34.1 DNA Is Replicated by Polymerases

✓ 3 Identify the enzymes that take part in the process of DNA replication.

The full replication machinery in a cell comprises more than 20 proteins engaged in intricate and coordinated interplay. The key enzymes are called DNA polymerases, which promote the formation of the phosphodiester linkages joining units of the DNA backbone. E. coli has five DNA polymerases, designated by roman numerals, that participate in DNA replication and repair (Table 34.1). We will focus our attention on two of the better understood polymerases: DNA polymerase I and DNA polymerase III.

Table 34.1 E. coli DNA polymerases
Note: Polymerases II, IV, and V can replicate through regions of damaged DNA (Chapter 35). They are called translesion polymerases or error-prone polymerases.

DNA Polymerase Catalyzes Phosphodiester-Linkage Formation

DNA polymerases catalyze the step-by-step addition of deoxyribonucleotides to a DNA strand (Figure 34.1). The reaction, in its simplest form, is

where dNTP stands for any deoxyribonucleotide and PPi is a pyrophosphate ion. DNA synthesis has the following characteristics:

DID YOU KNOW?

A template is a sequence of nucleic acids that determines the sequence of a complementary nucleic acid.

DID YOU KNOW?

A primer is the initial segment of a polymer that is to be extended on which elongation depends.

  1. The reaction requires all four activated precursors—that is, the deoxynucleoside 5′-triphosphates dATP, dGTP, dCTP, and TTP—as well as the Mg2+ ion.

  2. The new DNA strand is assembled directly on a preexisting DNA template. DNA polymerases catalyze the formation of a phosphodiester linkage efficiently only if the base on the incoming nucleoside triphosphate is complementary to the base on the template strand. Thus, DNA polymerase is a template-directed enzyme that synthesizes a product with a base sequence complementary to that of the template.

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    Figure 34.1: A polymerization reaction catalyzed by DNA polymerases.

  3. DNA polymerases require a primer to begin synthesis. A primer strand having a free 3′-OH group must be already bound to the template strand. The strand-elongation reaction catalyzed by DNA polymerases is a nucleophilic attack by the 3′-OH end of the growing strand on the innermost phosphorus atom of deoxynucleoside triphosphate (Figure 34.2). A phosphodiester bridge is formed and pyrophosphate is released. The subsequent hydrolysis of pyrophosphate to yield two ions of orthophosphate (Pi) by pyrophosphatase drives the polymerization forward. Elongation of the DNA strand proceeds in the 5′-to-3′ direction.

    Figure 34.2: Strand-elongation reaction. DNA polymerases catalyze the formation of a phosphodiester bridge. Elongation of the DNA strand proceeds in the 5′-to-3′ direction.

  4. Many DNA polymerases are able to correct mistakes in DNA by removing mismatched nucleotides. These polymerases have a distinct nuclease activity that allows them to excise incorrect bases by a separate reaction. For instance, DNA polymerase I has three distinct active sites: the polymerase site, a 3′ → 5′ exonuclease site, and a 5′ → 3′ exonuclease site. The 3′ → 5′ nuclease activity contributes to the remarkably high fidelity of DNA replication, which has an error rate of less than 10−8 per base pair. We will consider the function of the 5′ → 3′ nuclease activity shortly.

The three-dimensional structures of a number of DNA polymerase enzymes are known. The first such structure to be elucidated was a fragment of E. coli DNA polymerase I, called the Klenow fragment, that consisted of the polymerase and the 3′ → 5′ exonuclease. The shape approximates the shape of a right hand with domains that are referred to as the fingers, the thumb, and the palm (Figure 34.3). The finger and thumb domains wrap around DNA—much as your own fingers wrap around a baseball bat—and hold it across the enzyme’s active site, which is located in the palm domain.

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Figure 34.3: DNA polymerase structure. The first DNA polymerase structure determined was that of a fragment of E. coli DNA polymerase I called the Klenow fragment. Notice that, like other DNA polymerases, the polymerase unit resembles a right hand with fingers (blue), palm (yellow), and thumb (red). The Klenow fragment also includes an exonuclease domain that removes incorrect nucleotide bases.

