28.1 How Is DNA Replicated? 863 replication involves two replication forks that move in opposite directions. Bidirec- tional replication predicts that, if radioactively labeled nucleotides are provided as substrates for new DNA synthesis, both replication forks will become radioactively labeled. The experiment illustrated in Figure 28.2 confirms this prediction. Replication Requires Unwinding of the DNA Helix Semiconservative replication depends on unwinding the DNA double helix to ex- pose single-stranded templates to polymerase action. For a double helix to unwind, it must either rotate about its axis (while the ends of its strands are held fixed), or positive supercoils must be introduced, one for each turn of the helix unwound (see Chapter 11). If the chromosome is circular, as in E. coli, only the latter alternative is possible. Because DNA replication in E. coli proceeds at a rate approaching 1000 nucleotides per second and there are about 10 bp per helical turn, the chromosome would accumulate 100 positive supercoils per second! In effect, the DNA would be- come too tightly supercoiled to allow unwinding of the strands. DNA gyrase, a Type II topoisomerase, acts to overcome the torsional stress im- posed upon unwinding; DNA gyrase introduces negative supercoils at the expense of ATP hydrolysis. The unwinding reaction is driven by helicases (see also Chapter 16), a class of proteins that catalyze the ATP-dependent unwinding of DNA double he- lices. Unlike topoisomerases that alter the linking number of dsDNA through phos- phodiester bond breakage and reunion (see Chapter 11), helicases simply disrupt the hydrogen bonds that hold the two strands of duplex DNA together. A helicase molecule requires a single-stranded region for binding. It then moves along the sin- gle strand, unwinding the double-stranded DNA in an ATP-dependent process. SSB (single-stranded DNA-binding protein) binds to the unwound strands, preventing their re-annealing. At least ten distinct DNA helicases involved in different aspects of DNA and RNA metabolism have been found in E. coli alone. DnaB is the DNA heli- case acting in E. coli DNA replication. DnaB helicase assembles as a hexameric (␣ 6 ) “doughnut”-shaped protein ring, with DNA passing through its hole. DNA Replication Is Semidiscontinuous As shown in Figure 28.2, both parental DNA strands are replicated at each advancing replication fork. The enzyme that carries out DNA replication is DNA polymerase. A template is something whose edge is shaped in a particular way so that it can serve as a guide in making a similar object with a corresponding contour. Emerging p rogeny DNA AT GC TA A GC AT GC AT GC CG A A GC AT GC GC GCCG AT AT AT GC AT AT T GC AT AT GC AT AT GC Old OldNew New New Old Old FIGURE 28.1 DNA replication:Strand separation fol- lowed by the copying of each strand. (a) Labeled DNA Labeled DNA Unidirectional replication Bidirectional replication (b) FIGURE 28.2 Bidirectional replication. (a) Comparison of labeling during unidirectional versus bidirectional repli- cation. (b) An autoradiogram of E. coli chromosome replication in the presence of radioactive thymidine confirms bidirectional replication. (Photo courtesy of David M. Prescott, University of Colorado.) 864 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair This enzyme uses single-stranded DNA (ssDNA) as a template and makes a comple- mentary strand by polymerizing deoxynucleotides in the order specified by their base pairing with bases in the template. DNA polymerases synthesize DNA only in a 5Ј→3Ј direction, reading the antiparallel template strand in a 3Ј→5Ј sense. A dilemma arises: How does DNA polymerase copy the parent strand that runs in the 5Ј→3Ј direction at the replication fork? It turns out that replication is semidiscontinuous (Figure 28.3): As the DNA helix is unwound during its replication, the 3Ј→5Ј strand (as defined by the direction that the replication fork is moving) can be copied continuously by DNA polymerase synthesizing in the 5Ј→3Ј direction behind the replication fork. The other parental strand is copied only when a sufficient stretch of its