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Cell_Biology_Alberts_1110	Cell_Biology_Alberts	The cells of a sexually reproducing animal or plant are of two types: germ cells and somatic cells. The germ cells transmit genetic information from parent to offspring; the somatic cells form the body of the organism (Figure 5–1). We have seen that germ cells must be protected against high rates of mutation to maintain the species. However, the somatic cells of multicellular organisms must also be protected from genetic change to properly maintain the organized structure of the body. Nucleotide changes in somatic cells can give rise to variant cells, some of which, through “local” natural selection, proliferate rapidly at the expense of the rest of the organism. In an extreme case, the result is the uncontrolled cell proliferation that we know as cancer, a disease that causes (in Europe and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant	Cell_Biology_Alberts. The cells of a sexually reproducing animal or plant are of two types: germ cells and somatic cells. The germ cells transmit genetic information from parent to offspring; the somatic cells form the body of the organism (Figure 5–1). We have seen that germ cells must be protected against high rates of mutation to maintain the species. However, the somatic cells of multicellular organisms must also be protected from genetic change to properly maintain the organized structure of the body. Nucleotide changes in somatic cells can give rise to variant cells, some of which, through “local” natural selection, proliferate rapidly at the expense of the rest of the organism. In an extreme case, the result is the uncontrolled cell proliferation that we know as cancer, a disease that causes (in Europe and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant
Cell_Biology_Alberts_1111	Cell_Biology_Alberts	and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant increase in the mutation frequency would presumably cause a disastrous increase in the incidence of cancer by accelerating the rate at which somatic-cell variants arise. Thus, both for the perpetuation of a species with a large number of genes (germ-cell stability) and for the prevention of cancer resulting from mutations in somatic cells (somatic-cell stability), multicellular organisms like ourselves depend on the remarkably high fidelity with which their DNA sequences are replicated and maintained.	Cell_Biology_Alberts. and North America) more than 20% of human deaths each year. These deaths are due largely to an accumulation of changes in the DNA sequences of somatic cells, as discussed in Chapter 20. A significant increase in the mutation frequency would presumably cause a disastrous increase in the incidence of cancer by accelerating the rate at which somatic-cell variants arise. Thus, both for the perpetuation of a species with a large number of genes (germ-cell stability) and for the prevention of cancer resulting from mutations in somatic cells (somatic-cell stability), multicellular organisms like ourselves depend on the remarkably high fidelity with which their DNA sequences are replicated and maintained.
Cell_Biology_Alberts_1112	Cell_Biology_Alberts	In all cells, DNA sequences are maintained and replicated with high fidelity. The mutation rate, approximately one nucleotide change per 1010 nucleotides each time the DNA is replicated, is roughly the same for organisms as different as bacteria and humans. Because of this remarkable accuracy, the sequence of the human genome (approximately 3.2 × 109 nucleotide pairs) is unchanged or changed by only a few nucleotides each time a typical human cell divides. This allows most humans to pass accurate genetic instructions from one generation to the next, and also to avoid the changes in somatic cells that lead to cancer. All organisms duplicate their DNA with extraordinary accuracy before each cell division. In this section, we explore how an elaborate “replication machine” achieves this accuracy, while duplicating DNA at rates as high as 1000 nucleotides per second.	Cell_Biology_Alberts. In all cells, DNA sequences are maintained and replicated with high fidelity. The mutation rate, approximately one nucleotide change per 1010 nucleotides each time the DNA is replicated, is roughly the same for organisms as different as bacteria and humans. Because of this remarkable accuracy, the sequence of the human genome (approximately 3.2 × 109 nucleotide pairs) is unchanged or changed by only a few nucleotides each time a typical human cell divides. This allows most humans to pass accurate genetic instructions from one generation to the next, and also to avoid the changes in somatic cells that lead to cancer. All organisms duplicate their DNA with extraordinary accuracy before each cell division. In this section, we explore how an elaborate “replication machine” achieves this accuracy, while duplicating DNA at rates as high as 1000 nucleotides per second.
Cell_Biology_Alberts_1113	Cell_Biology_Alberts	As introduced in Chapter 1, DNA templating is the mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary DNA sequence (Figure 5–2). This process requires the separation of the DNA helix into two template strands, and entails the recognition of each nucleotide in the DNA template strands by a free (unpolymerized) complementary nucleotide. The separation of Figure 5–1 Germ-line cells and somatic cells carry out fundamentally different functions. In sexually reproducing organisms, the germ-line cells (red) propagate genetic information into the next generation. Somatic cells (blue), which form the body of the organism, are necessary for the survival of germ-line cells but do not themselves leave any progeny. Figure 5–2 The DNA double helix acts as a template for its own duplication.	Cell_Biology_Alberts. As introduced in Chapter 1, DNA templating is the mechanism the cell uses to copy the nucleotide sequence of one DNA strand into a complementary DNA sequence (Figure 5–2). This process requires the separation of the DNA helix into two template strands, and entails the recognition of each nucleotide in the DNA template strands by a free (unpolymerized) complementary nucleotide. The separation of Figure 5–1 Germ-line cells and somatic cells carry out fundamentally different functions. In sexually reproducing organisms, the germ-line cells (red) propagate genetic information into the next generation. Somatic cells (blue), which form the body of the organism, are necessary for the survival of germ-line cells but do not themselves leave any progeny. Figure 5–2 The DNA double helix acts as a template for its own duplication.
