Figure 5–4 DNA synthesis catalyzed by DNA polymerase. (A) DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3ʹ-OH end of a polynucleotide chain, the growing primer strand that is paired to an existing template strand. The newly synthesized DNA strand therefore polymerizes in the 5ʹ-to-3ʹ direction as shown also in the previous figure. Because each incoming deoxyribonucleoside triphosphate must pair with the template strand to be recognized by the DNA polymerase, this strand determines which of the four possible deoxyribonucleotides (A, C, G, or T) will be added. The reaction  is driven by a large, favorable free-energy change, caused by the release of pyrophosphate and its subsequent hydrolysis  to two molecules of inorganic phosphate. (B) Structure of DNA polymerase complexed wth DNA (orange), as determined by x-ray crystallography (Movie 5.1). The template DNA strand is the longer strand and the newly synthesized DNA is the shorter. (C) Schematic diagram of DNA polymerase, based on the structure in (B). The proper base-pair geometry of a correct incoming deoxyribonucleoside triphosphate causes the polymerase to tighten around the base pair, thereby initiating the nucleotide addition reaction (middle diagram (C)). Dissociation of pyrophosphate relaxes the polymerase, allowing translocation of the DNA by one nucleotide so the active site of the polymerase is ready to receive the next deoxyribonucleoside triphosphate.
Analyses carried out in the early 1960s on whole replicating chromosomes revealed a localized region of replication that moves progressively along the parental DNA double helix. Because of its Y-shaped structure, this active region is called a replication fork (Figure 5–6). At the replication fork, a multienzyme complex that contains the DNA polymerase synthesizes the DNA of both new daughter strands.
Initially, the simplest mechanism of DNA replication seemed to be the continuous growth of both new strands, nucleotide by nucleotide, at the replication fork as it moves from one end of a DNA molecule to the other. But because of the antiparallel orientation of the two DNA strands in the DNA double helix (see Figure 5–2), this mechanism would require one daughter strand to polymerize in the 5ʹ-to-3ʹ direction and the other in the 3ʹ-to-5ʹ direction. Such a replication fork would require two distinct types of DNA polymerase enzymes. However, as attractive as this model might be, the DNA polymerases at replication forks can synthesize only in the 5ʹ-to-3ʹ direction.
How, then, can a DNA strand grow in the 3ʹ-to-5ʹ direction? The answer was first suggested by the results of an experiment performed in the late 1960s. Researchers added highly radioactive 3H-thymidine to dividing bacteria for a few seconds, so that only the most recently replicated DNA—that just behind the replication fork—became radiolabeled. This experiment revealed the transient existence of pieces of DNA that were 1000–2000 nucleotides long, now commonly known as Okazaki fragments, at the growing replication fork. (Similar replication
Figure 5–5 The semiconservative nature of DNA replication. In a round of replication, each of the two strands of DNA is used as a template for the formation of a complementary DNA strand. The original strands therefore remain intact through many cell generations.
intermediates were later found in eukaryotes, where they are only 100–200 nucleotides long.) The Okazaki fragments were shown to be polymerized only in the 5ʹ-to-3ʹ chain direction and to be joined together after their synthesis to create long DNA chains.