LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 3. INFORMATION PATHWAYS - 2017

PART III. INFORMATION PATHWAYS

Class="center">There are no boring Enzymes.

— Arthur Kornberg, For the Love of Enzymes, 1975

25. DNA METABOLISM

As the repository of Genetic information, DNA occupies a central place among biological macromolecules. The nucleotide sequence of DNA encodes the Introduction/19.html">Primary Structure of all cellular Cell/22.html">RNA and Protein molecules and, via enzymes, indirectly influences the synthesis of all other cellular components. The flow of information from DNA to RNA and protein determines the size, shape, and vitality of all living things.

DNA is exceptionally well-suited for the stable storage of genetic information. However, METABOLISM/2.html">THE CONCEPT OF "stable storage" denotes a dynamic, active state rather than a static, unchanging picture. It fails to reflect The complexity of the processes by which genetic information is preserved and passed from one cell generation to the next. DNA metabolism encompasses both the precise copying of molecules (Replication) and the processes that affect the very structure carrying that information (repair and recombination). It is these processes that we will examine in this chapter.

DNA metabolism must proceed with exquisite fidelity. The Chemical Reactions of joining one nucleotide to another during DNA Replication are elegant and surprisingly simple. The complexity, as we shall see, lies in the enzymatic machinery that ensures the accurate transmission of genetic information. Errors occurring during DNA Synthesis can have severe consequences, not only because they alter or disrupt the function of a particular Gene, but also because such changes are heritable.

The enzymes involved in DNA synthesis copy DNA molecules containing millions of bases. They accomplish this with remarkable speed and precision, despite the fact that DNA is densely packaged and bound to various Proteins. The formation of phosphodiester bonds between NUCLEOTIDES in a growing DNA chain is only part of a complex process involving numerous Proteins and Enzymes.

The imperative to preserve genetic information lies at The Heart of DNA Repair. Chapter 8 (Vol. 1) details the susceptibility of DNA to various damaging agents. Such damaging events occur infrequently, yet they are critically important because organisms have very low tolerance for Changes in DNA sequence. DNA is the only macromolecule provided with a dedicated repair system; the sheer number, diversity, and complexity of repair mechanisms reflect the wide array of hazards that threaten DNA.

Cells can rearrange their stored genetic information through processes collectively known as "recombination." At first glance, recombination might seem to undermine the paramount principle of genetic stability and integrity. In reality, however, most DNA rearrangements play a constructive role in maintaining genome integrity by specifically influencing DNA replication, repair, and chromosome segregation.

This chapter focuses heavily on the Enzymes of DNA metabolism. They deserve careful study not only for their profound biological significance and pure scientific interest, but also for their growing role as therapeutic targets in medicine and as tools in a wide array of modern biochemical technologies. Many foundational discoveries in DNA metabolism were made using Escherichia coli cells; therefore, the well-characterized enzymes of this bacterium serve as our primary model to explain the fundamentals of metabolism. A glance at some of the key genes on the E. coli genetic map (Fig. 25-1) gives a sense of the sheer complexity of the enzyme systems involved in DNA metabolism.

Fig. 25-1. Chromosome map of Escherichia coli. The locations of genes encoding many proteins essential for DNA metabolism are shown. The large number of known genes involved in DNA metabolism underscores the complexity of these processes. The numbers from 0 to 100 within the circular chromosome correspond to genetic units called minutes. Each minute corresponds to a segment of the DNA molecule approximately 40,000 bp in length. Three-letter gene designations generally reflect aspects of their function; for example, mut indicates mutagenesis; dna, DNA replication; pol, DNA polymerase; rpo, RNA polymerase; uvr, UV resistance; rec, recombination; dam, adenine methylation; lig, DNA ligase; Ter, replication termination; and ori, THE ORIGIN OF replication (oriC in E. coli, as shown).

Before diving into the details of replication, let us take a brief detour to discuss the conventions used for naming bacterial genes and proteins, as we will encounter many of them throughout this and subsequent chapters. Similar conventions apply to eukaryotic gene designations, though specific Abbreviations may depend on the species, and no universal naming rule exists across all eukaryotic systems.

Key Conventions.

Bacterial genes are typically designated by three italicized lowercase letters that generally reflect their function. For example, the dna, uvr, and rec genes stand for DNA replication, UV damage resistance, and recombination, respectively. When multiple genes share the same function, they are further distinguished by capital letters A, B, C, etc. (e.g., dnaA, dnaB, dnaQ), which usually indicate the chronological order of their discovery rather than their sequential action in a pathway. ■

The Use of abbreviations for protein names is less straightforward. Genetic studies typically identify and characterize the protein product of each gene. Many bacterial genes were identified and named before The Significance of their protein products was understood. Sometimes, a gene product turns out to be a previously known protein, requiring a rename. Frequently, however, the gene product remains novel, possessing an activity that defies description by a conventional enzyme name.

Key Conventions.

Bacterial proteins often retain the names of their corresponding genes. E. coli protein names are written in roman type with an initial capital letter; for instance, the protein products of the dnaA and recA genes are designated DnaA and RecA, respectively. ■

25.1. DNA Replication

Long before The structure of DNA was elucidated, scientists marveled at the ability of organisms to recreate themselves and of cells to generate many identical copies of large, complex macromolecules. Thinking on this subject centered around the Concept of the template—a structure that allows molecules to line up in a specific order to form a macromolecule with a unique sequence and function. By the 1940s, it was widely appreciated that DNA carries genetic information, but only after James Watson and Francis Crick resolved its structure did it become clear how DNA serves as a template for replication and transmits genetic information: one strand is complementary to the other. Bases pair according to strict rules, and each strand acts as a template for a new strand with a predictable complementary sequence (see Figs. 8-14 and 8-15 in Vol. 1).

It has been established that the fundamental features of DNA Replication and its catalytic mechanisms are remarkably similar across all living species. We will emphasize this mechanistic unity as we progress from the General Principles of replication to the replication enzymes of E. coli, and finally to eukaryotic replication.

Basic principles of DNA Replication

Early studies of bacterial DNA replication and its enzymes established several foundational principles that govern DNA synthesis in All living organisms.

DNA replication is semiconservative.

Each DNA strand serves as a template for the synthesis of a new strand, yielding two new double-stranded DNA molecules, each consisting of one new and one old strand. For this reason, the process is called semiconservative replication.

Watson and Crick proposed the hypothesis of semiconservative replication shortly after publishing their 1953 paper on DNA Structure; in 1957, this hypothesis was confirmed by Matthew Meselson and Franklin Stahl through a brilliantly devised experiment. Meselson and Stahl cultivated E. coli cells for many generations in a medium where the sole nitrogen source (NH₄Cl) contained the heavy nitrogen isotope ¹⁵N instead of the most common light isotope ¹⁴N. The density of DNA isolated from these cells is about 1% greater than that of normal [¹⁴N] DNA (Fig. 25-2a). Despite this slight difference, a mixture of heavy [¹⁵N] DNA and light [¹⁴N] DNA can be separated by cesium chloride density-gradient centrifugation.