sequence has been exposed for DNA polymerase to read it in the 3Ј→5Ј sense. Thus, one parental strand is copied continuously to give a newly synthesized copy, called the leading strand, at each replication fork. The other parental strand is copied in an intermittent, or dis- continuous, mode to yield a set of fragments 1000 to 2000 nucleotides in length, called the Okazaki fragments (Figure 28.3a). These fragments are then joined to form an intact lagging strand. Because both strands are synthesized in concert by a dimeric DNA polymerase situated at the replication fork, the 5Ј→3Ј parental strand must wrap around in trombone fashion so that the unit of dimeric DNA polymerase replicating it can move along it in the 3Ј→5Ј direction (Figure 28.3b). Overall, each of the two DNA duplexes produced in DNA replication contains one “old” and one “new” DNA strand, and half of the new strand was formed by leading strand synthesis and the other half by lagging strand synthesis. The Lagging Strand Is Formed from Okazaki Fragments In 1968, Tuneko and Reiji Okazaki provided biochemical verification of the semi- discontinuous pattern of DNA replication just described. The Okazakis exposed a rapidly dividing E. coli culture to 3 H-labeled thymidine for 30 seconds, quickly col- lected the cells, and found that half of the label incorporated into nucleic acid ap- peared in short ssDNA chains just 1000 to 2000 nucleotides in length. (The other half of the radioactivity was recovered in very large DNA molecules.) Subsequent ex- periments demonstrated that with time, the newly synthesized short ssDNA Okazaki fragments became covalently joined to form longer polynucleotide chains, in ac- cord with a semidiscontinuous mode of replication. The generality of this mode of (a) 3Ј 5Ј 3Ј 5Ј 3Ј 5Ј 5Ј 3Ј Leading strand Parental strands Movement of replication fork Lagging strand (b) 3Ј 5Ј 3Ј 5Ј 5Ј 3Ј Okazaki fragments Parental strands Movement of replication fork Lagging strand Dimeric DNA polymerase 3Ј Leading strand Okazaki fragments FIGURE 28.3 The semidiscontinuous model for DNA replication. Newly synthesized DNA is shown as red. (a) Leading and lagging strand synthesis. (b) Synthesis of both strands carried out by a dimeric DNA poly- merase situated at the replication fork. Because DNA polymerase must read the template strand in the 3Ј→5Ј direction, the 5Ј→3Ј parental strand must wrap around in trombone fashion. 28.2 What Are the Properties of DNA Polymerases? 865 replication has been corroborated with electron micrographs of DNA undergoing replication in eukaryotic cells. 28.2 What Are the Properties of DNA Polymerases? The enzymes that replicate DNA are called DNA polymerases. All DNA polymerases, whether from prokaryotic or eukaryotic sources, share the following properties: 1. The incoming base is selected within the DNA polymerase active site, as deter- mined by Watson–Crick geometric interactions with the corresponding base in the template strand. 2. Chain growth is in the 5Ј→3Ј direction and is antiparallel to the template strand. 3. DNA polymerases cannot initiate DNA synthesis de novo—all require a primer oligonucleotide with a free 3Ј-OH to build upon. Despite these commonalities, DNA replication in bacterial cells is simpler than in eukaryotes and thus will be considered first. E. coli Cells Have Several Different DNA Polymerases Table 28.1 compares the properties of the principal DNA polymerases in E. coli. These enzymes are nicknamed pol and numbered I through V in order of their discovery. DNA polymerases I, II, and V function principally in DNA repair; DNA polymerase III is the chief DNA-replicating enzyme of E. coli. Only 40 or so copies of this enzyme are present per cell. The First DNA Polymerase Discovered Was E. coli DNA Polymerase I In 1957, Arthur Kornberg and his colleagues discovered the first DNA polymerase, DNA polymerase I. DNA polymerase I catalyzed the synthesis of DNA in vitro if provided with all four deoxynucleoside-5Ј-triphosphates (dATP, dTTP, dCTP, dGTP), a template DNA strand to copy, and a primer. A primer is essential because DNA polymerases can elongate only preexisting