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Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman; 2000.

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An Introduction to Genetic Analysis. 7th edition.

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Mechanism of DNA replication

Watson and Crick first reasoned that complementary base pairing provides the basis of fidelity in DNA replication; that is, that each base in the template strand dictates the complementary base in the new strand. However, we now know that the process of DNA replication is very complex and requires the participation of many different components. Let’s examine each of these components and see how they fit together to produce our current picture of DNA synthesis in E. coli, the best-studied cellular replication system. In the preceding section, we introduced the concept of the replication fork. Figure 8-20 gives a detailed schematic view of fork movement during DNA replication; we can refer to this illustration as we consider each component of the process.

Figure 8-20. DNA replication fork.

Figure 8-20

DNA replication fork.

DNA polymerases

In the late 1950s, Arthur Kornberg successfully identified and purified the first DNA polymerase, an enzyme that catalyzes the replication reaction.

Image ch8e2.jpg

This reaction works only with the triphosphate forms of the nucleotides (such as deoxyadenosine triphosphate, or dATP). The total amount of DNA at the end of the reaction can be as much as 20 times the amount of original input DNA, so most of the DNA present at the end must be progeny DNA. Figure 8-21 depicts the chain-elongation reaction, or polymerization reaction, catalyzed by DNA polymerases. We now know that there are three DNA polymerases in E. coli. The first enzyme that Kornberg purified is called DNA polymerase I or pol I. This enzyme has three activities, which appear to be located in different parts of the molecule:

Figure 8-21. Chain-elongation reaction catalyzed by DNA polymerase.

Figure 8-21

Chain-elongation reaction catalyzed by DNA polymerase.

1.

a polymerase activity, which catalyzes chain growth in the 5′ → 3′ direction;

2.

a 3′ → 5′ exonuclease activity, which removes mismatched bases; and

3.

A 5′ → 3′ exonuclease activity, which degrades double-stranded DNA.

Subsequently, two additional polymerases, pol II and pol III, were identified in E. coli. Pol II may repair damaged DNA, although no particular role has been assigned to this enzyme. Pol III, together with pol I, has a role in the replication of E. coli DNA (Figure 8-20). The complete complex, or holoenzyme, of pol III contains at least 20 different polypeptide subunits, although the catalytic “core” consists of only three subunits, alpha (α), epsilon (ϵ), and theta (θ). The pol III complex will complete the replication of single-stranded DNA if there is at least a short segment of duplex already present. The short oligonucleotide that creates the duplex is termed a primer.

Prokaryotic origins of replication

E. coli replication begins from a fixed origin but then proceeds bidirectionally (with moving forks at both ends of the replicating piece), as shown in Figure 8-22, ending at a site called the terminus. The unique origin is termed oriC and is located at 83 minutes on the genetic map. It is 245 bp long and has several components, as illustrated in Figure 8-23. First, there is a side-by-side, or tandem, set of 13-bp sequences, which are nearly identical. There is also a set of binding sites for a protein, the DnaA protein. An initial step in DNA synthesis is the unwinding of the DNA at the origin in response to binding of the DnaA protein. The consequences of bidirectional replication can be seen in Figure 8-24, which gives a larger view of DNA replication.

Figure 8-22. Chain-elongation reaction catalyzed by DNA polymerase.

Figure 8-22

Chain-elongation reaction catalyzed by DNA polymerase. (From L. Stryer, Biochemistry, 4th ed. Copyright © 1995 by Lubert Stryer.)

Figure 8-24. Bidirectional replication of a circular DNA molecule.

Figure 8-24

Bidirectional replication of a circular DNA molecule.

Eukaryotic origins of replication

Bacteria such as E. coli usually require a 40-minute replication-division cycle, but, in eukaryotes, the cycle can vary from 1.4 hours in yeast to 24 hours in cultured animal cells and may last from 100 to 200 hours in some cells. Eukaryotes have to solve the problem of coordinating the replication of more than one chromosome, as well as replicating the complex structure of the chromosome itself (see Chapter 3 for a description of chromosome structure).