Fig. 25-2, a — several cell generations were grown in a medium containing only heavy nitrogen ¹⁵N, so the DNA of these cells contained exclusively ¹⁵N, as indicated by a single (blue) band formed upon density-gradient centrifugation in CsCl. b — cells were transferred to a medium containing only light nitrogen ¹⁴N, and after the first Cell Division, DNA was isolated which, in the cesium chloride gradient, formed a higher (purple) band; c — after the second round of replication, the DNA separated into two bands: a hybrid (purple) band and an even lighter (red) band containing only DNA with ¹⁴N, thereby confirming the semiconservative nature of replication.

E. coli cells grown on a ¹⁵N medium were transferred to a fresh nutrient medium containing only the ¹⁴N isotope, where the cells continued to grow until the population size doubled. DNA isolated from this first cell generation was centrifuged in a CsCl gradient, revealing a single band whose position indicated that the double-helical DNA of the daughter cells is a hybrid containing one new strand with ¹⁴N and one parental strand with ¹⁵N (Fig. 25-2b).

These results disproved the alternative conservative replication theory, according to which one daughter DNA molecule contains two newly synthesized strands and the other daughter molecule contains two parental strands; had this theory been correct, no hybrid DNA would have been observed in the Meselson-Stahl experiment. The semiconservative replication hypothesis was further confirmed in the next stage of the experiment (Fig. 25-2c). Following a second cell division and doubling in the ¹⁴N medium, DNA was isolated and separated in a cesium chloride gradient into two bands: one corresponding in density to light DNA, and the other to the hybrid DNA obtained after the first cell division.

Replication begins at an origin of replication and usually proceeds bidirectionally.

Following the proof of the semiconservative mechanism of replication, numerous questions arose. Does the parental DNA unwind completely before the replication of each strand begins? Does replication start at a random site or at a specific locus? Once initiated at a given point, does replication proceed in one direction or two?

Early studies by John Cairns, using autoradiography, demonstrated that replication is a highly coordinated process in which parental strands simultaneously unwind and replicate. Cairns obtained radioactive E. coli DNA by cultivating the Bacteria in a medium containing tritium-labeled (³H) thymidine. The DNA was carefully extracted, coated with a photographic emulsion, and left for several weeks; during this time, the radioactive thymidine left "traces" of silver grains in the photoemulsion, creating a photographic imprint of the DNA molecule. These autoradiographs show that the intact E. coli chromosome is a single giant circular structure 1.7 mm in length. In the radioactive DNA isolated from cells during replication, an additional loop was also detected (Fig. 25-3a). Cairns concluded that this loop arises from the formation of two radioactive daughter strands, each complementary to a parental strand. At one or both ends of the loop moves a Replication fork, where the parental DNA unwinds, allowing the separated strands to replicate. According to Cairns's data, both DNA strands replicate simultaneously, and various modifications of his experiment (Fig. 25-3b) showed that bacterial Chromosome replication is bidirectional: active replication forks are located at both ends of the loop.

Fig. 25-3. Demonstration of the bidirectional Nature of DNA replication. a — during the replication of a circular chromosome, a structure resembling the Greek letter theta (θ) is formed because both strands replicate simultaneously (the new strand is shown in red). b — replication can proceed in one or both directions, which can be determined by autoradiography: when ³H is introduced for a short period just before stopping the reaction, the label (marked in red) is detected in one or two replication forks, respectively. This demonstrated the bidirectionality of replication in E. coli, Bacillus subtilis, and other bacteria. The autoradiogram shows a replication "eye" in the DNA of B. subtilis. The highest density of silver grains (arrows) is observed precisely at the two sites where replication is taking place. The non-replicated portion of the chromosome located outside the eye contains no label and is therefore invisible.

To determine whether replication forks originate at a specific site on the DNA, it was necessary to establish reference points along the entire length of the molecule. This was accomplished using Denaturation mapping, developed by Ross Inman and colleagues. Using the bacteriophage λ chromosome (48,502 bp in length), it was shown that selective DNA denaturation can be induced in sequences with an unusually high content of A = T pairs, yielding a reproducible pattern of single-stranded eyes (see Fig. 8-28 in Vol. 1). Similarly, isolated DNA containing replication loops can be partially denatured. This makes it possible to measure and map the position and movement of replication forks by using the denatured regions as reference points. Using this method, it was shown that replication loops in this system always originate at specific sites, which were termed origins of replication. The previously established bidirectional nature of replication was also confirmed. In circular DNA molecules, the two replication forks meet at a point opposite the origin of replication. Specific origins of replication have since been identified and characterized in bacteria and lower eukaryotes.

DNA synthesis is semidiscontinuous and proceeds in the 5'—> 3' direction.

A new DNA strand is always synthesized in the 5' —> 3' direction, meaning that DNA chain growth occurs at the free OH group of the 3' end (for the STRUCTURE OF THE 5' and 3' ends of a DNA chain, see Fig. 8-7 in Vol. 1). Because the two DNA strands are antiparallel, the template strand is read from its 3' end toward its 5' end.

However, if DNA synthesis always proceeds in the 5' —> 3' direction, how can both strands be synthesized simultaneously? If both strands were synthesized continuously as the replication fork advances, one strand would have to be synthesized in the 3' —> 5' direction. This problem was resolved in the 1960s by Reiji Okazaki and his coworkers. They discovered that one of the new DNA strands is synthesized as short segments, which are now called Okazaki fragments. Their research made it clear that one strand is synthesized continuously and the other discontinuously (Fig. 25-4). The direction of Synthesis of the continuous, or leading, strand coincides with the direction of movement of the replication fork. The direction of synthesis of the discontinuous, or lagging, strand is opposite to the movement of the fork. Okazaki fragments vary in length from several hundred to several thousand nucleotides, depending on The Cell type. As we will see below, the synthesis of the leading and lagging strands is tightly coordinated.

Fig. 25-4. Strand relationships at the replication fork. The new DNA strand (red) is always synthesized in the 5' —> 3' direction. The template is read in the opposite direction, 3' —> 5'. The leading strand is synthesized continuously in the Direction of replication fork movement. The other strand, the lagging strand, is synthesized discontinuously as short segments (Okazaki fragments) in the direction opposite to that in which the replication fork moves. Okazaki fragments are joined by DNA ligase. In bacteria, Okazaki fragments are approximately 1,000 to 2,000 nucleotides long. In Eukaryotic cells, they are shorter: 150 to 200 nucleotides.