The Specificity of Replication Is Dictated by the Complementarity of Bases

Because DNA is the repository of genetic information, it must be replicated with high accuracy. Each base added to the growing strand should be the Watson–Crick complement of the base in the corresponding position in the template strand. The binding of the dNTP containing the proper base is favored by the formation of a base pair, which is stabilized by specific hydrogen bonds. The binding of a noncomplementary base is less likely because the interactions are energetically weaker. Although the hydrogen bonds linking two complementary bases make a significant contribution to the fidelity of DNA replication, overall shape complementarity also is crucial. Studies show that a nucleotide with a base that is very similar in shape to adenine but lacks the ability to form base-pairing hydrogen bonds can still direct the incorporation of thymidine, both in vitro and in vivo (Figure 34.4). However, DNA polymerases replicate DNA even more faithfully than can be accounted for by these interactions alone.

Figure 34.4: Shape complementarity. The base analog on the right has the same shape as adenosine, but groups that form hydrogen bonds between base pairs have been replaced by groups (shown in red) not capable of hydrogen bonding. Nonetheless, studies reveal that, when incorporated into the template strand, this analog directs the insertion of thymidine in DNA replication.

What is the basis of DNA polymerase’s low error rate? The answer to this question is complex, but one important factor is induced fit—the change in the structure of the enzyme when it binds the correct nucleotide. DNA polymerases close down around the incoming nucleoside triphosphate (dNTP), as shown in Figure 34.5. The binding of a dNTP into the active site of a DNA polymerase triggers a conformational change: the finger domain rotates to form a tight pocket into which only a properly shaped base pair will readily fit.

Figure 34.5: Shape selectivity. The binding of a deoxyribonucleoside triphosphate (dNTP) to DNA polymerase induces a conformational change, generating a tight pocket for the base pair consisting of the dNTP and its partner on the template strand. Such a conformational change is possible only when the dNTP corresponds to the Watson–Crick partner of the template base. Only the part of the polymerase directly participating in nucleotide binding is displayed (yellow ribbons). The green ball represents the Mg2+ ion.

!clinic! CLINICAL INSIGHT: The Separation of DNA Strands Requires Specific Helicases and ATP Hydrolysis

For a double-stranded DNA molecule to replicate, the two strands of the double helix must be separated from each other, at least at the site of replication. This separation allows each strand to act as a template on which a new DNA strand can be assembled. Specific enzymes, termed helicases, utilize the energy of ATP hydrolysis to power strand separation. Pathological conditions that result from defects in helicase activity attest to the importance of helicases. For instance, Werner syndrome, which is characterized by premature aging, is due to a defect in helicase activity (Figure 34.6).

Figure 34.6: Werner syndrome. A very rare disorder, Werner syndrome is characterized by premature aging. (A) people afflicted by Werner syndrome develop normally until adolescence, at which time they begin to age rapidly. (B) By age 40, they look several decades older.

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Helicases are a large and diverse family of enzymes taking part in many biological processes. The helicases in DNA replication are typically oligomers containing six subunits that form a ring structure. Each subunit has a loop that extends toward the center of the ring structure and interacts with DNA. A possible mechanism for the action of a helicase is shown in Figure 34.7. Two subunits are bound to ATP, two to ADP and Pi, and two are initially free of nucleotides. One of the strands of the double helix passes through the hole in the center of the helicase, bound to the loops on two adjacent subunits, one of which has bound ATP and the other of which has bound ADP + Pi. The binding of ATP to the subunit that initially had no bound nucleotides results in a conformational change within the entire hexamer, leading to the release of ADP + Pi from two subunits and the binding of the single-stranded DNA by one of the subunits that has just bound ATP. This conformational change pulls the DNA through the center of the hexamer. The protein acts as a wedge, forcing the two strands of the double helix apart. This cycle then repeats itself, moving two bases along the DNA strand with each cycle.

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Figure 34.7: Helicase mechanism. One of the strands of the double helix passes through the hole in the center of the helicase, bound to the loops of two adjacent subunits. Two of the subunits do not contain bound nucleotides. On the binding of ATP to these two subunits and the release of ADP + Pi from two other subunits, the helicase hexamer undergoes a conformational change, pulling the DNA through the helicase. The helicase acts as a wedge to force separation of the two strands of DNA.