In eukaryotes, replication proceeds from multiple points of origin. This process can be demonstrated by a procedure in which a eukaryotic cell is briefly exposed to [3H]thymidine, in a step called a pulse exposure, and is then provided an excess of “cold” (unlabeled) thymidine, in a step called the chase; the DNA is then extracted, and autoradiographs are made. Figure 8-25 shows the results of such a procedure, with what appear to be distinct, simultaneously replicating regions along the DNA molecule. Replication appears to begin at several different sites on these eukaryotic chromosomes. Similarly, a pulse-and-chase study of DNA replication in polytene (giant) chromosomes of Drosophila by autoradiography reveals many replication regions within single chromosome arms (Figure 8-26). As yet there is no firm proof that these regions are indeed different starting points on a single DNA molecule. However, experiments in yeast indicate the existence of approximately 400 replication origins distributed among the 17 yeast chromosomes, and in humans there are estimated to be more than 10,000 growing forks.

Figure 8-25. A replication pattern in DNA revealed by autoradiography.

Figure 8-25

A replication pattern in DNA revealed by autoradiography. A cell is briefly exposed to [3H]thymidine (pulse) and then provided with an excess of nonradioactive (“cold”) thymidine (chase). DNA is spread on a slide and autoradiographed. (more...)

Figure 8-26. Replication pattern in a Drosophila polytene chromosome revealed by autoradiography.

Figure 8-26

Replication pattern in a Drosophila polytene chromosome revealed by autoradiography. Several points of replication are seen within a single chromosome, as indicated by the arrows.

Priming DNA synthesis

DNA polymerases can extend a chain but cannot start a chain. Therefore, as already mentioned, DNA synthesis must first be initiated with a primer, or short oligonucleotide, that generates a segment of duplex DNA. The primer in DNA replication can be seen in Figure 8-27 (see also Figure 8-20). RNA primers are synthesized either by RNA polymerase or by an enzyme termed primase. Primase synthesizes a short (approximately 30 bp long) stretch of RNA complementary to a specific region of the chromosome. The RNA chain is then extended with DNA by DNA polymerase. E. coli primase forms a complex with the template DNA, and additional proteins, such as DnaB, DnaT, Pri A, Pri B, and Pri C. The entire complex is termed a primosome (see Figure 8-20).

Figure 8-27. Initiation of DNA synthesis by an RNA primer.

Figure 8-27

Initiation of DNA synthesis by an RNA primer.

Leading strand and lagging strand

DNA polymerases synthesize new chains only in the 5′ → 3′ direction and therefore, because of the antiparallel nature of the DNA molecule, move in a 3′ → 5′ direction on the template strand. The consequence of this polarity is that while one new strand, the leading strand, is synthesized continuously, the other, the lagging strand, must be synthesized in short, discontinuous segments, as can be seen in Figure 8-28 (see also Figure 8-20). The addition of nucleotides along the template for the lagging strand must proceed toward the template’s 5′ end (because replication always moves along the template in a 3′ → 5′ direction so that the new strand can grow 5′ → 3′). Thus, the new strand must grow in a direction opposite that of the movement of the replication fork. As fork movement exposes a new section of lagging-strand template, a new lagging-strand fragment is begun and proceeds away from the fork until it is stopped by the preceding fragment. In E. coli, pol III carries out most of the DNA synthesis on both strands, and pol I fills in the gaps left in the lagging strand, which are then sealed by the enzyme DNA ligase. DNA ligases join broken pieces of DNA by catalyzing the formation of a phosphodiester bond between the 5′ phosphate end of a hydrogen-bonded nucleotide and an adjacent 3′ OH group, as shown in Figure 8-29. It is the only enzyme that can seal DNA chains. Figure 8-30 shows the lagging-strand synthesis and gap repair in detail. The primers for the discontinuous synthesis on the lagging strand are synthesized by primase (step a). The primers are extended by DNA polymerase (step b) to yield DNA fragments that were first detected by Reiji Okazaki and are termed Okazaki fragments. The 5′ → 3′ exonuclease activity of pol I removes the primers (step c) and fills in the gaps with DNA, which are sealed by DNA ligase (step d). One proposed mechanism that allows the same dimeric holoenzyme molecule to participate in both leading- and lagging-strand synthesis is shown in Figure 8-31. Here, the looping of the template for the lagging strand allows a single pol III dimer to generate both daughter strands. After approximately 1000 base pairs, pol III will release the segment of lagging-strand duplex and allow a new loop to be formed.

