed by replication-fork initiation. This diagram outlines the major steps in the initiation of replication forks at replication origins. The structure formed at the last step, in which both strands of the parental DNA helix have been separated from each other and serve as templates for DNA synthesis, is called a replication bubble.
Figure 5–24 DNA replication of a bacterial genome. It takes E. coli about 30 minutes to duplicate its genome of 4.6 × 106 nucleotide pairs. For simplicity, no Okazaki fragments are shown on the lagging strand. What happens as the two replication forks approach each other and collide at the end of the replication cycle is not well understood, although the replication machines are disassembled as part of the process.
The positions at which the DNA helix is first opened are called replication origins (Figure 5–23). In simple cells like those of bacteria or yeast, origins are specified by DNA sequences several hundred nucleotide pairs in length. This DNA contains both short sequences that attract initiator proteins and stretches of DNA that are especially easy to open. We saw in Figure 4–4 that an A-T base pair is held together by fewer hydrogen bonds than a G-C base pair. Therefore, DNA rich in A-T base pairs is relatively easy to pull apart, and regions of DNA enriched in A-T base pairs are typically found at replication origins.
Although the basic process of replication-fork initiation depicted in Figure 5–23 is fundamentally the same for bacteria and eukaryotes, the detailed way in which this process is performed and regulated differs between these two groups of organisms. We first consider the simpler and better-understood case in bacteria and then turn to the more complex situation found in yeasts, mammals, and other eukaryotes.
Bacterial Chromosomes Typically Have a Single Origin of DNA Replication