life11e_ch13

The two DNA strands grow differently at the replication fork

The DNA at the replication fork—the site where DNA unwinds to expose the bases so that they can act as templates—opens up like a zipper in one direction. Study Figure 13.14 and try to imagine what is happening over a short period of time. Remember that the two DNA strands are antiparallel; that is, the 3′ end of one strand is paired with the 5′ end of the other.

Figure 13.14 The Two New Strands Form in Different Ways As the parent DNA unwinds, both new strands are synthesized in the 5′-to-3′ direction, although their template strands are antiparallel. The leading strand grows continuously forward, but the lagging strand grows in short discontinuous stretches called Okazaki fragments. Eukaryotic Okazaki fragments are hundreds of nucleotides long, with gaps between them.

Animation 13.4 Leading and Lagging Strand Synthesis

www.life11e.com/a13.4

One newly replicating strand (the leading strand) is oriented so that it can grow continuously at its 3′ end as the fork opens up.
The other new strand (the lagging strand) is oriented so that as the fork opens up, its exposed 3′ end gets farther and farther away from the fork, and an unreplicated gap is formed. This gap would get bigger and bigger if there were not a special mechanism to overcome this problem.

Synthesis of the lagging strand requires the synthesis of relatively small, discontinuous stretches of DNA (100–200 nucleotides in eukaryotes; 1,000–2,000 nucleotides in prokaryotes). These discontinuous stretches are synthesized just as the leading strand is, by the addition of new nucleotides one at a time to the 3′ end of the new strand, but the synthesis of this new strand moves in the direction opposite to that in which the replication fork is moving. These stretches of new DNA are called Okazaki fragments (after their discoverer, the Japanese biochemist Reiji Okazaki). While the leading strand grows continuously “forward,” the lagging strand grows in shorter, “backward” stretches with gaps between them.

279

A single primer is needed for synthesis of the leading strand, but each Okazaki fragment requires its own primer to be synthesized by the primase. In bacteria, DNA polymerase III then synthesizes an Okazaki fragment by adding nucleotides to one primer until it reaches the primer of the previous fragment (Figure 13.15). At this point, DNA polymerase I removes the old primer and replaces it with DNA. Left behind is a tiny nick—the final phosphodiester linkage between the adjacent Okazaki fragments is missing. The enzyme DNA ligase catalyzes the formation of that bond, linking the fragments and making the lagging strand whole.

Figure 13.15 The Lagging Strand Story In bacteria, DNA polymerase I and DNA ligase cooperate with DNA polymerase III to complete the complex task of synthesizing the lagging strand.

DNA replication involves remarkable teamwork among various proteins that act on the DNA strands. Let’s review the proteins involved in DNA replication in the order of their activity at the replication fork:

DNA helicase unwinds the double helix and separates the two strands.
Single-strand binding proteins bind to separated strands and prevent them from re-forming the double helix.
DNA primase makes RNA primers.
DNA polymerase links new nucleotides to form the new DNA strands and removes the primers.
DNA ligase connects Okazaki fragments made by DNA polymerase to one another.

280

Working together, these proteins make new DNA at a rate in excess of 1,000 base pairs per second, committing errors in fewer than 1 base in a million.

A SLIDING CLAMP INCREASES THE RATE OF DNA REPLICATION How do DNA polymerases work so fast? We saw in Key Concept 8.3 that an enzyme catalyzes a chemical reaction:

Substrate binds to enzyme → one product is formed → enzyme is released → cycle repeats

DNA replication would not proceed as rapidly as it does if it went through such a cycle for each nucleotide. Instead, DNA polymerases are processive—that is, they catalyze the formation of many phosphodiester linkages each time they bind to a DNA molecule:

Substrates bind to enzyme → many products are formed → enzyme is released → cycle repeats

281

The DNA polymerase–DNA complex is stabilized by a sliding DNA clamp, which has multiple identical subunits assembled into a doughnut shape (Figure 13.16). The doughnut’s “hole” is just large enough to encircle the DNA double helix, along with a thin layer of water molecules for lubrication. The clamp binds to the DNA polymerase–DNA complex, keeping the enzyme and the DNA associated tightly with each other. If the clamp is absent, DNA polymerase dissociates from DNA after forming 20 to 100 phosphodiester linkages. With the clamp, it can polymerize up to 50,000 nucleotides before it detaches.

Figure 13.16 A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization The clamp increases the efficiency of polymerization by keeping the enzyme bound to the substrate, so the enzyme does not have to repeatedly bind to template and substrate.

DNA IS THREADED THROUGH A REPLICATION COMPLEX So far, you are probably envisioning DNA replication as a locomotive (the replication complex) moving along a railroad track (the DNA). But this is not so. Commonly in eukaryotes, the replication complexes seem to be stationary, attached at specific positions in the nucleus. It is the DNA that moves, essentially sliding into the replication complex as one double-stranded molecule and emerging as two double-stranded molecules.