DNA is degraded by Nucleases

To understand the enzymology of DNA replication, we first examine enzymes that degrade rather than synthesize DNA. These enzymes are called nucleases, or DNases if they exhibit greater Specificity for DNA than for RNA. Every cell contains several different nucleases belonging to two major classes: exonucleases and endonucleases. Exonucleases degrade Nucleic Acids from one end of the molecule. Many of them operate exclusively in either the 5' —> 3' or 3' —> 5' direction, removing nucleotides from either the 5' or 3' end of one strand of a double-stranded nucleic acid molecule or from single-stranded DNA. Endonucleases initiate nucleic acid degradation at specific internal sites, cleaving the strand into smaller fragments. Some exonucleases and endonucleases degrade only single-stranded DNAs. There are several important classes of endonucleases that cleave only specific nucleotide sequences (for example, Restriction Endonucleases, which are of paramount importance in biotechnology; see Chapter 9, Fig. 9-2 in Vol. 1). We will encounter many types of nucleases in this and subsequent chapters.

DNA is synthesized by DNA polymerases

The search for an enzyme capable of synthesizing DNA began in 1955. Arthur Kornberg and his colleagues succeeded in purifying and characterizing DNA polymerase from E. coli cells. This single-polypeptide enzyme is now called DNA polymerase I (M_r = 103,000; encoded by the polA gene). Much later, researchers discovered that E. coli contains at least four other DNA polymerases, described below.

Detailed studies of DNA polymerase I established characteristics of DNA synthesis common to all DNA polymerases. The primary reaction involves phosphoryl group transfer. In this process, the 3'-hydroxyl group of the nucleotide at the 3' end of the growing chain acts as a nucleophile, attacking the α-phosphorus of the incoming deoxynucleoside 5'-triphosphate (Fig. 25-5). This releases inorganic pyrophosphate. The basic reaction is as follows:

where dNMP and dNTP are deoxyribonucleoside 5'-monophosphate and deoxyribonucleoside 5'-triphosphate, respectively. The reaction proceeds with a minimal change in Free energy because one phosphodiester bond is formed at the expense of a less stable phosphoanhydride bond. Noncovalent base-stacking interactions and base pairing provide additional stabilization to the elongating DNA chain compared to free nucleotides. Furthermore, DNA synthesis results in the release of 19 kJ/mol of energy within The Cell as a consequence of the subsequent Hydrolysis of pyrophosphate by inorganic pyrophosphatase (p. 38, vol. 2).

Fig. 25-5. REACTION MECHANISM. DNA chain elongation. (a) DNA polymerase I requires a single-stranded DNA template and a primer with a free 3'-hydroxyl group at its terminus to which a new nucleotide unit is added. Each incoming nucleotide is selected by base-pairing complementarity to the corresponding nucleotide in the template strand. The reaction product retains a free 3'-hydroxyl group, enabling The addition of the next nucleotide. (b) Catalysis presumably requires two Mg²⁺ ions coordinated to the two phosphate groups of the incoming nucleoside triphosphate and to three Asp residues, two of which are conserved across all DNA polymerases. The Mg²⁺ ion shown on the right facilitates the attack of the primer's 3'-hydroxyl group on the α-phosphate of the nucleoside triphosphate, whereas the other Mg²⁺ ion assists in pyrophosphate removal. Both ions stabilize the structure of the pentacoordinate Transition State. RNA polymerase operates via a similar mechanism (see Fig. 26-1b).

Early studies on DNA polymerase I already established two fundamental requirements for DNA polymerization. First, all DNA polymerases require a template. The polymerization reaction proceeds on a DNA template in accordance with the Watson-Crick base-pairing rules: if guanine is present in the template, a deoxycytidine nucleotide is incorporated into the new strand, and so forth. This discovery is of paramount importance not only because it explains the chemical basis for accurate semi-conservative DNA replication, but also because it represents the first documented example of template-directed Biosynthesis.

Second, polymerases require a primer. A primer is a strand segment (complementary to the template) with a free 3'-hydroxyl group to which a nucleotide can be added. In other words, prior to the initiation of synthesis, a portion of the new chain must already exist; all DNA polymerases can only add nucleotides to a pre-existing strand. Many primers consist of RNA oligonucleotides rather than DNA, and specialized enzymes synthesize these primers precisely where and when they are needed.

Following the addition of a nucleotide to the growing DNA chain, DNA polymerase either dissociates or translocates along the template to add the next nucleotide. Dissociation and re-association of the polymerase can limit the overall rate of polymerization—the process typically proceeds much faster when the polymerase incorporates nucleotides without leaving the template. The average number of nucleotides added before the polymerase dissociates defines its processivity. DNA polymerases vary widely in processivity; some add only a few nucleotides before dissociating, whereas others incorporate many thousands.

Replication is a highly accurate process

Replication proceeds with exceptional fidelity. In E. coli, only one error occurs per 10⁹ to 10¹⁰ incorporated nucleotides. For the E. coli chromosome, which is approximately 4.6 × 10⁶ bp in size, this implies that only a single mistake happens every 1,000 to 10,000 replication cycles. During polymerization, the Selection between a correct and an incorrect nucleotide is governed not only by the specificity of hydrogen bonding between complementary Base Pairs, but also by the geometry of standard A=T and G=C base pairs (Fig. 25-6). The Active Site of DNA polymerase I can accommodate only base pairs with the correct geometry. An incorrect nucleotide may form Hydrogen Bonds with the template, but such a mismatched pair generally fails to fit into the enzyme's active site, thereby screening out erroneous bases before the phosphodiester bond is formed.

Fig. 25-6. The Role of base-pair geometry in The fidelity of DNA replication. (a) Standard A=T and G=C base pairs are nearly identical in geometry; if the enzyme's active site can accommodate one pair (blue box), it typically accommodates the other as well. (b) The geometry of anomalous base pairs differs significantly, preventing them from fitting into the active site of DNA polymerase I.

However, the intrinsic accuracy of the polymerization reaction alone is insufficient to account for the exceptionally high fidelity of replication. In vitro measurements have demonstrated that DNA polymerase incorporates one incorrect nucleotide for every 10⁴ to 10⁵ correct ones. Occasionally, these errors arise because a base transiently adopts a rare tautomeric form (see Fig. 8-9, vol. 1), allowing it to hydrogen-bond with an incorrect partner. In vivo error rates are further reduced by auxiliary enzymatic mechanisms.