Cell_Biology_Alberts_1114	Cell_Biology_Alberts	Figure 5–2 The DNA double helix acts as a template for its own duplication. Because the nucleotide A will pair successfully only with T, and G only with C, each strand of DNA can serve as a template to specify the sequence of nucleotides in its complementary strand by DNA base-pairing. In this way, a double-helical DNA molecule can be copied precisely. the DNA helix exposes the hydrogen-bond donor and acceptor groups on each DNA base for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization into a new DNA chain. The first nucleotide-polymerizing enzyme, DNA polymerase, was discovered in 1957. The free nucleotides that serve as substrates for this enzyme were found to be deoxyribonucleoside triphosphates, and their polymerization into DNA required a single-strand DNA template. Figure 5–3 and Figure 5–4 illustrate the stepwise mechanism of this reaction. The DNA Replication Fork Is Asymmetrical	Cell_Biology_Alberts. Figure 5–2 The DNA double helix acts as a template for its own duplication. Because the nucleotide A will pair successfully only with T, and G only with C, each strand of DNA can serve as a template to specify the sequence of nucleotides in its complementary strand by DNA base-pairing. In this way, a double-helical DNA molecule can be copied precisely. the DNA helix exposes the hydrogen-bond donor and acceptor groups on each DNA base for base-pairing with the appropriate incoming free nucleotide, aligning it for its enzyme-catalyzed polymerization into a new DNA chain. The first nucleotide-polymerizing enzyme, DNA polymerase, was discovered in 1957. The free nucleotides that serve as substrates for this enzyme were found to be deoxyribonucleoside triphosphates, and their polymerization into DNA required a single-strand DNA template. Figure 5–3 and Figure 5–4 illustrate the stepwise mechanism of this reaction. The DNA Replication Fork Is Asymmetrical
Cell_Biology_Alberts_1115	Cell_Biology_Alberts	The DNA Replication Fork Is Asymmetrical During DNA replication inside a cell, each of the two original DNA strands serves as a template for the formation of an entire new strand. Because each of the two daughters of a dividing cell inherits a new DNA double helix containing one original and one new strand (Figure 5–5), the DNA double helix is said to be replicated “semiconservatively.” How is this feat accomplished? 3˜ end of strand 5˜ end of strand Figure 5–3 The chemistry of DNA synthesis. The addition of a deoxyribonucleotide to the 3ʹ end of a polynucleotide chain (the primer strand) is the fundamental reaction by which DNA is synthesized. As shown, base-pairing between an incoming deoxyribonucleoside triphosphate and an existing strand of DNA (the template strand) guides the formation of the new strand of DNA and causes it to have a complementary nucleotide sequence. The way in which complementary nucleotides base-pair is shown in Figure 4–4. direction of chain growth	Cell_Biology_Alberts. The DNA Replication Fork Is Asymmetrical During DNA replication inside a cell, each of the two original DNA strands serves as a template for the formation of an entire new strand. Because each of the two daughters of a dividing cell inherits a new DNA double helix containing one original and one new strand (Figure 5–5), the DNA double helix is said to be replicated “semiconservatively.” How is this feat accomplished? 3˜ end of strand 5˜ end of strand Figure 5–3 The chemistry of DNA synthesis. The addition of a deoxyribonucleotide to the 3ʹ end of a polynucleotide chain (the primer strand) is the fundamental reaction by which DNA is synthesized. As shown, base-pairing between an incoming deoxyribonucleoside triphosphate and an existing strand of DNA (the template strand) guides the formation of the new strand of DNA and causes it to have a complementary nucleotide sequence. The way in which complementary nucleotides base-pair is shown in Figure 4–4. direction of chain growth
Cell_Biology_Alberts_1116	Cell_Biology_Alberts	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	Cell_Biology_Alberts. 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
Cell_Biology_Alberts_1117	Cell_Biology_Alberts	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.	Cell_Biology_Alberts. 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.
Cell_Biology_Alberts_1118	Cell_Biology_Alberts	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.	Cell_Biology_Alberts. 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.
Cell_Biology_Alberts_1119	Cell_Biology_Alberts	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.	Cell_Biology_Alberts. 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.
Cell_Biology_Alberts_1120	Cell_Biology_Alberts	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.	Cell_Biology_Alberts. 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.
Cell_Biology_Alberts_1121	Cell_Biology_Alberts	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. A replication fork therefore has an asymmetric structure (Figure 5–7). The DNA daughter strand that is synthesized continuously is known as the leading strand. Its synthesis slightly precedes the synthesis of the daughter strand that is synthesized discontinuously, known as the lagging strand. For the lagging strand, the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth. The synthesis of this strand by a discontinuous “backstitching” mechanism means that DNA replication requires only the 5ʹ-to-3ʹ type of DNA polymerase. The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms	Cell_Biology_Alberts. 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. A replication fork therefore has an asymmetric structure (Figure 5–7). The DNA daughter strand that is synthesized continuously is known as the leading strand. Its synthesis slightly precedes the synthesis of the daughter strand that is synthesized discontinuously, known as the lagging strand. For the lagging strand, the direction of nucleotide polymerization is opposite to the overall direction of DNA chain growth. The synthesis of this strand by a discontinuous “backstitching” mechanism means that DNA replication requires only the 5ʹ-to-3ʹ type of DNA polymerase. The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms
Cell_Biology_Alberts_1122	Cell_Biology_Alberts	The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms As discussed above, the fidelity of copying DNA during replication is such that only about one mistake occurs for every 1010 nucleotides copied. This fidelity is much higher than one would expect from the accuracy of complementary base-pairing. The standard complementary base pairs (see Figure 4–4) are not the only ones possible. For example, with small changes in helix geometry, two hydrogen bonds can form between G and T in DNA. In addition, rare tautomeric forms of the four DNA bases occur transiently in ratios of 1 part to 104 or 105. These forms mispair without a change in helix geometry: the rare tautomeric form of C pairs with A instead of G, for example.	Cell_Biology_Alberts. The High Fidelity of DNA Replication Requires Several Proofreading Mechanisms As discussed above, the fidelity of copying DNA during replication is such that only about one mistake occurs for every 1010 nucleotides copied. This fidelity is much higher than one would expect from the accuracy of complementary base-pairing. The standard complementary base pairs (see Figure 4–4) are not the only ones possible. For example, with small changes in helix geometry, two hydrogen bonds can form between G and T in DNA. In addition, rare tautomeric forms of the four DNA bases occur transiently in ratios of 1 part to 104 or 105. These forms mispair without a change in helix geometry: the rare tautomeric form of C pairs with A instead of G, for example.