chains; they cannot join two deoxyribonucleoside-5Ј-phosphates together to make the initial phosphodiester bond. The primer base pairs with the template DNA, forming a short, double- stranded region. This primer must possess a free 3Ј-OH end to which an incoming deoxynucleoside monophosphate is added. One of the four dNTPs is selected as substrate, pyrophosphate (PP i ) is released, and the dNMP is linked to the 3Ј-OH of the primer chain through formation of a phosphoester bond (Figure 28.4). The deoxynucleotide selected as substrate is chosen through its geometric fit with the template base to form a Watson–Crick base pair. As DNA polymerase I catalyzes the successive addition of deoxynucleotide units to the 3Ј-end of the primer, the chain is elongated in the 5Ј→3Ј direction, forming a polynucleotide sequence that is antiparallel and complementary to the template. DNA polymerase I can proceed Property Pol I Pol II Pol III (core)* Mass (kD) 103 88 130(␣), 27.5(⑀), 8.6() Molecules/cell 400 40 Turnover number † 20 40 1000 Polymerization 5Ј⎯→3Ј Yes Yes Yes Exonuclease 3Ј⎯→5Ј Yes Yes Yes Exonuclease 5Ј⎯→3Ј Yes No No *␣-, ⑀-, and -subunits. † Nucleotides polymerized at 37°C/second/molecule of enzyme. TABLE 28.1 Properties of the DNA Polymerases of E. coli 866 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair along the template strand, synthesizing a complementary strand of 3 to 200 bases before it “falls off” (dissociates from) the template. The degree to which the en- zyme remains associated with the template through successive cycles of nucleotide addition is referred to as its processivity. As DNA polymerases go, DNA polymerase I is a modestly processive enzyme. Arthur Kornberg was awarded the Nobel Prize in Physiology or Medicine in 1959 for his discovery of this DNA polymerase. DNA polymerase I is the best characterized of these enzymes. E. coli DNA Polymerase I Has Three Active Sites on Its Single Polypeptide Chain In addition to its 5Ј→3Ј polymerase activity, E. coli DNA polymerase I has two other catalytic functions: a 3Ј→5Ј exonuclease (3Ј-exonuclease) activity and a 5Ј→3Ј exonu- clease (5Ј-exonuclease) activity. The three distinct catalytic activities of DNA polym- erase I reside in separate active sites in the enzyme. E. coli DNA Polymerase I Is Its Own Proofreader and Editor The exonuclease activities of E. coli DNA polymerase I are functions that enhance the accuracy of DNA replication. The 3Ј-exonuclease activity removes nucleotides from the 3Ј-end of the growing chain (Figure 28.5), an action that negates the action of the polymerase activity. Its purpose, however, is to remove incorrect (mismatched) bases. Primer strand CH 2 O O – O O O P O – – OO O P O – P OH Base O Base OH O Base 3 Ј 5 Ј Base CH 2 O 5Ј 3Ј 2Ј 4Ј 5Ј 3Ј 4Ј 5Ј Template strand FIGURE 28.4 The chain elongation reaction catalyzed by DNA polymerase.The 3Ј-OH carries out a nucleophilic attack on the ␣-phosphoryl group of the incoming dNTP to form a phosphoester bond, and PP i is released. The subsequent hydrolysis of PP i by inorganic pyro- phosphatase renders the reaction effectively irreversible. G C T A G T T T A C A A C G C A T G A G C G G C T T Mismatched bases 5Ј 3Ј Template DNA p olymerase I 3 Ј Exonuclease hydrolysis site 5 Ј 3' FIGURE 28.5 The 3Ј→5Ј exonuclease activity of DNA polymerase I removes nucleotides from the 3Ј-end of the growing DNA chain. 28.2 What Are the Properties of DNA Polymerases? 867 Although the 3Ј-exonuclease works slowly compared to the polymerase, the poly- merase cannot elongate an improperly base-paired primer terminus. Thus, the rela- tively slow 3Ј-exonuclease has time to act and remove the mispaired nucleotide. There- fore, the polymerase active site is a proofreader, and the 3Ј-exonuclease activity is an editor. This check on the accuracy of base pairing enhances the overall precision of the process. The 5Ј-exonuclease of DNA polymerase I acts upon duplex DNA, degrading it from the 5Ј-end by releasing mononucleotides and oligonucleotides. It can remove distorted (mispaired) segments lying in the path of the advancing polymerase. Its biological roles depend