One major error-correction mechanism, shared by nearly all DNA polymerases, relies on an independent 3' → 5' exonuclease activity that proofreads every newly added nucleotide. This nuclease activity is extremely sensitive to mismatched base pairs, enabling the enzyme to excise incorrectly incorporated nucleotides (Fig. 25-7). If the polymerase inserts a wrong nucleotide, its translocation to the next position is temporarily halted. This pause provides an opportunity for error correction. Utilizing its 3' → 5' exonuclease activity, the polymerase removes the mismatched nucleotide and resumes chain elongation. This activity, known as proofreading, is not simply the reverse of the polymerization reaction (Equation 25-1) because pyrophosphate is not involved. The polymerization and proofreading activities of DNA polymerase can be measured independently. Proofreading enhances the overall accuracy of the polymerization reaction by an additional factor of 10² to 10³. In monomeric DNA polymerase I, both the polymerase and proofreading activities reside in distinct active sites within the same polypeptide chain.

Fig. 25-7. Error correction via the 3' → 5' exonuclease activity of DNA polymerase I. Structural Analysis of the enzyme reveals that when oriented along the direction of DNA movement, the exonuclease active site is positioned ahead of the polymerase active site. A mismatch (in this case, a C–A error) blocks the translocation of DNA polymerase I to the next position. The enzyme slips backward, corrects the error via its 3' → 5' exonuclease activity, and subsequently resumes 5' → 3' polymerization.

Through precise base selection coupled with proofreading, DNA polymerase makes only about one error per 10⁶ to 10⁸ incorporated nucleotides. In reality, the experimentally measured replication fidelity of E. coli is even higher. Additional accuracy is conferred by a separate enzymatic system that corrects mismatched base pairs escaping replication-associated proofreading. This Mismatch Repair process, alongside other DNA repair pathways, is discussed in Section 25.2.

E. coli contains at least five DNA polymerases

Over 90% of the DNA polymerase activity in E. coli extracts is attributable to DNA polymerase I. However, shortly after the isolation of this enzyme in 1955, accumulating evidence indicated that it could not be responsible for replicating the large E. coli chromosome. First, the rate at which the enzyme incorporates nucleotides (600 nucleotides/min) is far too slow to match the progression rate of the replication fork in the bacterial cell (which is at least 100 times faster). Second, DNA polymerase I exhibits relatively low processivity. Third, genetic studies demonstrated that replication involves numerous genes and, consequently, many proteins, making it clear that DNA polymerase I does not act alone. Fourth, and most importantly, in 1969 John Cairns isolated a bacterial mutant with a defective DNA polymerase I gene that synthesized an inactive enzyme. Although this strain was hypersensitive to DNA damage, it was nonetheless viable!

The search for other DNA polymerases led to the discovery of DNA polymerase II and DNA polymerase III in E. coli in the early 1970s. DNA polymerase II participates in one of the DNA repair pathways (Section 25.3), whereas DNA polymerase III serves as the primary replicative enzyme in E. coli. The properties of the three E. coli DNA polymerases are compared in Table 25-1. DNA polymerases IV and V, identified in 1999, are involved in a specialized pathway of error-prone translesion DNA synthesis (Section 25.2).

Table 25-1. Comparison of E. coli DNA polymerases

^a For multimeric enzymes, the gene encoding the subunit with polymerase activity is listed. The dnaE gene is now designated as polC.

^b Polymerase subunit only. DNA polymerase II shares several subunits with DNA polymerase III, specifically the β, γ, δ, δ', χ, and ψ subunits (see Table 25-2).

Thus, DNA polymerase I is not the primary replicative enzyme; rather, it plays a housekeeping and maintenance role during replication, recombination, and DNA repair. These specialized Functions are complemented by its 5' → 3' exonuclease activity. Distinct from the 3' → 5' proofreading exonuclease (Fig. 25-7), this activity resides in a structural domain that can be cleaved from the enzyme by mild protease Treatment. Upon removal of the 5' → 3' exonuclease domain, the remaining fragment (M_r = 68,000), known as the Klenow fragment (Fig. 25-8), retains both the polymerization and proofreading activities. Intact DNA polymerase I utilizes its 5' → 3' exonuclease activity in a process termed nick Translation to replace a segment of DNA (or RNA) annealed to a template strand (Fig. 25-9). Most other DNA polymerases lack a 5' → 3' exonuclease activity.

Fig. 25-8. The large fragment of DNA polymerase I (Klenow fragment). This polymerase is widely distributed among bacteria. The Klenow fragment, generated by proteolytic Cleavage of the polymerase, retains the polymerization and proofreading activities. The Klenow fragment shown here is from the thermophilic bacterium Bacillus stearothermophilus (PDB ID 3BDP). The polymerase active site is deeply embedded in a cleft near the distal end of the bound DNA (shown in blue, with the template strand in dark blue).

Fig. 25-9. Nick translation. During this process, an RNA or DNA strand paired with a template DNA is simultaneously degraded by the 5' —> 3' exonuclease activity of DNA polymerase I and replaced by a new strand synthesized via the polymerase activity of the same enzyme. This function is employed in DNA repair and the removal of RNA primers during replication (both processes are described below). The nucleic acid strand (DNA or RNA) to be removed is shown in green, and the new strand in red. DNA synthesis begins at a nick (a phosphodiester bond break that releases 3'-hydroxyl and 5'-phosphate groups). Polymerase I extends the non-template DNA strand and moves the nick along the DNA. This process is called nick translation. The nick persists at the site of DNA polymerase dissociation and is later sealed by another enzyme.

DNA polymerase III has a much more complex architecture than DNA polymerase I; it consists of ten Different types of subunits (Table 25-2). Its polymerase and proofreading activities are associated with the α and ε subunits, respectively. Upon association of the θ subunit with the α and ε subunits, a core enzyme is formed, which is capable of DNA synthesis but exhibits limited processivity. Two polymerase core enzymes can associate with one another via another subunit complex called the clamp loader, or the y complex, which consists of five subunits of four different types, Ƭ_2yδδ'. The core enzymes are linked via the Ƭ subunits. Two additional units, χ and Ψ, are attached to the clamp loader. The complete set of 13 protein subunits (of nine different types) is referred to as DNA polymerase III* (Fig. 25-10a).

Table 25-2. Subunits of E. coli DNA Polymerase III

^а The y subunit is encoded by a portion of the Ƭ subunit gene, such that 66% of the Ƭ subunit from the N-terminus shares the same Amino Acid Sequence as the y subunit. The y subunit is produced As a result of a translational frameshift (see Fig. 27-9), leading to premature Translation termination.

DNA polymerase III* is capable of polymerizing DNA, but with a much lower processivity than is required for the organized replication of an entire chromosome. The necessary increase in processivity is achieved by the addition of four β subunits to form the complete DNA polymerase III holoenzyme. The β subunits associate in pairs to form doughnut-shaped structures that encircle DNA molecules like clamps (Fig. 25-10b). Each dimer binds to a polymerase III* core enzyme (one dimeric clamp per core enzyme) and slides along the DNA during replication. The sliding β clamp prevents the dissociation of DNA polymerase III from the DNA complex, dramatically increasing processivity to 500,000 nucleotides and beyond (Table 25-1).