Cell_Biology_Alberts_1123	Cell_Biology_Alberts	If the DNA polymerase did nothing special when a mispairing occurred between an incoming deoxyribonucleoside triphosphate and the DNA template, the wrong nucleotide would often be incorporated into the new DNA chain, producing frequent mutations. The high fidelity of DNA replication, however, depends not only on the initial base-pairing but also on several “proofreading” mechanisms that act sequentially to correct any initial mispairings that might have occurred. Figure 5–6 Two replication forks moving in opposite directions on a circular chromosome. An active zone of DNA replication moves progressively along a replicating DNA molecule, creating a Y-shaped DNA structure known as a replication fork: the two arms of each Y are the two daughter DNA molecules, and the stem of the Y is the parental DNA helix. In this diagram, parental strands are orange; newly synthesized strands are red. 1 µm (Micrograph courtesy of Jerome Vinograd.)	Cell_Biology_Alberts. If the DNA polymerase did nothing special when a mispairing occurred between an incoming deoxyribonucleoside triphosphate and the DNA template, the wrong nucleotide would often be incorporated into the new DNA chain, producing frequent mutations. The high fidelity of DNA replication, however, depends not only on the initial base-pairing but also on several “proofreading” mechanisms that act sequentially to correct any initial mispairings that might have occurred. Figure 5–6 Two replication forks moving in opposite directions on a circular chromosome. An active zone of DNA replication moves progressively along a replicating DNA molecule, creating a Y-shaped DNA structure known as a replication fork: the two arms of each Y are the two daughter DNA molecules, and the stem of the Y is the parental DNA helix. In this diagram, parental strands are orange; newly synthesized strands are red. 1 µm (Micrograph courtesy of Jerome Vinograd.)
Cell_Biology_Alberts_1124	Cell_Biology_Alberts	Figure 5–7 The structure of a DNA replication fork. Left, replication fork with newly synthesized DNA in red and arrows indicating the 5ʹ-to-3ʹ direction of DNA synthesis. Because both daughter DNA strands are polymerized in the 5ʹ-to-3ʹ direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments, named after the scientist who discovered them. Right, the same fork a short time later. On the lagging strand, the Okazaki fragments are synthesized sequentially, with those nearest the fork being the most recently made.	Cell_Biology_Alberts. Figure 5–7 The structure of a DNA replication fork. Left, replication fork with newly synthesized DNA in red and arrows indicating the 5ʹ-to-3ʹ direction of DNA synthesis. Because both daughter DNA strands are polymerized in the 5ʹ-to-3ʹ direction, the DNA synthesized on the lagging strand must be made initially as a series of short DNA molecules, called Okazaki fragments, named after the scientist who discovered them. Right, the same fork a short time later. On the lagging strand, the Okazaki fragments are synthesized sequentially, with those nearest the fork being the most recently made.
Cell_Biology_Alberts_1125	Cell_Biology_Alberts	DNA polymerase performs the first proofreading step just before a new nucleotide is covalently added to the growing chain. Our knowledge of this mechanism comes from studies of several different DNA polymerases, including one produced by a bacterial virus, T7, that replicates inside E. coli. The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favorable. Moreover, after nucleotide binding, but before the nucleotide is covalently added to the grow-AAAAAAAAA ing chain, the enzyme must undergo a conformational change in which its “grip” tightens around the active site (see Figure 5–4). Because this change occurs more readily with correct than incorrect base-pairing, it allows the polymerase to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before	Cell_Biology_Alberts. DNA polymerase performs the first proofreading step just before a new nucleotide is covalently added to the growing chain. Our knowledge of this mechanism comes from studies of several different DNA polymerases, including one produced by a bacterial virus, T7, that replicates inside E. coli. The correct nucleotide has a higher affinity for the moving polymerase than does the incorrect nucleotide, because the correct pairing is more energetically favorable. Moreover, after nucleotide binding, but before the nucleotide is covalently added to the grow-AAAAAAAAA ing chain, the enzyme must undergo a conformational change in which its “grip” tightens around the active site (see Figure 5–4). Because this change occurs more readily with correct than incorrect base-pairing, it allows the polymerase to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before
Cell_Biology_Alberts_1126	Cell_Biology_Alberts	to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before the polymerase can mistakenly add them.	Cell_Biology_Alberts. to “double-check” the exact base-pair geometry before it catalyzes the addition of the nucleotide. Incorrectly paired nucleotides are harder to add and therefore more likely to diffuse away before the polymerase can mistakenly add them.