Figure 8-28. DNA synthesis proceeds by continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand.

Figure 8-28

DNA synthesis proceeds by continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand.

Figure 8-29. The reaction catalyzed by DNA ligase (Enz) joins the 3′-OH end of one fragment to the 5′ phosphate of the adjacent fragment (From H.

Figure 8-29

The reaction catalyzed by DNA ligase (Enz) joins the 3′-OH end of one fragment to the 5′ phosphate of the adjacent fragment (From H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell Biology, (more...)

Figure 8-30. The overall structure of a growing fork (top) and steps in the synthesis of the lagging strand.

Figure 8-30

The overall structure of a growing fork (top) and steps in the synthesis of the lagging strand. (From H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J. Darnell, Molecular Cell Biology, 3d ed. Copyright © 1995 by Scientific (more...)

Figure 8-31. The looping of the template for the lagging strand enables a dimeric DNA polymerase III holoenzyme at the replication fork to synthesize both daughter strands.

Figure 8-31

The looping of the template for the lagging strand enables a dimeric DNA polymerase III holoenzyme at the replication fork to synthesize both daughter strands. (Courtesy of A. Kornberg. From L. Stryer, Biochemistry, 4th ed. Copyright © 1995 by Lubert (more...)

Replication at chromosome tips

The ends of chromosomes present a special problem for the replication process. Figure 8-32 shows the problem: for the leading strand, the polynucleotide addition during replication can always extend to the end because it is automatically primed from behind. However, at the tip, the lagging strand reaches a point where its system of RNA priming cannot work, and an unpolymerized section remains and a shortened chromosome would be the result. To solve this problem, the tips of chromosomes, called telomeres, have adjacent repeats of simple DNA sequences. For example, in the ciliate Tetrahymena, there is repetition of the sequence TTGGGG; in humans, it is TTAGGG. These repeats do not code for an RNA or a protein product but nevertheless serve a definite function in replication. An enzyme called telomerase adds these simple repeat units to the chromosome ends. The telomerase protein is a member of a class of enzymes called reverse transcriptases, which are used in specialized situations to synthesize DNA from RNA. The telomerase carries a small RNA molecule, part of which acts as a template for the polymerization of the telomeric repeat unit that is added to the 3′ end. For example, in Tetrahymena, the RNA is 3′-AACCCC-5′, which acts as the template for the repeat unit, which is 5′-TTGGGG-3′ (Figure 8-33). The additional DNA is then able to act as template for synthesis on the lagging strand. This process counteracts the tendency to shorten at replication. Figure 8-34 demonstrates the positions of the telomeric DNA through in situ hybridization. An age-dependent decline in telomere length has been found in several somatic tissues in humans. In addition, human fibroblasts in culture show progressive telo-mere shortening up to their eventual death. Such observations have led to the telomere theory of aging, and the validity of this theory is now being tested.

Figure 8-32. The replication problem at chromosome ends.

Figure 8-32

The replication problem at chromosome ends. There is no way of priming the last section of the lagging strand, and a shortened chromosome would result.

Figure 8-33. Telomerase carries a short RNA molecule that acts as a template for the addition of the complementary DNA sequence at the 3′ end of the double helix.

Figure 8-33

Telomerase carries a short RNA molecule that acts as a template for the addition of the complementary DNA sequence at the 3′ end of the double helix. In the ciliate Tetrahymena, the DNA sequence added is TTGGGG.

Figure 8-34. Chromosomes probed in situ with a telomere-specific DNA probe that has been coupled to a substance that can fluoresce yellow under the microscope.

Figure 8-34

Chromosomes probed in situ with a telomere-specific DNA probe that has been coupled to a substance that can fluoresce yellow under the microscope. Each sister chromatid binds the probe at both ends. An unbroken nucleus is shown at the bottom of the photograph. (more...)

Helicases and topoisomerases

Helicases are enzymes that disrupt the hydrogen bonds that hold the two DNA strands together in a double helix. Hydrolysis of ATP drives the reaction. Among E. coli helicases are the DnaB protein and the Rep protein. The Rep protein may help to unwind the double helix ahead of the polymerase (refer to Figure 8-20). The unwound DNA is stabilized by the single-stranded binding (SSB) protein, which binds to the single-stranded DNA and retards reformation of the duplex.