Fig. 25-10. DNA polymerase III. (a) Structure of bacterial DNA polymerase III. Two central domains (core enzymes), consisting of the α, ε, and θ subunits, are linked to the y complex (clamp loader), which consists of five subunits, Ƭ_2yδδ'. The y and Ƭ subunits are encoded by the same gene. The y subunit represents a truncated Ƭ subunit: the Ƭ subunit contains a domain identical to y plus an additional segment that interacts with the core enzyme. Two other subunits, χ and Ψ (not shown), of DNA polymerase III* are also associated with the y complex. Two β clamps attach to the two core enzymes; each clamp is a dimer of the β subunit. The complex interacts with the DnaB helicase via the Ƭ subunit. (b) Two β subunits of E. coli polymerase III form a ring-shaped clamp that encircles the DNA. The clamp slides along the DNA molecule, increasing the processivity of the DNA polymerase III holoenzyme to 500,000 nucleotides and beyond, while preventing the DNA from dissociating. The bottom view shows two β subunits, depicted as cyan and gray ribbons, surrounding a space-filling model of the DNA molecule. The side view shows the surface contour of the β subunits (gray) surrounding the DNA double helix (cyan and blue), shown as a stick model (based on PDB ID 2PОL).

Numerous enzymes and protein factors participate in DNA replication

The replication of E. coli requires not only DNA polymerase but also at least 20 different enzymes and proteins, each performing a specific task. This entire complex has been named the DNA replication system, or replisome. The complexity of the enzymatic replication machinery stems from the structure of DNA and the requirement for high-fidelity synthesis. The Major Classes of replication enzymes are listed below according to their functions.

To gain access to the DNA strands that serve as templates, the two parental strands must be separated. This process is typically carried out by helicases—enzymes that move along the DNA and unwind its strands utilizing the energy of ATP. Strand Separation creates topological tension in the helical structure of DNA (see Fig. 24-12), which is relieved by topoisomerases. The separated strands are stabilized by single-stranded DNA-binding proteins. As noted previously, before DNA polymerases can initiate DNA synthesis, primers must be present on the template—typically short RNA segments synthesized by enzymes called primases. Subsequently, the RNA primers are removed and replaced with DNA; in E. coli, this action is one of the primary functions of DNA polymerase I. Following the excision of the RNA primer and the incorporation of DNA, a nick in the form of a broken phosphodiester bond remains within the DNA sequence. These nicks are sealed by DNA ligases. All of these processes require coordination and regulation, which have been thoroughly studied in the E. coli system.

E. coli chromosome replication proceeds in stages

The synthesis of a DNA molecule can be divided into three stages: initiation, elongation, and termination, which differ in both their chemical nature and enzymatic composition. This and the two following chapters examine these three general Stages of the synthesis of large informational polymers (DNA, RNA, and proteins), as well as the Specific features of each biosynthetic pathway. The events described below are based on information originally obtained from in vitro experiments using purified E. coli proteins, although replication principles are highly conserved across all systems.

Initiation.

The E. coli origin of replication (oriC) consists of 245 bp and contains sequence elements that are conserved among many bacterial origins of replication. The standard set of conserved sequences is shown in Fig. 25-11. Of greatest interest are Two Types of sequences: five 9-bp repeats (R sites), which serve as binding sites for the key initiator protein DnaA, and an A=T-rich region called the DNA unwinding element (DUE). In addition, there are three auxiliary DnaA binding sites (I sites) and binding sites for the proteins IHF (integration host factor) and FIS (factor for inversion stimulation). These two proteins are required for certain recombination reactions described later in this chapter, and their names reflect their functions. Another DNA-binding protein, HU (a histone-like bacterial protein formerly known as factor U), also participates in the process but lacks a specific binding site.

Fig. 25-11. Sequence arrangement at the E. coli origin of replication (oriC). Consensus sequences (see p. 156 in vol. 1) of the key repeats are shown. N denotes any of the four nucleotides. Horizontal arrows indicate the orientation of The nucleotide sequences (left-to-right arrows point to the sequence in the top strand, and right-to-left arrows to the bottom strand). The FIS and IHF sites are binding sites for the proteins described in the text. The DnaA protein is bound to the R sites. The I sites are additional DnaA binding sites (with different sequences). DnaA interacts with them only when complexed with ATP.

At least 10 different enzymes and proteins participate in replication initiation (see Table 25-3). They unwind the DNA helix at the origin and assemble a prepriming complex for subsequent reactions. A key element in initiation is the DnaA protein, which belongs to the family of AAA+ ATPases (ATPases associated with diverse cellular activities). Many AAA+ ATPases, including DnaA, form oligomeric structures and hydrolyze ATP relatively slowly. ATP hydrolysis acts as a switch mediating transitions between different protein Conformations. In the case of DnaA, the ATP-bound form is active, whereas the ADP-bound form is inactive.

Eight molecules of the DnaA protein, each bound to ATP, form a spiral complex that envelops the R and I sites within oriC (Fig. 25-12). DnaA has a higher affinity for R sites than for I sites, and this protein binds to R sites equally well in both its ATP and ADP forms. In contrast, the I sites, which bind exclusively to ATP-bound DnaA, make it possible to distinguish between the active and inactive forms of DnaA. The DNA molecule is tightly wrapped in a right-handed direction around this complex, forming a positive supercoil (see Chapter 24). The resulting tension in adjacent DNA regions induces the denaturation of the A=T-rich DUE region. Furthermore, the complex formed at the origin also contains several DNA-binding proteins—HU, IHF, and FIS—which facilitate DNA bending.

Fig. 25-12. Model of replication initiation at the E. coli origin of replication (oriC). Eight ATP-bound molecules of the DnaA protein bind to the origin at the R and I sites (see Fig. 25-11). DNA is wrapped around this complex to form a right-handed helix. The A=T-rich DUE region denatures due to the strain generated in the strand by DnaA binding. The Formation of the spiral complex with DnaA is facilitated by the HU, IHF, and FIS proteins (not shown here, as their structures and exact roles are not yet fully defined). DnaB protein hexamers bind to each DNA strand with the aid of the DnaC protein. Through its helicase activity, the DnaB protein unwinds the DNA, preparing it for primer deposition and DNA synthesis.

Next, another AAA+ ATPase, DnaC, loads the DnaB protein onto the separated DNA strands at the denatured region. A hexamer consisting of DnaC molecules, each bound to ATP, forms a stable complex with the hexameric ring-shaped DnaB helicase. Upon interaction of DnaC with DnaB, the DnaB ring opens, a process also facilitated by further interaction between DnaB and DnaA. Two ring-shaped DnaB hexamers bind to the DUE—one on each DNA strand. The ATP molecule bound to DnaC is hydrolyzed, resulting in the release of DnaC, while DnaB remains bound to the DNA.