Cell_Biology_Alberts_1127	Cell_Biology_Alberts	The next error-correcting reaction, known as exonucleolytic proofreading, takes place immediately after those rare instances in which an incorrect nucle otide is covalently added to the growing chain. DNA polymerase enzymes are unpaired or mispaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3ʹ-OH ter minus that can prime DNA synthesis. In this way, DNA polymerase functions as a attached to DNA polymerase “self-correcting” enzyme that removes its own polymerization errors as it moves chews back to create a base- along the DNA (Figure 5–8 and Figure 5–9). paired 3˜-OH end on the primer	Cell_Biology_Alberts. The next error-correcting reaction, known as exonucleolytic proofreading, takes place immediately after those rare instances in which an incorrect nucle otide is covalently added to the growing chain. DNA polymerase enzymes are unpaired or mispaired residues at the primer terminus, continuing until enough nucleotides have been removed to regenerate a correctly base-paired 3ʹ-OH ter minus that can prime DNA synthesis. In this way, DNA polymerase functions as a attached to DNA polymerase “self-correcting” enzyme that removes its own polymerization errors as it moves chews back to create a base- along the DNA (Figure 5–8 and Figure 5–9). paired 3˜-OH end on the primer
Cell_Biology_Alberts_1128	Cell_Biology_Alberts	paired 3˜-OH end on the primer The self-correcting properties of the DNA polymerase depend on its require ment for a perfectly base-paired primer terminus, and it is apparently not possible for such an enzyme to start synthesis de novo, without an existing primer. By contrast, the RNA polymerase enzymes involved in gene transcription do not need such an efficient exonucleolytic proofreading mechanism: errors in making RNA are not passed on to the next generation, and the occasional defective RNA molecule that is produced has no long-term significance. RNA polymerases are thus able to start new polynucleotide chains without a primer. DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand	Cell_Biology_Alberts. paired 3˜-OH end on the primer The self-correcting properties of the DNA polymerase depend on its require ment for a perfectly base-paired primer terminus, and it is apparently not possible for such an enzyme to start synthesis de novo, without an existing primer. By contrast, the RNA polymerase enzymes involved in gene transcription do not need such an efficient exonucleolytic proofreading mechanism: errors in making RNA are not passed on to the next generation, and the occasional defective RNA molecule that is produced has no long-term significance. RNA polymerases are thus able to start new polynucleotide chains without a primer. DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand
Cell_Biology_Alberts_1129	Cell_Biology_Alberts	DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand Figure 5–8 Exonucleolytic proofreading by DNA polymerase during DNA replication. In this example, a C is accidentally incorporated at the growing 3ʹ-OH end of a DNA chain. The part of DNA polymerase that removes the misincorporated nucleotide is a specialized member of a large class of enzymes, known as exonucleases, that cleave nucleotides one at a time from the ends of polynucleotides. Figure 5–9 Editing by DNA polymerase. DNA polymerase complexed with the DNA template in the polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E) and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNA transiently unpairs from the template and enters the editing site where the most recently added nucleotide is catalytically removed.	Cell_Biology_Alberts. DNA polymerase resumes the process of adding nucleotides to the base-paired 3˜-OH end of the primer strand Figure 5–8 Exonucleolytic proofreading by DNA polymerase during DNA replication. In this example, a C is accidentally incorporated at the growing 3ʹ-OH end of a DNA chain. The part of DNA polymerase that removes the misincorporated nucleotide is a specialized member of a large class of enzymes, known as exonucleases, that cleave nucleotides one at a time from the ends of polynucleotides. Figure 5–9 Editing by DNA polymerase. DNA polymerase complexed with the DNA template in the polymerizing mode (left) and the editing mode (right). The catalytic sites for the exonucleolytic (E) and the polymerization (P) reactions are indicated. In the editing mode, the newly synthesized DNA transiently unpairs from the template and enters the editing site where the most recently added nucleotide is catalytically removed.
Cell_Biology_Alberts_1130	Cell_Biology_Alberts	There is an error frequency of about one mistake for every 104 polymerization events both in RNA synthesis and in the separate process of translating mRNA sequences into protein sequences. This error rate is over 100,000 times greater than that in DNA replication, where, as we have seen, a series of proofreading processes makes the process unusually accurate (Table 5–1). Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction	Cell_Biology_Alberts. There is an error frequency of about one mistake for every 104 polymerization events both in RNA synthesis and in the separate process of translating mRNA sequences into protein sequences. This error rate is over 100,000 times greater than that in DNA replication, where, as we have seen, a series of proofreading processes makes the process unusually accurate (Table 5–1). Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction
Cell_Biology_Alberts_1131	Cell_Biology_Alberts	Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction The need for accuracy probably explains why DNA replication occurs only in the 5ʹ-to-3ʹ direction. If there were a DNA polymerase that added deoxyribonucleoside triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, rather than the incoming mononucleotide, would have to provide the activating triphosphate needed for the covalent linkage. In this case, the mistakes in polymerization could not be simply hydrolyzed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis (see Figure 5–3). It is therefore possible to correct a mismatched base only if it has been added to the 3ʹ end of a DNA chain. Although the backstitching mechanism for DNA replication seems complex, it preserves the 5ʹ-to-3ʹ direction of polymerization that is required for exonucleolytic proofreading.	Cell_Biology_Alberts. Only DNA Replication in the 5ʹ-to-3ʹ Direction Allows Efficient Error Correction The need for accuracy probably explains why DNA replication occurs only in the 5ʹ-to-3ʹ direction. If there were a DNA polymerase that added deoxyribonucleoside triphosphates in the 3ʹ-to-5ʹ direction, the growing 5ʹ end of the chain, rather than the incoming mononucleotide, would have to provide the activating triphosphate needed for the covalent linkage. In this case, the mistakes in polymerization could not be simply hydrolyzed away, because the bare 5ʹ end of the chain thus created would immediately terminate DNA synthesis (see Figure 5–3). It is therefore possible to correct a mismatched base only if it has been added to the 3ʹ end of a DNA chain. Although the backstitching mechanism for DNA replication seems complex, it preserves the 5ʹ-to-3ʹ direction of polymerization that is required for exonucleolytic proofreading.