The action of helicases during DNA replication generates twists in the circular DNA that need to be removed to allow replication to continue. Circular DNA can be twisted and coiled, much like the extra coils that can be introduced into a rubber band. This supercoiling can be created or relaxed by enzymes termed topoisomerases, an example of which is DNA gyrase (Figure 8-35). Topoisomerases can also induce (catenate) or remove (decatenate) knots, or links in a chain. There are two basic types of isomerases. Type I enzymes induce a single-stranded break into the DNA duplex. Type II enzymes cause a break in both strands. In E. coli, topo I and topo III are examples of type I enzymes, whereas gyrase is an example of a type II enzyme.

Figure 8-35. DNA-gyrase-catalyzed supercoiling.

Figure 8-35

DNA-gyrase-catalyzed supercoiling. Replicating DNA generates “positive” supercoils, depicted at the bottom of the diagram, as a result of rapid rotation of the DNA at the replication fork. DNA gyrase can nick and close phosphodiester bonds, (more...)

Untwisting of the DNA strands to open the replication fork causes extra twisting at other regions, and the supercoiling releases the strain of the extra twisting (Figure 8-36). During replication, gyrase is needed to remove positive supercoils ahead of the replication fork.

Figure 8-36. Swivel function of topoisomerase during replication.

Figure 8-36

Swivel function of topoisomerase during replication. Extra-twisted (positively supercoiled) regions accumulate ahead of the fork as the parental strands separate for replication. A topoisomerase is required to remove these regions, acting as a swivel (more...)

Exonuclease editing

Both DNA polymerase I and DNA polymerase III also possess 3′ → 5′ exonuclease activity, which serves a “proofreading” and “editing” function by searching for mismatched bases that were inserted erroneously during polymerization and excising them. The proofreading activity of pol III is the ϵ subunit, which must be bound to α for full proofreading activity (Figure 8-37). Strains lacking a functional ϵ have a higher mutation rate (see Chapter 16). Figure 8-38 shows the excision of a cytosine residue that has erroneously been paired with an adenine. As can be seen, hydrolysis takes place at the 5′ end of the mismatched base; removal of the incorrect base leaves a 3′-OH group on the preceding base, which is then free to continue the growing strand by accepting the correct nucleotide triphosphate (thymidine, in this case).

Figure 8-37. Proofreading by the pol III α–ϵ complex.

Figure 8-37

Proofreading by the pol III α–ϵ complex. (From A. Kornberg and T. A Baker, DNA Replication, 2d ed. Copyright © 1992 by W. H. Freeman and Company.)

Figure 8-38. The 3′ → 5′ exonuclease action of DNA polymerase III.

Figure 8-38

The 3′ → 5′ exonuclease action of DNA polymerase III.

Note that this exonuclease activity takes place at the 3′ end of the growing strand (and is therefore 3′ → 5′). The coordination of exonuclease activity with strand growth helps to explain why replication is in the 5′ → 3′ direction. As we saw earlier, new bases are added when the 3′ OH on the terminal deoxyribose of the growing strand attacks the high-energy phosphate of the nucleotide triphosphate that is being added (see Figure 8-21). Chain growth is thus 5′ → 3′. It is conceivable that replication could be in the 3′ → 5′ direction (in Figure 8-21, the 5′ triphosphate at the bottom would be the last base on the chain, and the 3′ OH that attacks it would be on the free nucleotide triphosphate about to be added to the strand). However, if replication were in this direction, there would be exonuclease excisions at the 5′ end of the strand. When a mismatched base was removed, a 5′ OH would be left at the end of the growing strand. The 3′ OH of an incoming nucleotide triphosphate would thus be facing this 5′ OH instead of the high-energy 5′ triphosphate necessary for bond formation. No bond would form and strand growth would stop. Therefore, replication is not in the 3′ → 5′ direction.

Eukaryotic DNA polymerases

There are at least five DNA polymerases, α, β, γ, δ, and ϵ, in higher eukaryotes. Polymerases α and δ in the nucleus have roles similar to pol I in E. coli. Polymerase β has a role in DNA repair and gap filling. The γ polymerase is found in mitochondria and appears to take part in replication of mitochondrial DNA.