on the ability of DNA polymerase I to bind at nicks (single- stranded breaks) in dsDNA and move in the 5Ј→3Ј direction, removing successive nucleotides with its 5Ј-exonucleolytic activity. (This overall process is known as nick translation, because the nick is translated [that is, moved] down the DNA.) This 5Ј-exonuclease activity plays an important role in primer removal during DNA repli- cation, as we shall soon see. DNA polymerase I is also involved in DNA repair processes (see Section 28.8). E. coli DNA Polymerase III Holoenzyme Replicates the E. coli Chromosome In its holoenzyme form, DNA polymerase III is the enzyme responsible for replica- tion of the E. coli chromosome. The simplest form of DNA polymerase III showing any DNA-synthesizing activity in vitro, “core” DNA polymerase III, is 165 kD in size and consists of three polypeptides: ␣ (130 kD), ⑀ (27.5 kD), and (8.6 kD). In vivo, core DNA polymerase III functions as part of a multisubunit complex, the DNA polymerase III holoenzyme, which is composed of ten different kinds of subunits (Table 28.2). The various auxiliary subunits increase both the polymerase activity of the core enzyme and its processivity. DNA polymerase III holoenzyme synthesizes DNA strands at a speed of nearly 1 kb/sec. DNA polymerase III holoenzyme is organized in the following way: Two core (␣⑀) DNA polymerase III units and one ␥-complex are attached to DnaB helicase via two -subunits to form a structure known as DNA polymerase III*. In turn, each core polymerase within DNA po- lymerase III* binds to a -subunit dimer to create DNA polymerase III holoenzyme, a 17-subunit ((␣⑀) 2 2 2 2 ␥␦␦Ј) complex (Figure 28.6). The ␥-complex is respon- sible for assembly of the DNA polymerase III holoenzyme complex onto DNA. The ␥-complex of the holoenzyme acts as a clamp loader by catalyzing the ATP- dependent transfer of a pair of -subunits to each strand of the DNA template. Each -subunit dimer forms a closed ring around a DNA strand and acts as a tight clamp that can slide along the DNA (Figure 28.7). Each  2 -sliding clamp tethers a Subunit Mass (kD) Function ␣ 130 Polymerase ⑀ 27.5 3Ј-Exonuclease 8.6 ⑀-subunit stabilization 71 DNA template binding; core enzyme dimerization  41 Sliding clamp, processivity ␥ 47.5 Part of the ␥-complex* ␦ 39 Part of the ␥-complex* ␦Ј 37 Part of the ␥-complex* 17 Interaction with SSB and the ␥-complex 15 Interaction with and the ␥-complex *Subunits , ␥, ␦, ␦Ј, , and form the so-called ␥-complex responsible for adding -subunits (the sliding clamp) to DNA and anchoring the sliding clamp to the two core DNA polymerase III structures.The ␥-complex is referred to as the clamp loader. TABLE 28.2 Subunits of E. coli DNA Polymerase III Holoenzyme 868 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair core polymerase to the template, accounting for the great processivity of the DNA polymerase holoenzyme. This complex can replicate an entire strand of the E. coli genome (more than 4.6 megabases) without dissociating. Compare this to the pro- cessivity of DNA polymerase I, which is only 20! The core polymerase synthesizing the lagging strand must release from the DNA template when synthesis of an Okazaki fragment is completed and rejoin the template at the next RNA primer to begin synthesis of the next Okazaki fragment. The -subunit serves as a “processivity switch” that accomplishes this purpose. The -subunit is usually “off ” and is turned “on” only on the lagging strand and only when synthesis of an Okazaki fragment is completed. When activated, ejects the  2 -sliding clamp bound to the lagging strand core polymerase. Almost immediately, the lagging strand core polymerase is reloaded onto a new  2 -sliding clamp at the 3Ј-end of next RNA primer, and synthesis of the next Okazaki fragment commences. A DNA Polymerase III Holoenzyme Sits at Each Replication Fork We now can present a snapshot of the enzymatic apparatus assembled at a replication fork (Figure 28.8 and Table 28.3). DNA gyrase (topoisomerase) and DnaB helicase unwind the DNA double helix, and the unwound, single-stranded regions of DNA are RNA primer DnaB helicase