Cell_Biology_Alberts_1132	Cell_Biology_Alberts	Despite these safeguards against DNA replication errors, DNA polymerases occasionally make mistakes. However, as we shall see later, cells have yet another Figure 5–10 RNA primer synthesis. A schematic view of the reaction catalyzed by DNA primase, the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template. Unlike DNA polymerase, this enzyme can start a new polynucleotide chain by joining two nucleoside triphosphates together. The primase synthesizes a short polynucleotide in the 5ʹ-to-3ʹ direction and then stops, making the 3ʹ end of this primer available for the DNA polymerase. chance to correct these errors by a process called strand-directed mismatch repair. Before discussing this mechanism, however, we describe the other types of proteins that function at the replication fork. A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand	Cell_Biology_Alberts. Despite these safeguards against DNA replication errors, DNA polymerases occasionally make mistakes. However, as we shall see later, cells have yet another Figure 5–10 RNA primer synthesis. A schematic view of the reaction catalyzed by DNA primase, the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template. Unlike DNA polymerase, this enzyme can start a new polynucleotide chain by joining two nucleoside triphosphates together. The primase synthesizes a short polynucleotide in the 5ʹ-to-3ʹ direction and then stops, making the 3ʹ end of this primer available for the DNA polymerase. chance to correct these errors by a process called strand-directed mismatch repair. Before discussing this mechanism, however, we describe the other types of proteins that function at the replication fork. A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand
Cell_Biology_Alberts_1133	Cell_Biology_Alberts	A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand For the leading strand, a primer is needed only at the start of replication: once a replication fork is established, the DNA polymerase is continuously presented with a base-paired chain end on which to add new nucleotides. On the lagging side of the fork, however, each time the DNA polymerase completes a short DNA Okazaki fragment (which takes a few seconds), it must start synthesizing a completely new fragment at a site further along the template strand (see Figure 5–7). A special mechanism produces the base-paired primer strand required by the DNA polymerase molecules. The mechanism depends on an enzyme called DNA primase, which uses ribonucleoside triphosphates to synthesize short RNA primers on the lagging strand (Figure 5–10). In eukaryotes, these primers are about 10 nucleotides long and are made at intervals of 100–200 nucleotides on the lagging strand.	Cell_Biology_Alberts. A Special Nucleotide-Polymerizing Enzyme Synthesizes Short RNA Primer Molecules on the Lagging Strand For the leading strand, a primer is needed only at the start of replication: once a replication fork is established, the DNA polymerase is continuously presented with a base-paired chain end on which to add new nucleotides. On the lagging side of the fork, however, each time the DNA polymerase completes a short DNA Okazaki fragment (which takes a few seconds), it must start synthesizing a completely new fragment at a site further along the template strand (see Figure 5–7). A special mechanism produces the base-paired primer strand required by the DNA polymerase molecules. The mechanism depends on an enzyme called DNA primase, which uses ribonucleoside triphosphates to synthesize short RNA primers on the lagging strand (Figure 5–10). In eukaryotes, these primers are about 10 nucleotides long and are made at intervals of 100–200 nucleotides on the lagging strand.
Cell_Biology_Alberts_1134	Cell_Biology_Alberts	The chemical structure of RNA was introduced in Chapter 1 and is described in detail in Chapter 6. Here, we note only that RNA is very similar in structure to DNA. A strand of RNA can form base pairs with a strand of DNA, generating a DNA–RNA hybrid double helix if the two nucleotide sequences are complementary. Thus, the same templating principle used for DNA synthesis guides the synthesis of RNA primers. Because an RNA primer contains a properly base-paired nucleotide with a 3ʹ-OH group at one end, it can be elongated by the DNA polymerase at this end to begin an Okazaki fragment. The synthesis of each Okazaki fragment ends when this DNA polymerase runs into the RNA primer attached to the 5ʹ end of the previous fragment. To produce a continuous DNA chain from the many DNA fragments made on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the	Cell_Biology_Alberts. The chemical structure of RNA was introduced in Chapter 1 and is described in detail in Chapter 6. Here, we note only that RNA is very similar in structure to DNA. A strand of RNA can form base pairs with a strand of DNA, generating a DNA–RNA hybrid double helix if the two nucleotide sequences are complementary. Thus, the same templating principle used for DNA synthesis guides the synthesis of RNA primers. Because an RNA primer contains a properly base-paired nucleotide with a 3ʹ-OH group at one end, it can be elongated by the DNA polymerase at this end to begin an Okazaki fragment. The synthesis of each Okazaki fragment ends when this DNA polymerase runs into the RNA primer attached to the 5ʹ end of the previous fragment. To produce a continuous DNA chain from the many DNA fragments made on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the
Cell_Biology_Alberts_1135	Cell_Biology_Alberts	on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the 5ʹend of the previous one to complete the process (Figure 5–11 and Figure 5–12).	Cell_Biology_Alberts. on the lagging strand, a special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA. An enzyme called DNA ligase then joins the 3ʹend of the new DNA fragment to the 5ʹend of the previous one to complete the process (Figure 5–11 and Figure 5–12).
Cell_Biology_Alberts_1136	Cell_Biology_Alberts	Why might an erasable RNA primer be preferred to a DNA primer that would not need to be erased? The argument that a self-correcting polymerase cannot start chains de novo also implies the converse: an enzyme that starts chains anew cannot be efficient at self-correction. Thus, any enzyme that primes the synthesis of Okazaki fragments will of necessity make a relatively inaccurate copy (at least one error in 105). Even if the copies retained in the final product constituted as little as 5% of the total genome (for example, 10 nucleotides per 200-nucleotide DNA fragment), the resulting increase in the overall mutation rate would be enormous. It therefore seems likely that the use of RNA rather than DNA for priming brings a powerful advantage to the cell: the ribonucleotides in the primer automatically mark these sequences as “suspect copy” to be efficiently removed and replaced.	Cell_Biology_Alberts. Why might an erasable RNA primer be preferred to a DNA primer that would not need to be erased? The argument that a self-correcting polymerase cannot start chains de novo also implies the converse: an enzyme that starts chains anew cannot be efficient at self-correction. Thus, any enzyme that primes the synthesis of Okazaki fragments will of necessity make a relatively inaccurate copy (at least one error in 105). Even if the copies retained in the final product constituted as little as 5% of the total genome (for example, 10 nucleotides per 200-nucleotide DNA fragment), the resulting increase in the overall mutation rate would be enormous. It therefore seems likely that the use of RNA rather than DNA for priming brings a powerful advantage to the cell: the ribonucleotides in the primer automatically mark these sequences as “suspect copy” to be efficiently removed and replaced.