The attachment of the DnaB helicase is a pivotal moment in replication initiation. Subsequently, the replicative DnaB helicase moves along the single-stranded DNA in the 5' —> 3' direction, unwinding the DNA as it advances. Thus, the DnaB helicase molecules bound to the two DNA strands move in opposite directions, generating two replication forks. All other replication fork proteins are also directly or indirectly associated with DnaB. The DNA polymerase III holoenzyme is linked via its Ʈ subunits. Additional interactions of DnaB are described below. At the onset of replication, as the DNA strands separate in the region of the replication fork, numerous molecules of single-stranded DNA-binding protein (SSB) bind to them to stabilize them, while DNA gyrase (DNA topoisomerase II) relieves the topological strain generated ahead of the fork by DNA unwinding.

Initiation is the only phase of DNA replication known to be regulated, ensuring that replication occurs only once per Cell Cycle. The precise regulatory mechanism has not yet been fully elucidated, but genetic and biochemical studies have identified several independent regulatory elements.

As soon as DNA polymerase III and the β-subunits bind to DNA (signaling the completion of the initiation phase), the Hda protein binds to the β-subunits, interacting with DnaA and stimulating the hydrolysis of its bound ATP. The Hda protein (whose name stands for “DnaA-homologous”) is another AAA+ ATPase related to DnaA. ATP hydrolysis leads to the disassembly of the DnaA complex at the origin of replication. The slow release of ADP and the binding of new ATP molecules complete the cycle of transformations between the inactive (ADP-bound) and active (ATP-bound) forms of the protein, which takes from 20 to 40 min.

The course of replication initiation is influenced by DNA Methylation and interactions with the bacterial Plasma Membrane. DNA in the oriC region is methylated by Dam methylase (DNA adenine methylation; Table 25-3), which introduces a methyl group at the N⁶ position of the adenine residue within the palindromic sequence (5’) GATC. The oriC origin of replication in E. coli is particularly rich in GATC repeats—containing 11 of them in a 245 bp sequence, whereas across the entire E. coli chromosome, this repeat occurs on average only once every 256 bp.

Table 25-3. Proteins Required for Initiation of Replication at the E. coli Origin

* In this case, the subunits are identical.

Immediately after replication, the DNA is hemimethylated; the parent strands in the oriC sequences are methylated, whereas the newly synthesized strands are not. These hemimethylated oriC sequences are sequestered through interactions with The Plasma Membrane (by an unknown mechanism) and binding to the SeqA protein. After some time, the oriC sequence detaches from the plasma membrane, SeqA dissociates, and Dam methylase fully methylates the DNA before it can bind DnaA again for a new round of replication.

Elongation.

The elongation phase of replication involves two tightly coupled processes: leading-strand synthesis and lagging-strand synthesis. Multiple enzymes must be present at the replication fork for the synthesis of both strands. First, the parental DNA is unwound by DNA helicase, and the resulting topological stress is relieved by topoisomerases. Then, the SSB protein stabilizes each separated single strand. From this point onward, leading-strand and lagging-strand synthesis diverge dramatically.

Leading-strand synthesis is relatively straightforward: it begins with the synthesis of a short (10 to 60 nucleotides) RNA primer at the origin of replication by primase (the DnaG protein). To carry out this reaction, DnaG interacts with the DnaB helicase, with the primer being synthesized in the direction opposite to the movement of the DnaB helicase. In fact, the DnaB helicase moves along the strand that will become the lagging strand during DNA synthesis. However, the first primer, synthesized during the initial DnaG-DnaB interaction, serves to launch leading-strand DNA synthesis in the opposite direction. Deoxyribonucleotides are added to this primer by DNA polymerase III, which is associated with the DnaB helicase located on the opposite DNA strand. Leading-strand synthesis proceeds continuously at a rate that matches The rate of DNA unwinding at the replication fork.

As noted previously, the lagging strand is synthesized discontinuously in short Okazaki fragments (Fig. 25-13a). First, primase synthesizes An RNA primer. Then, much like in leading-strand synthesis, DNA polymerase III binds to the primer and extends it by adding deoxyribonucleotides (Fig. 25-13b). While synthesizing each Okazaki fragment appears simple at this stage, the overall process is actually quite complex. The complexity lies in coordinating the synthesis of the leading and lagging strands: both strands are synthesized by a single asymmetric DNA polymerase III dimer, a feat accomplished by looping the lagging-strand template (Fig. 25-14) to bring the two polymerization sites into close proximity.

Fig. 25-13. Synthesis of Okazaki fragments. (a) At a certain distance, primase synthesizes an RNA primer for a new Okazaki fragment. When viewing the two parallel template strands, lagging-strand synthesis formally occurs in the direction opposite to the movement of the fork. (b) Each primer is elongated by DNA polymerase III. (c) DNA synthesis continues until the polymerase reaches the primer of the previously synthesized Okazaki fragment. A new primer is synthesized near the replication fork to restart the process.

Okazaki fragment synthesis on the lagging strand relies on a remarkable enzymatic apparatus. The DnaB helicase and DnaG primase form a distinct functional unit, the primosome, as part of the replication complex. DNA polymerase III uses one set of core enzymes (the core polymerase) for continuous leading-strand synthesis, while another set of core enzymes carries out cycles of Okazaki fragment synthesis on the looped lagging strand. The DnaB helicase, positioned ahead of DNA polymerase III, unwinds the DNA at the replication fork (Fig. 25-14a), moving along the lagging-strand template in the 5' —> 3' direction. DNA primase periodically associates with the DnaB helicase to synthesize a short RNA primer (Fig. 25-14b). Next, the DNA polymerase III clamp loader places a new sliding β-clamp onto the primer (Fig. 25-14c). When synthesis of an Okazaki fragment is complete, replication pauses; the DNA polymerase III core enzyme dissociates from its clamp (and from the completed Okazaki fragment) and binds to a new clamp (Fig. 25-14d, e). This triggers the initiation of a new Okazaki fragment. As mentioned earlier, the entire complex responsible for coordinated DNA synthesis at the replication fork is called the replisome. The proteins involved at the replication fork are listed in Table 25-4.

Table 25-4. E. coli Replisome Proteins

Fig. 25-14. DNA synthesis on the leading and lagging strands. The events occurring at the replication fork are coordinated by a single DNA polymerase III dimer complexed with the DnaB helicase. Replication is shown in progress here (stages a through e are discussed in the text). The lagging strand is looped so that DNA synthesis occurs simultaneously on both the leading and lagging templates. Red arrows indicate the 3'-ends of the two new strands and the direction of DNA synthesis; thick black arrows show the direction of parental DNA movement through the complex. An Okazaki fragment is beginning to be synthesized on the lagging strand. Subunit color coding and clamp loader functions are explained in Fig. 25-15.

