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Modern Genetic Analysis Chapter 4. The Inheritance of Genes
DNA Replication

In both prokaryotes and eukaryotes, DNA replication occurs as a prelude to cell division. This DNA replication phase is called the S (synthesis) phase. The two daughter DNA molecules formed from replication eventually become chromosomes in their own right in the daughter cells.

As with all phenomena that involve nucleic acids, the basic machinery of DNA replication depends on complementarity of DNA molecules and on the ability of proteins to form specific interactions with DNA of specific sequences.

Semiconservative Replication

Figure 4-1 diagrams the mechanism for DNA replication in prokaryotes and eukaryotes. Imagine that the double helix is like a zipper that unzips, starting at one end (the bottom in this figure). We can see that the unwinding of the two strands exposes single bases on each strand, and each exposed base has the potential to pair with free nucleotides in solution. (These newly added nucleotides come from a pool that has been chemically synthesized in the cytoplasm.) Because base-pairing rules are strict, each exposed base can pair only with its complementary base. Because of this base complementarity, each of the two single strands acts as a template (an alignment guide) to re-form a double helix identical with the original. This type of replication is termed semiconservative because each daughter double helix contains one parental and one newly synthesized strand. If DNA molecules are allowed to replicate in nucleotides containing rare isotopes (which act as tags), it can be shown experimentally that the daughter molecules contain one strand with the normal isotope and one strand with the rare form (see Genetics in Process 4-1).

The Polymerization Process

First, let's explore a general overview of the polymerization events occurring during the replication process. The enzyme that catalyzes the polymerization of nucleotides is DNA polymerase. This enzyme works by adding deoxyribonucleotides to the 3 end of a growing nucleotide chain, using a single-stranded DNA template (Figure 4-2). Recall from Chapter 3 that RNA polymerase acts in a similar way, adding ribonucleotides to a 3-growing RNA molecule. The substrates for DNA polymerase are the triphosphate forms of the deoxynucleotides dATP, dGTP, dCTP, and dTTP. DNA polymerase acts at the replication fork, the zone where the DNA is unwinding and exposing single strands to act as templates. Because the nucleotide polymerization catalyzed by DNA polymerase is always at the 3 growing tip, new synthesis can occur in a smooth, continuous manner on one template only; this new strand is called the leading strand (Figure 4-3). Synthesis on the other template also takes place at 3 growing tips but in short stretches running the "wrong way" (since for this strand, the 5 3 direction is away from the replication fork). In essence, for this strand DNA polymerase runs out of exposed template and has to wait until the replication fork exposes more DNA before it can synthesize another short stretch of DNA. These short stretches, called Okazaki fragments, are later joined together by an enzyme called DNA ligase. The new strand thus formed is called the lagging strand.

Many active proteins are needed to carry out the general replication process just described. The interacting components of replication in the bacterium Escherichia coli are shown in Figure 4-4 on the next page. The main polymerase is DNA polymerase III (pol III), which catalyzes 3 nucleotide addition at the replication fork. However, polymerases of this type need to add nucleotides to an already existing nucleotide chain. Therefore at the beginning of replication on both the leading and the lagging strands, short stretches of RNA are synthesized to act as polymerization starters or primers. On the leading strand, only one initial primer is needed because after the initial priming, continuous addition can use the growing DNA strand as the primer. However, on the lagging strand every Okazaki fragment needs its own primer. The primers are synthesized by a set of proteins called a primosome, of which a central component is an enzyme primase, a type of RNA polymerase. Removal of the RNA primers and filling in of the gaps left by their removal with DNA is performed by a different DNA polymerase, pol I. After pol I has done its job, ligase joins the 3 end of the gap-filling DNA to the 5 end of the downstream Okazaki fragment.

The movement of the replication fork is accomplished by the enzyme helicase, which breaks hydrogen bonds between the paired bases and unwinds the double helix ahead of the advancing DNA polymerase. The single strands of DNA so created are prevented from rejoining by single-strand binding proteins. As the DNA is unwound, it tends to become supercoiled, a process similar to the one we observe when trying to pull apart two strands of a piece of string or rope. The double helix is returned to its relaxed state by the action of another enzyme, gyrase, which is a type of topoisomerase. This class of enzymes can cut and rejoin DNA strands, allowing them to "pass through" each other, looking like a magician interlocking and separating steel rings.

Origins of Replication

In the genomes of prokaryotes and eukaryotes, replication begins from specific nucleotide sequences recognized by the replication apparatus; these are called origins of replication. Synthesis then proceeds bidirectionally, with two forks moving outward in opposite directions, as shown in Figure 4-5a. The replicated double helices that are being produced by each origin of replication elongate and eventually join each other. When replication of the two strands is complete, two identical daughter molecules of DNA result. In the eukaryotic chromosome these replicas are called sister chromatids (Figure 4-5b). Note that the term chromatid is applied only temporarily. Chromatids are in fact bona fide chromosomes, and they reassume this identity after cell division.

MESSAGE
DNA replicates semiconservatively. With separated single strands of the double helix as templates, nucleotides are polymerized at the 3 ends of new chains. Addition proceeds continuously in the leading strand but occurs in short bursts in the lagging strand.

© 1999 by W. H. Freeman and Company. All rights reserved.