Cell_Biology_Alberts_1137	Cell_Biology_Alberts	Figure 5–11 The synthesis of one of many DNA fragments on the lagging strand. In eukaryotes, RNA primers are made at intervals spaced by about 200 nucleotides on the lagging strand, and each RNA primer is approximately 10 nucleotides long. This primer is erased by a special DNA repair enzyme (an RNAse H) that recognizes an RNA strand in an RNA/DNA helix and fragments it; this leaves gaps that are filled in by DNA polymerase and DNA ligase. DNA polymerase adds to new RNA primer to start new Okazaki fragment sealing by DNA ligase joins new Okazaki fragment to the growing chain Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork	Cell_Biology_Alberts. Figure 5–11 The synthesis of one of many DNA fragments on the lagging strand. In eukaryotes, RNA primers are made at intervals spaced by about 200 nucleotides on the lagging strand, and each RNA primer is approximately 10 nucleotides long. This primer is erased by a special DNA repair enzyme (an RNAse H) that recognizes an RNA strand in an RNA/DNA helix and fragments it; this leaves gaps that are filled in by DNA polymerase and DNA ligase. DNA polymerase adds to new RNA primer to start new Okazaki fragment sealing by DNA ligase joins new Okazaki fragment to the growing chain Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork
Cell_Biology_Alberts_1138	Cell_Biology_Alberts	Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork For DNA synthesis to proceed, the DNA double helix must be opened up (“melted”) ahead of the replication fork so that the incoming deoxyribonucleoside triphosphates can form base pairs with the template strands. However, the DNA double helix is very stable under physiological conditions; the base pairs are locked in place so strongly that it requires temperatures approaching that of boiling water to separate the two strands in a test tube. For this reason, two additional types of replication proteins—DNA helicases and single-strand DNA-binding proteins—are needed to open the double helix and provide the appropriate single-strand DNA templates for the DNA polymerase to copy.	Cell_Biology_Alberts. Special Proteins Help to Open Up the DNA Double Helix in Front of the Replication Fork For DNA synthesis to proceed, the DNA double helix must be opened up (“melted”) ahead of the replication fork so that the incoming deoxyribonucleoside triphosphates can form base pairs with the template strands. However, the DNA double helix is very stable under physiological conditions; the base pairs are locked in place so strongly that it requires temperatures approaching that of boiling water to separate the two strands in a test tube. For this reason, two additional types of replication proteins—DNA helicases and single-strand DNA-binding proteins—are needed to open the double helix and provide the appropriate single-strand DNA templates for the DNA polymerase to copy.
Cell_Biology_Alberts_1139	Cell_Biology_Alberts	DNA helicases were first isolated as proteins that hydrolyze ATP when they are bound to single strands of DNA. As described in Chapter 3, the hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work. DNA helicases use this principle to propel themselves rapidly along a DNA single strand. When they encounter a region of double helix, they continue to move along their strand, thereby prying apart the helix at rates of up to 1000 nucleotide pairs per second (Figure 5–13 and Figure 5–14). The two strands of DNA have opposite polarities, and, in principle, a helicase	Cell_Biology_Alberts. DNA helicases were first isolated as proteins that hydrolyze ATP when they are bound to single strands of DNA. As described in Chapter 3, the hydrolysis of ATP can change the shape of a protein molecule in a cyclical manner that allows the protein to perform mechanical work. DNA helicases use this principle to propel themselves rapidly along a DNA single strand. When they encounter a region of double helix, they continue to move along their strand, thereby prying apart the helix at rates of up to 1000 nucleotide pairs per second (Figure 5–13 and Figure 5–14). The two strands of DNA have opposite polarities, and, in principle, a helicase
Cell_Biology_Alberts_1140	Cell_Biology_Alberts	The two strands of DNA have opposite polarities, and, in principle, a helicase Figure 5–12 The reaction catalyzed by DNA ligase. This enzyme seals a broken phosphodiester bond. As shown, DNA ligase uses a molecule of ATP to activate the 5ʹ end at the nick (step 1) before forming the new bond (step 2). In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis. could unwind the DNA double helix by moving in the 5ʹ-to-3ʹ direction along one 5˜ 3˜ strand or in the 3ʹ-to-5ʹ direction along the other. In fact, both types of DNA helicase exist. In the best-understood replication systems in bacteria, a helicase moving 5ʹ to 3ʹ along the lagging-strand template appears to have the predominant role, for reasons that will become clear shortly.	Cell_Biology_Alberts. The two strands of DNA have opposite polarities, and, in principle, a helicase Figure 5–12 The reaction catalyzed by DNA ligase. This enzyme seals a broken phosphodiester bond. As shown, DNA ligase uses a molecule of ATP to activate the 5ʹ end at the nick (step 1) before forming the new bond (step 2). In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the energetically favorable process of ATP hydrolysis. could unwind the DNA double helix by moving in the 5ʹ-to-3ʹ direction along one 5˜ 3˜ strand or in the 3ʹ-to-5ʹ direction along the other. In fact, both types of DNA helicase exist. In the best-understood replication systems in bacteria, a helicase moving 5ʹ to 3ʹ along the lagging-strand template appears to have the predominant role, for reasons that will become clear shortly.