The DNA polymerase III clamp loader—composed of portions of two τ subunits, along with the γ, δ, and δ' subunits—also functions as an AAA+ ATPase. This complex binds ATP and a new sliding β-clamp. This binding generates strain within the dimeric clamp, causing the ring to open at the interface of one of the subunits (Fig. 25-15). The lagging strand, complete with its newly attached primer, slips into the ring through the resulting gap. The clamp loader then hydrolyzes ATP, releasing the sliding β-clamp and allowing it to snap shut around the DNA.

Fig. 25-15. The DNA polymerase III clamp loader complex. The complex consists of five subunits: the γ, δ, and δ' subunits, as well as the N-terminal domains of both τ subunits (see Fig. 25-10). The complex binds three ATP molecules and the dimeric β-clamp. This binding causes the β-clamp to open at the interface between two of its subunits. Hydrolysis of the bound ATP allows the β-clamp to re-close around the DNA.

The replisome carries out DNA synthesis rapidly, adding roughly 1,000 nucleotides per second to each strand (leading and lagging). Once an Okazaki fragment has been assembled, its RNA primer is removed and replaced with a DNA sequence by DNA polymerase I, and the remaining nick is sealed by DNA ligase (Fig. 25-16).

Fig. 25-16. Final stages of lagging-strand fragment synthesis. DNA polymerase I uses its 5' —> 3' exonuclease activity to remove RNA primers from the lagging strand and replace them with DNA. The remaining nick (break) is sealed by DNA ligase. The roles of ATP and NAD⁺ are shown in Fig. 25-17.

DNA ligase catalyzes the formation of a phosphodiester bond between a 3’-hydroxyl group at the end of One DNA strand and a 5’-phosphate at the end of another. The phosphate must first be activated by adenylylation. Viral and eukaryotic DNA ligases use ATP for this purpose. Bacterial DNA ligases (Fig. 25-17) differ in that they use NAD⁺ as the AMP source—a cofactor typically involved in hydride-transfer reactions (see Fig. 13-24). DNA ligase is yet another enzyme of DNA metabolism that has become an essential tool in recombinant DNA experiments (see Fig. 9-1).

Fig. 25-17. Reaction mechanism of DNA ligase action. At each of the three stages, one phosphodiester bond is formed at the expense of another. Stages ① and ② result in the activation of the 5'-phosphate group at the nick. AMP is first transferred to a Lysine residue in the enzyme molecule and then to the 5'-phosphate at the nick. In stage ③, the 3'-hydroxyl group attacks this phosphate and displaces AMP, forming a phosphodiester bond and thereby sealing the break. In E. coli, the AMP in the DNA ligase reaction originates from NAD⁺. DNA ligases isolated from certain viral and eukaryotic sources utilize ATP rather than NAD⁺, and in stage ① they release pyrophosphate instead of nicotinamide mononucleotide (NMN).

Termination.

Finally, the two replication forks of the circular E. coli chromosome meet at a termination region containing multiple copies of a 20 bp sequence called Ter (from the Latin terminus, meaning end) (Fig. 25-18). The Ter sequences are oriented on the chromosome to act as a trap: a replication fork can enter but cannot leave. The Ter sequences serve as binding sites for the Tus protein (short for terminus utilization substance). The Tus-Ter complex can stall a replication fork moving in only one direction. In each replication cycle, only one Tus-Ter complex is active—specifically, the first one encountered by the leading fork. Given that replication forks moving in opposite directions normally halt upon meeting, Ter sequences might seem redundant. However, they prevent over-replication by one fork in case the other fork is delayed or stalled by DNA damage or another obstacle.

Fig. 25-18. Termination of E. coli chromosome replication. The Ter sequences (from TerA to TerF) are arranged on the chromosome in two clusters with opposite orientations.

Thus, when one replication fork encounters the functional Tus-Ter complex, it halts; the other fork then stops when it meets the first (stalled) fork. Next, the final few hundred base pairs of DNA between these large Protein Complexes are replicated (via an as-yet-unknown mechanism), yielding two topologically linked (catenated) circular Chromosomes (Fig. 25-19). Circular DNA molecules linked in this manner are called catenanes. In E. coli, the unwinding and separation of catenated rings is carried out by topoisomerase IV (a type II topoisomerase). Subsequently, upon cell division, the separated chromosomes segregate into the daughter cells. The final phase of replication in other circular chromosomes, including many DNA Viruses that infect eukaryotic cells, follows a similar pathway.

Fig. 25-19. The role of topoisomerases in replication termination. DNA replication at two oppositely directed replication forks produces complete chromosomes linked as catenanes (topological interlinked rings). The rings are not covalently joined, but because they are intertwined and each is covalently closed, they cannot be separated without the aid of topoisomerases. In E. coli, type II topoisomerase (DNA topoisomerase IV) plays the primary role in separating catenated chromosomes by introducing a transient double-strand break in one chromosome, allowing the other chromosome to pass through the break.

Replication in eukaryotic cells follows a similar but more complex pathway

DNA molecules in Eukaryotic cells are significantly larger than those in bacteria and are packaged into complex nucleoprotein structures (Chromatin; see Section 24.3). The basic mechanism of DNA replication in eukaryotes is conserved with that of bacteria, and many of the protein complexes share Structural and functional Homology. However, eukaryotic replication is tightly regulated and coordinated with the cell cycle, which adds a layer of complexity.

Origins of replication are relatively well-characterized in lower eukaryotes, but less so in higher eukaryotes. In vertebrates, initiation can occur at diverse A+T-rich sequences, and the specific sites of initiation may shift with each cell division. Replication in the Yeast Saccharomyces cerevisiae initiates at defined regions known as autonomously replicating sequences (ARS) or replicators. Yeast replicators are roughly 150 bp long and contain several essential conserved sequence elements. About 400 replicators are distributed across the 16 chromosomes of the haploid yeast genome.

Regulatory mechanisms ensure that the entire cellular DNA is replicated precisely once per cell cycle. Cyclins and cyclin-dependent Kinases (CDKs), with which cyclins form active complexes, play a pivotal role in this regulation (p. 660, Vol. 1). Cyclins are rapidly degraded by ubiquitin-dependent proteolysis at the end of M phase (mitosis), and their absence permits the assembly of prereplicative complexes (pre-RCs) at replication origins. In rapidly dividing cells, pre-RC assembly occurs at the end of M phase. In slower-dividing cells, this assembly is delayed until late G1 phase. The formation of pre-RCs licenses the cell to begin replication (a stage sometimes referred to as licensing).