Experimental applications of base-sequence complementarity

In 1960, Paul Doty and Julius Marmur observed that, when DNA is heated to 100°C, all the hydrogen bonds between the complementary strands are destroyed, and the DNA becomes single stranded (Figure 8-39). If the solution is cooled slowly, some double-stranded DNA is formed. This reannealing process occurs when two single strands happen to collide in such a way that the complementing base sequences can align and reconstitute the original double helix. The annealing of complementary strands is very specific, as shown in Figure 8-39, and forms the basis of many important techniques in molecular biology, such as the identification of specific DNA segments by hybridization and the isolation of specific DNA fragments that are used in cloning, as explained in Chapter 12. Figure 8-40a shows the basic profile of a typical DNA strand as the temperature increases. At a temperature characteristic for each DNA segment, the DNA starts to denature. The melting temperature, T m, is defined as the temperature at which half the molecules are denatured into single strands. The melting temperature depends on the proportion of G:C base pairs, because they are held together by three hydrogen bonds, whereas A:T base pairs are held together by two hydrogen bonds. The higher the G:C content, the higher the melting temperature, as shown in Figure 8-40b.

Figure 8-39. The denaturation and renaturation of double-stranded DNA molecules.

Figure 8-39

The denaturation and renaturation of double-stranded DNA molecules.

Figure 8-40. (a) The absorption of ultraviolet light of 260-nm wavelength by solutions of single-stranded and double-stranded DNA.

Figure 8-40

(a) The absorption of ultraviolet light of 260-nm wavelength by solutions of single-stranded and double-stranded DNA. As regions of double-stranded DNA unpair, the absorption of light by those regions increases almost twofold. The temperature at which (more...)

Doty and Marmur’s finding that in solutions the separated single strands of a double helix will find each other because of complementary base pairing led to several experimental applications that have had an enormous effect on research in molecular genetics.

1.

Analysis of genome structure. If total genomic DNA is melted and allowed to reanneal, several distinct stages are observed in the annealing process. First, there is a stage of very rapid annealing. This stage represents highly repetitive DNA because, for this type of DNA, there are many copies per genome. These copies can find each other faster than can unique genes that are present in only one copy per haploid genome. Later annealing fractions contain progressively less repetitive DNA, and unique sequences anneal last. Hence this property allowed scientists to isolate and characterize the various repetitive categories. This characterization in turn allowed the overall characterization of genomes from organisms of most taxonomic groups, providing another approach to evolutionary comparison at the genetic level.

2.

Gene isolation. Many of the techniques for isolating genes (gene cloning; see Chapter 12) are based on DNA hybridization by base complementarity. The most common type uses a radioactive denatured DNA fragment as a probe to find a clone of some specific gene of interest in a mixture of clones representing the whole genome.

3.

Southern and Northern hybridization. We learned in Chapter 1 that a denatured labeled probe can be used to identify specific genomic fragments in a mixture separated on an electrophoretic gel (the technique of Southern hybridization); in a parallel technique, specific RNA transcripts can be detected on electrophoretic gels (Northern hybridization). Because of the incisiveness of these techniques, they now form part of the everyday technology used by geneticists everywhere.

4.

Chromosome mapping. Hybridization by probes has allowed the identification of DNA markers at specific chromosomal locations. [One type of DNA marker is the restriction fragment length polymorphism (RFLP); see Chapter 13.] DNA markers have provided many thousands of new loci to saturate the chromosomal map. Furthermore, such markers have provided linked markers for the diagnosis of disease alleles in humans. In a related technique, labeled probes can be added to partly denatured DNA still in chromosomes, revealing the chromosomal position of the DNA homologous to the probe (in situ hybridization; see Chapter 14).

Hence we see that the structure of DNA provides not only two key properties for biological function (replication and information storage), but also key techniques for the genetic dissection of organisms and their cells.

MESSAGE

The specificity of base complementarity forms the foundation for the continuity of life through replication and the foundation for information transfer from DNA into protein—the main determinant of biological form. This same specificity is used by geneticists as a tool to investigate gene and genome structure and function.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21862

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