Cell_Biology_Alberts_1141	Cell_Biology_Alberts	Single-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins, bind tightly and cooperatively to exposed single-strand DNA without covering the bases, which therefore remain available as templates. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-strand conformation. In addition, through cooperative binding, they coat and straighten out the regions of single-strand DNA, which occur routinely in the lagging-strand template, thereby preventing the formation of the short hairpin helices that readily form in single-strand DNA (Figure 5–15 and Figure 5–16). If not removed, these hairpin helices can impede the DNA synthesis catalyzed by DNA polymerase. A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA	Cell_Biology_Alberts. Single-strand DNA-binding (SSB) proteins, also called helix-destabilizing proteins, bind tightly and cooperatively to exposed single-strand DNA without covering the bases, which therefore remain available as templates. These proteins are unable to open a long DNA helix directly, but they aid helicases by stabilizing the unwound, single-strand conformation. In addition, through cooperative binding, they coat and straighten out the regions of single-strand DNA, which occur routinely in the lagging-strand template, thereby preventing the formation of the short hairpin helices that readily form in single-strand DNA (Figure 5–15 and Figure 5–16). If not removed, these hairpin helices can impede the DNA synthesis catalyzed by DNA polymerase. A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA
Cell_Biology_Alberts_1142	Cell_Biology_Alberts	A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA On their own, most DNA polymerase molecules will synthesize only a short string of nucleotides before falling off the DNA template. The tendency to dissociate quickly from a DNA molecule allows a DNA polymerase molecule that has just Figure 5–13 An assay for DNA helicase enzymes. A short DNA fragment is annealed to a long DNA single strand to form a region of DNA double helix. The double helix is melted as the helicase runs along the DNA single strand, releasing the short DNA fragment in a reaction that requires the presence of both the helicase protein and ATP. The rapid stepwise movement of the helicase is powered by its ATP hydrolysis (shown schematically in Figure 3–75A). As indicated, many DNA helicases are composed of six subunits.	Cell_Biology_Alberts. A Sliding Ring Holds a Moving DNA Polymerase Onto the DNA On their own, most DNA polymerase molecules will synthesize only a short string of nucleotides before falling off the DNA template. The tendency to dissociate quickly from a DNA molecule allows a DNA polymerase molecule that has just Figure 5–13 An assay for DNA helicase enzymes. A short DNA fragment is annealed to a long DNA single strand to form a region of DNA double helix. The double helix is melted as the helicase runs along the DNA single strand, releasing the short DNA fragment in a reaction that requires the presence of both the helicase protein and ATP. The rapid stepwise movement of the helicase is powered by its ATP hydrolysis (shown schematically in Figure 3–75A). As indicated, many DNA helicases are composed of six subunits.
Cell_Biology_Alberts_1143	Cell_Biology_Alberts	Figure 5–14 The structure of a DNA helicase. (A) Diagram of the protein 5˜ as a hexameric ring drawn to scale with a replication fork. (B) Detailed structure of the bacteriophage T7 replicative helicase, as determined by x-ray diffraction. Six identical subunits bind and hydrolyze ATP in an ordered fashion to propel this molecule, like a rotary engine, along a DNA single strand that passes through the central hole. Red indicates bound ATP molecules in the structure (Movie 5.2). (PDB code: 1E0J.) finished synthesizing one Okazaki fragment on the lagging strand to be recycled quickly, so as to begin the synthesis of the next Okazaki fragment on the same strand. This rapid dissociation, however, would make it difficult for the polymerase to synthesize the long DNA strands produced at a replication fork were it not for an accessory protein (called PCNA in eukaryotes) that functions as a regulated sliding clamp. This clamp keeps the polymerase firmly on the DNA when it is moving, but	Cell_Biology_Alberts. Figure 5–14 The structure of a DNA helicase. (A) Diagram of the protein 5˜ as a hexameric ring drawn to scale with a replication fork. (B) Detailed structure of the bacteriophage T7 replicative helicase, as determined by x-ray diffraction. Six identical subunits bind and hydrolyze ATP in an ordered fashion to propel this molecule, like a rotary engine, along a DNA single strand that passes through the central hole. Red indicates bound ATP molecules in the structure (Movie 5.2). (PDB code: 1E0J.) finished synthesizing one Okazaki fragment on the lagging strand to be recycled quickly, so as to begin the synthesis of the next Okazaki fragment on the same strand. This rapid dissociation, however, would make it difficult for the polymerase to synthesize the long DNA strands produced at a replication fork were it not for an accessory protein (called PCNA in eukaryotes) that functions as a regulated sliding clamp. This clamp keeps the polymerase firmly on the DNA when it is moving, but
Cell_Biology_Alberts_1144	Cell_Biology_Alberts	replication fork were it not for an accessory protein (called PCNA in eukaryotes) that functions as a regulated sliding clamp. This clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-strand region of DNA.	Cell_Biology_Alberts. replication fork were it not for an accessory protein (called PCNA in eukaryotes) that functions as a regulated sliding clamp. This clamp keeps the polymerase firmly on the DNA when it is moving, but releases it as soon as the polymerase runs into a double-strand region of DNA.
Cell_Biology_Alberts_1145	Cell_Biology_Alberts	How can a sliding clamp prevent the polymerase from dissociating without at the same time impeding the polymerase’s rapid movement along the DNA molecule? The three-dimensional structure of the clamp protein, determined by x-ray diffraction, revealed it to be a large ring around the DNA double helix. One face of the ring binds to the back of the DNA polymerase, and the whole ring slides freely along the DNA as the polymerase moves. The assembly of the clamp around the DNA requires ATP hydrolysis by a special protein complex, the clamp loader, which hydrolyzes ATP as it loads the clamp on to a primer–template junction (Figure 5–17).	Cell_Biology_Alberts. How can a sliding clamp prevent the polymerase from dissociating without at the same time impeding the polymerase’s rapid movement along the DNA molecule? The three-dimensional structure of the clamp protein, determined by x-ray diffraction, revealed it to be a large ring around the DNA double helix. One face of the ring binds to the back of the DNA polymerase, and the whole ring slides freely along the DNA as the polymerase moves. The assembly of the clamp around the DNA requires ATP hydrolysis by a special protein complex, the clamp loader, which hydrolyzes ATP as it loads the clamp on to a primer–template junction (Figure 5–17).