As in bacteria, a crucial step in the initiation of eukaryotic replication is the loading of the replicative helicase, a heterohexameric MCM protein complex (derived from minichromosome maintenance; subunits MCM2 through MCM7). The ring-shaped MCM2-7 helicase functions analogously to the bacterial DnaB helicase and is recruited to DNA by another hexameric assembly called the origin recognition complex (ORC) (Fig. 25-20). The ORC consists of five AAA+ ATPase domains and is functionally homologous to the bacterial DnaA complex. In addition, loading of the MCM2-7 complex requires two accessory proteins, CDC6 (cell division cycle) and CDT1 (CDC10-dependent transcript 1), with the yeast CDC6 protein itself functioning as another AAA+ ATPase.

Fig. 25-20. Assembly of the prereplicative complex at the eukaryotic origin of replication. ORC, CDC6, and CDT1 proteins bind to the replication origin. Many of these proteins are AAA+ ATPases that facilitate the loading of the MCM2-7 replicative helicase in a reaction analogous to the loading of the bacterial DnaB helicase by DnaC. Binding of the MCM helicase complex to DNA forms the prereplicative complex (pre-RC), a critical milestone for the initiation of replication.

To trigger replication during S phase, specific cyclin-CDK complexes (such as cyclin E-CDK2; see Fig. 12-45, Vol. 1) and CDC7-DBF4 are synthesized and activated. Both types of complexes promote replication initiation by binding to and phosphorylating select pre-RC components. Other cyclins and CDKs act to inhibit the assembly of new pre-RCs once replication has begun. For example, CDK2 associates with cyclin A as cyclin E levels decline during S phase, which modulates CDK2 activity and prevents the formation of additional pre-RCs.

The replication fork speed in eukaryotes (approximately 50 nucleotides per second) is roughly 20-fold slower than in E. coli. If human chromosomal replication initiated from a single origin, replicating an average human chromosome at this rate would take well over 500 hours. In reality, human chromosomal replication is bidirectional and initiates from multiple origins spaced 30 to 300 kbp apart. Because eukaryotic chromosomes are generally much larger than bacterial chromosomes, multiple initiation sites are likely a universal feature of eukaryotic cells.

Like bacteria, eukaryotes possess multiple DNA polymerases, some of which serve specialized roles, such as Mitochondrial DNA replication. Nuclear DNA replication relies on DNA polymerase α operating in a complex with DNA polymerase δ. Subunit DNA polymerase α maintains a highly conserved structure and set of properties across all eukaryotic cells. One of its subunits functions as a primase, while the largest subunit (M_r ≈ 180,000) bears the polymerase activity. However, this polymerase lacks a proofreading 3'—>5' exonuclease activity and cannot ensure high replication fidelity. DNA polymerase α is therefore thought to function primarily in synthesizing short primers (either RNA or DNA) for Okazaki fragments on the lagging strand. Elongation of these primers is carried out by multisubunit DNA polymerase δ. This enzyme is stimulated by and associates with proliferating cell nuclear antigen (PCNA; M_r = 29,000), a protein abundant in the nuclei of dividing cells. The three-dimensional structure of PCNA bears a striking resemblance to the β subunit of E. coli DNA polymerase III (Fig. 25-10b), despite a lack of significant primary Sequence homology. Functionally, PCNA acts as a sliding clamp that dramatically increases polymerase processivity. DNA polymerase δ possesses proofreading 3'—>5' exonuclease activity and is believed to synthesize both the leading and lagging strands within a dimeric replication complex reminiscent of bacterial DNA polymerase III.

Another enzyme, DNA polymerase ε, can substitute for DNA polymerase δ in certain contexts, such as DNA repair. DNA polymerase ε may also operate at the replication fork—analogously to bacterial DNA polymerase I—by removing the RNA primers of Okazaki fragments on the lagging strand.

Two additional protein complexes are involved in eukaryotic DNA replication. Replication protein A (RPA) binds single-stranded DNA, functioning similarly to the SSB protein in E. coli cells. Replication factor C (RFC) serves as the clamp loader for PCNA, facilitating the assembly of active replication complexes. The Amino acid sequences of the RFC subunits show substantial similarity to those of the bacterial γ complex.

To achieve the termination of replication in linear eukaryotic chromosomes, specialized structures called telomeres are synthesized at the ends of each chromosome (see Chapter 26).

Viral DNA polymerases are targets for Antiviral Therapy

Many DNA viruses encode their own DNA polymerases, several of which serve as targets for pharmacological intervention. For instance, the DNA polymerase of the Herpes simplex virus is inhibited by acyclovir, a drug developed by Gertrude Elion (see p. 558, Vol. 2). Acyclovir consists of a guanine base attached to an incomplete ribose ring.

Acyclovir is phosphorylated by viral thymidine kinase. Its affinity for the viral enzyme is roughly 200 times greater than its affinity for cellular thymidine kinase, ensuring that phosphorylation occurs preferentially in virus-infected cells. Cellular kinases then convert the resulting acyclo-GMP into acyclo-GTP, which acts as both an inhibitor and a substrate for DNA polymerases. Acyclolo-GTP inhibits herpesvirus DNA polymerase much more strongly than cellular DNA polymerases. Because acyclo-GTP lacks a 3'-hydroxyl group, its incorporation into a DNA chain acts as a chain-terminating signal. Consequently, viral replication is blocked at multiple stages. ■

Summary of Section 25.1 DNA Replication

■ DNA replication occurs with exceptionally high fidelity and during a specific phase of the cell cycle. Replication is semi-conservative, with each strand serving as a template for a new daughter strand. The process unfolds in three stages: initiation, elongation, and termination. In bacteria, the reaction begins at the origin of replication and typically proceeds bidirectionally.

■ DNA is synthesized by DNA polymerases in the 5' —> 3' direction. At the replication fork, the leading strand is synthesized continuously in the direction of fork movement, whereas the lagging strand is synthesized discontinuously as Okazaki fragments, which are subsequently joined together.

■ The fidelity of DNA replication is ensured by (1) base selection by the polymerase, (2) the proofreading 3' —> 5' exonuclease activity inherent to most DNA polymerases, and (3) specific mismatch repair systems that correct errors persisting after replication.

■ Most cells possess multiple DNA polymerases. In E. coli, the primary replicative enzyme is DNA polymerase III, while DNA polymerase I performs specialized functions during replication, recombination, and repair.

■ Numerous enzymes and protein factors operate during the initiation, elongation, and termination Stages of DNA replication, many of which belong to the AAA+ ATPase family.

■ Bacterial replication proteins are organized into large complexes where the DNA template is pulled through two replisomes anchored to the plasma membrane.