Primase ␥ ␦Ј␦ dsDNA Direction of polymerase movement Leading strand ␣⑀ core   3Ј 5Ј dsDNA 3Ј 5Ј Lagging strand ␣⑀ core   Direction of polymerase movement RNA primer Okazaki fragment Primase FIGURE 28.6 DNA polymerase III holoenzyme is a dimeric polymerase. One unit of polymerase synthesizes the leading strand, and the other synthesizes the lag- ging strand. Because DNA synthesis always proceeds in the 5Ј→3Ј direction as the template strand is read in the 3Ј→5Ј direction, lagging-strand synthesis must take place on a looped-out template. Lagging-strand synthe- sis requires repeated priming. Primase bound to the DnaB helicase carries out this function, periodically forming new RNA primers on the lagging strand. All single-stranded regions of DNA are coated with SSB (not shown). (a) (b) ᮤ FIGURE 28.7 (a) Ribbon diagram of the -subunit dimer of the DNA polymerase III holoenzyme on B-DNA, viewed down the axis of the DNA. One monomer of the -subunit dimer is colored blue and the other yellow. The centrally located DNA is multicolored.(b) Space-filling model of the -subunit dimer of the DNA polymerase III holoenzyme on B-DNA.The hole formed by the -subunits (diameter Ϸ 3.5 nm) is large enough to easily accommodate DNA (diameter Ϸ 2.5 nm) with no steric repulsion (pdb id ϭ 2POL).The rest of polymerase III holoenzyme (“core”polymerase ϩ ␥-complex) associates with this sliding clamp to form the replicative poly- merase (not shown). 28.2 What Are the Properties of DNA Polymerases? 869 maintained through interaction with SSB. Primase (DnaG) synthesizes an RNA primer on the lagging strand; the leading strand, which needs priming only once, was primed when replication was initiated. The lagging strand template is looped around, and each replicative DNA polymerase moves 5Ј→3Ј relative to its strand, copying tem- plate and synthesizing a new DNA strand. Each replicative polymerase is tethered to the DNA by its -subunit sliding clamp. The DNA polymerase III ␥-complex periodi- cally unclamps and then reclamps -subunits on the lagging strand as the primer for each new Okazaki fragment is encountered. Downstream on the lagging strand, DNA polymerase I excises the RNA primer and replaces it with DNA, and DNA ligase seals the remaining nick. DNA Ligase Seals the Nicks Between Okazaki Fragments DNA ligase (see Chapter 12) seals nicks in double-stranded DNA where a 3Ј-OH and a 5Ј-phosphate are juxtaposed. This enzyme is responsible for joining Okazaki frag- ments together to make the lagging strand a covalently contiguous polynucleotide chain. DNA Replication Terminates at the Ter Region Located diametrically opposite from oriC on the E. coli circular map is a terminus re- gion, the Ter, or t, locus. The oppositely moving replication forks meet here, and repli- cation is terminated. The Ter region contains a number of short DNA sequences, with DNA polymerase I DNA ligase 5Ј 3Ј 5Ј 3Ј Old Okazaki fragment Primer Lagging strand template DNA gyrase 5Ј 3Ј Primase Helicase Okazaki fragment Primer Leading strand template SSB Newly synthesized leading strand Dimeric replicative DNA polymerase -Subunit “sliding clamp” Primer FIGURE 28.8 General features of a replication fork.The DNA duplex is unwound by the action of DNA gyrase and helicase, and the single strands are coated with SSB (ssDNA-binding protein). Primase periodically primes synthesis on the lagging strand. Each half of the dimeric replicative polymerase is a “core”polymerase bound to its template strand by a -subunit sliding clamp. DNA polymerase I and DNA ligase act downstream on the lagging strand to remove RNA primers, replace them with DNA, and ligate the Okazaki fragments. Protein Function DNA gyrase Unwinding DNA SSB Single-stranded DNA binding DnaA Initiation factor; origin-binding protein DnaB 5Ј⎯→3Ј helicase (DNA unwinding) DnaC DnaB chaperone; loading DnaB on DNA Primase (DnaG) Synthesis of RNA primer DNA polymerase III holoenzyme Elongation (DNA synthesis) DNA polymerase I Excises RNA primer, fills in with DNA DNA ligase Covalently links Okazaki fragments Tus Termination TABLE 28.3 Proteins Involved in DNA Replication in E. coli 870 