Cell_Biology_Alberts_1146	Cell_Biology_Alberts	On the leading-strand template, the moving DNA polymerase is tightly bound to the clamp, and the two remain associated for a very long time. The DNA polymerase on the lagging-strand template also makes use of the clamp, but each time the polymerase reaches the 5ʹ end of the preceding Okazaki fragment, the polymerase releases itself from the clamp and dissociates from the template. This polymerase molecule then associates with a new clamp that is assembled on the RNA primer of the next Okazaki fragment. single-stranded region of DNA template with short regions of base-paired “hairpins”	Cell_Biology_Alberts. On the leading-strand template, the moving DNA polymerase is tightly bound to the clamp, and the two remain associated for a very long time. The DNA polymerase on the lagging-strand template also makes use of the clamp, but each time the polymerase reaches the 5ʹ end of the preceding Okazaki fragment, the polymerase releases itself from the clamp and dissociates from the template. This polymerase molecule then associates with a new clamp that is assembled on the RNA primer of the next Okazaki fragment. single-stranded region of DNA template with short regions of base-paired “hairpins”
Cell_Biology_Alberts_1147	Cell_Biology_Alberts	single-stranded region of DNA template with short regions of base-paired “hairpins” Figure 5–15 The effect of single-strand DNA-binding proteins (SSb proteins) on the structure of single-strand DNA. Because each protein molecule prefers to bind next to a previously bound molecule, long rows of this protein form on a DNA single strand. This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process. The “hairpin helices” shown in the bare, single-strand DNA result from a chance matching of short regions of complementary nucleotide sequence; they are similar to the short helices that typically form in RNA molecules (see Figure 1–6).	Cell_Biology_Alberts. single-stranded region of DNA template with short regions of base-paired “hairpins” Figure 5–15 The effect of single-strand DNA-binding proteins (SSb proteins) on the structure of single-strand DNA. Because each protein molecule prefers to bind next to a previously bound molecule, long rows of this protein form on a DNA single strand. This cooperative binding straightens out the DNA template and facilitates the DNA polymerization process. The “hairpin helices” shown in the bare, single-strand DNA result from a chance matching of short regions of complementary nucleotide sequence; they are similar to the short helices that typically form in RNA molecules (see Figure 1–6).
Cell_Biology_Alberts_1148	Cell_Biology_Alberts	Figure 5–16 Human single-strand binding protein bound to DNA. (A) Front view of the two DNA-binding domains of the protein (called RPA) which cover a total of eight nucleotides. Note that the DNA bases remain exposed in this protein–DNA complex. (B) Diagram showing the three-dimensional structure, with the DNA strand (orange) viewed end-on. (PDB code: 1JMC.)	Cell_Biology_Alberts. Figure 5–16 Human single-strand binding protein bound to DNA. (A) Front view of the two DNA-binding domains of the protein (called RPA) which cover a total of eight nucleotides. Note that the DNA bases remain exposed in this protein–DNA complex. (B) Diagram showing the three-dimensional structure, with the DNA strand (orange) viewed end-on. (PDB code: 1JMC.)
Cell_Biology_Alberts_1149	Cell_Biology_Alberts	Figure 5–17 The regulated sliding clamp that holds DNA polymerase on the DNA. (A) The structure of the clamp protein from E. coli, as determined by x-ray crystallography, with a DNA helix added to indicate how the protein fits around DNA (Movie 5.3). (B) Schematic illustration showing how the clamp (with red and yellow subunits) is loaded onto DNA to serve as a tether for a moving DNA polymerase molecule. The structure of the clamp loader (dark green) resembles a screw nut, (A) (B) sliding clamp clamp loader 5˜ATPADPPi ATPATP+ DNA + DNA polymerase 3˜5˜5˜3˜3˜RECYCLING OF RELEASED CLAMP LOADER ATP BINDING TO CLAMP LOADER OPENS SLIDING CLAMP DNA ENGAGED IN CLAMP ATP HYDROLYSIS LOCKS SLIDING CLAMP AROUND DNA AND RELEASES CLAMP LOADER DNA POLYMERASE BINDS TO SLIDING CLAMP + with its threads matching the grooves of double-stranded DNA. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. The clamp loader, thanks to	Cell_Biology_Alberts. Figure 5–17 The regulated sliding clamp that holds DNA polymerase on the DNA. (A) The structure of the clamp protein from E. coli, as determined by x-ray crystallography, with a DNA helix added to indicate how the protein fits around DNA (Movie 5.3). (B) Schematic illustration showing how the clamp (with red and yellow subunits) is loaded onto DNA to serve as a tether for a moving DNA polymerase molecule. The structure of the clamp loader (dark green) resembles a screw nut, (A) (B) sliding clamp clamp loader 5˜ATPADPPi ATPATP+ DNA + DNA polymerase 3˜5˜5˜3˜3˜RECYCLING OF RELEASED CLAMP LOADER ATP BINDING TO CLAMP LOADER OPENS SLIDING CLAMP DNA ENGAGED IN CLAMP ATP HYDROLYSIS LOCKS SLIDING CLAMP AROUND DNA AND RELEASES CLAMP LOADER DNA POLYMERASE BINDS TO SLIDING CLAMP + with its threads matching the grooves of double-stranded DNA. The loader binds to a free clamp molecule, forcing a gap in its ring of subunits so that this ring is able to slip around DNA. The clamp loader, thanks to