Topoisomerases Prepare the Double Helix for Unwinding

Most naturally occurring DNA molecules are negatively supercoiled. What is the basis for this prevalence? As discussed in Chapter 33, negative supercoiling arises from the unwinding or underwinding of DNA. In essence, negative supercoiling prepares DNA for processes requiring separation of the DNA strands, such as replication. The presence of supercoils in the immediate area of unwinding would, however, make unwinding difficult (Figure 34.8). Therefore, negative supercoils must be continuously removed, and the DNA relaxed, as the double helix unwinds.

Figure 34.8: Consequences of strand separation. DNA must be locally unwound to expose single-stranded templates for replication. This unwinding puts a strain on the molecule by causing the overwinding of nearby regions.

DID YOU KNOW?

To gyrate is to move in a circle or spiral or to revolve, usually about a fixed point or on an axis.

Enzymes called topoisomerases introduce or eliminate supercoils by temporarily cleaving DNA. Type I topoisomerases catalyze the relaxation of supercoiled DNA, a thermodynamically favorable process. Type II topoisomerases utilize free energy from ATP hydrolysis to add negative supercoils to DNA. In bacteria, type II topoisomerase is called DNA gyrase.

!clinic! CLINICAL INSIGHT: Bacterial Topoisomerase Is a Therapeutic Target

DNA gyrase is the target of several antibiotics that inhibit this bacterial topoisomerase much more than the eukaryotic one. Novobiocin blocks the binding of ATP to gyrase. Nalidixic acid and ciprofloxacin, in contrast, interfere with the breakage and rejoining of DNA strands. These two gyrase inhibitors are widely used to treat urinary-tract and other infections.

Ciprofloxacin, more commonly known as “cipro,” became a “celebrity” in the United States, owing to the anthrax poisonings in the fall of 2001 (Figure 34.9). It is a potent broad-spectrum antibiotic that prevents anthrax poisoning by preventing the growth of Bacillus anthracis if taken early enough after infection.

Figure 34.9: Anthrax poisoning in the United States in 2001. A mailbox in Hamilton, New Jersey, is covered in plastic because of possible contamination with B. anthracis spores. In the background, the flag flies at half-staff to honor two Hamilton postal workers who died from anthrax poisoning.

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Many Polymerases Proofread the Newly Added Bases and Excise Errors

Many polymerases further enhance the fidelity of replication by the use of proofreading mechanisms. One polymerase from E. coli, DNA polymerase I used in DNA replication and repair, displays an exonuclease activity in addition to the polymerase activity. The exonuclease removes mismatched nucleotides from the 3′ end of DNA by hydrolysis. If the wrong nucleotide is inserted, the malformed product is not held as tightly in the polymerase active site. It is likely to flop about because of the weaker hydrogen bonding and to find itself in the exonuclease active site, where the trespassing nucleotide is removed (Figure 34.10). This flopping is the result of Brownian motion.

Figure 34.10: Proofreading. The growing DNA strand occasionally leaves the polymerase site, especially if there is improper base-pairing, and migrates to the active site of exonuclease. There, one or more nucleotides are excised from the newly synthesized strand, removing potentially incorrect bases.

!quickquiz! QUICK QUIZ 1

What are the three key enzymes required for DNA synthesis, and what biochemical challenges to replication do they address?

How does the enzyme sense whether a newly added base is correct? First, an incorrect base will not pair correctly with the template strand and will be unlikely to be linked to the new strand. Second, even if an incorrect base is inserted into the new strand, it is likely to be deleted. After the addition of a new nucleotide, the DNA is pulled by one base pair into the enzyme. If an incorrect base is incorporated, the enzyme stalls owing to the structural disruption caused by the presence of a non-Watson–Crick base pair in the enzyme, and the pause provides additional time for the strand to wander into the exonuclease site. There is a cost to this editing function, however: DNA polymerase I removes approximately 1 correct nucleotide in 20. Although the removal of correct nucleotides is slightly wasteful energetically, proofreading increases the accuracy of replication by a factor of approximately 1000.