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair a consensus core element 5Ј-GTGTGTTGT. These Ter sequences act as terminators; clusters of three or four Ter sequences are organized into two sets inversely oriented with respect to one another. One set blocks the clockwise-moving replication fork, and its inverted counterpart blocks the counterclockwise-moving replication fork. Termi- nation requires binding of a specific replication termination protein, Tus protein, to Ter. Tus protein is a contrahelicase. That is, Tus protein prevents the DNA duplex from unwinding by blocking progression of the replication fork and inhibiting the ATP- dependent DnaB helicase activity. Final synthesis of both duplexes is completed. DNA Polymerases Are Immobilized in Replication Factories Most drawings of DNA replication (such as Figure 28.8) suggest that the DNA polymerases are tracking along the DNA, like locomotives along train tracks, syn- thesizing DNA as they go. Experimental evidence, however, favors the view that the DNA polymerases are immobilized, either via attachment to the cell membrane in prokaryotic cells or to the nuclear matrix in eukaryotic cells. All the associated pro- teins of DNA replication, as well as proteins necessary to hold DNA polymerase at its fixed location, constitute replication factories. The DNA is then fed through the DNA polymerases within the replication factory, much like tape is fed past the heads of a tape player, with all four strands of newly replicated DNA looping out from this fixed structure (Figure 28.9). 28.3 Why Are There So Many DNA Polymerases? Cells Have Different Versions of DNA Polymerase, Each for a Particular Purpose A host of different DNA polymerases have been discovered, and even simple bacte- ria such as Escherichia coli have more than one. Based on sequence homology, poly- merases can be grouped into seven different families. The families differ in terms of A DEEPER LOOK A Mechanism for All Polymerases Thomas A. Steitz of Yale University has suggested that biosynthesis of nucleic acids proceeds by an enzymatic mechanism that is uni- versal among polymerases. His suggestion is based on structural studies indicating that DNA polymerases use a “two-metal-ion” mechanism to catalyze nucleotide addition during elongation of a growing polynucleotide chain (see accompanying figure). The in- coming nucleotide has two Mg 2ϩ ions coordinated to its phosphate groups, and these metal ions interact with two aspartate residues that are highly conserved in DNA (and RNA) polymerases. These residues in phage T7 DNA polymerase are D705 and D882. One metal ion, designated A, interacts with the O atom of the free 3Ј-OH group on the polynucleotide chain, lowering its affinity for its hydrogen. This interaction promotes nucleophilic attack of the 3Ј-O on the phosphorus atom in the ␣-phosphate of the incoming nucleotide. The second metal ion (B in the figure) assists depar- ture of the product pyrophosphate group from the incoming nu- cleotide. Together, the two metal ions stabilize the pentacovalent transition state on the ␣-phosphorus atom. Adapted from Steitz, T., 1998. A mechanism for all polymerases. Nature 391:231–232. (See also Doublié, S., et al., 1998. Crystal structure of bacte- riophage T7 DNA replication complex at 2.2 Å resolution. Nature 391: 251–258; and Kiefer, J. R., et al., 1998. Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal. Nature 391:304–307.) – – – O O O O O O O O O O O – O O – O O – O O O C C C BaseЈ Base Base BaseЈ Primer B D882 A Me 2+ Me 2+ P P P D705 ␣ C Template OH dNTP  ␥ . . . C Replication factory FIGURE 28.9 A replication factory “fixed”to a cellular sub- structure extrudes loops of newly synthesized DNA as parental DNA duplex is fed in from the sides.Parental DNA strands are green; newly synthesized strands are blue; small circles indicate origins of replication. 28.4 How Is DNA Replicated in Eukaryotic Cells? 871 the biological function served by family members. For example, family A includes DNA polymerases involved in DNA repair in bacteria; family B polymerases include the eukaryotic DNA polymerases predominantly involved in replication of chromo- somal DNA; family C has the bacterial chromosomal DNA-replicating enzymes; members of families X and Y act in DNA repair pathways; and RT designates the DNA polymerases of retroviruses (such as HIV) and the telomerases that renew the ends of eukaryotic chromosomes. RT polymerases are novel in that they use RNA as the template. The Common Architecture of DNA Polymerases Despite sequence variation, the various DNA polymerase structures more or less fol- low a common architectural pattern that is reminiscent of a right hand, with distinct structural domains referred to as fingers, palm, and thumb (Figure 28.10). The ac- tive site of the polymerase, where deoxynucleotide addition to the growing chain is catalyzed, is located in the crevice within the palm domain that lies between the fin- gers and thumb domains. The fingers domain acts in deoxynucleotide recognition and binding, and the thumb is responsible for DNA binding, in the following man- ner: When the DNA polymerase binds to template-primer duplex DNA, its thumb domain closes around the DNA so that the DNA is bound in a groove formed by the thumb and palm. A dNTP substrate is then selected by the polymerase, and dNTP binding induces a conformational change in the fingers, which now rotate toward the polymerase active site in the palm. Catalysis ensues and a dNMP is added to the 3Ј-end of the growing primer strand; pyrophosphate is released, and the polymerase translocates one base farther along the template strand. In essence, all DNA polym- erases are molecular motors that synthesize DNA, using dNTP substrates to add dNMP units to the primer strand, as they move along the template strand, reading its base sequence. 28.4 How Is DNA Replicated in Eukaryotic Cells? DNA replication in eukaryotic cells shows strong parallels with prokaryotic DNA replication, but it is vastly more complex. First, eukaryotic DNA is organized into chromosomes which are compartmentalized within the nucleus. Furthermore, these chromosomes must be duplicated with high fidelity once (and only once!) each cell cycle. For example, in a dividing human cell, a carefully choreographed replication of 6 billion bp of DNA distributed among 46 chromosomes occurs. The C-terminus Fingers Palm Thumb Polymerase active site Exonuclease active site Exonuclease domain N-terminal domain 5Ј 5Ј 3Ј FIGURE 28.10 A structural paradigm for DNA poly- merases, bacteriophage RB69 DNA polymerase.Ternary complex formed between the RB69 DNA polymerase, DNA, and dNTP.The N-terminal domain of the protein (residues 1–108 and 340–382) is in yellow, the exonucle- ase domain (residues 109–339) is in red, the palm (residues 383–468 and 573–729) is magenta, the fingers (residues 469–572) are blue, and the thumb (residues 730–903) is green.The DNA is given in stick representa- tion, with the primer in gold and the template in blue- gray. A dNTP substrate (red) is shown at the active site, as are the two Ca 2ϩ ions (light blue spheres). Note also the calcium ion (blue sphere) at the exonuclease active site. (Adapted from Figure 1 in Franklin, M. C., Wang, J., and Steitz,T. A., 2001. Structure of the replicating complex of a Pol ␣ family DNA polymerase. Cell 105:657–667. Courtesy of Thomas A. Steitz.) 872 Chapter 28 DNA Metabolism: Replication, Recombination, and Repair events associated with cell growth and division in eukaryotic cells fall into a general sequence having four distinct phases: M, G 1 , S, and G 2 (Figure 28.11). Eukaryotic cells have solved the problem of replicating their enormous genomes in the few hours allotted to the S phase by initiating DNA replication at multiple origins of replication distributed along each chromosome. Depending on the organism and cell type, replication origins are DNA regions 0.5 to 2 kbp in size that occur every 3 to 300 kbp (for example, an average human chromosome has several hundred replication origins). Since eukaryotic DNA replication proceeds concomitantly throughout the genome, each eukaryotic chromosome must contain many units of replication, called replicons. The Cell Cycle Controls the Timing of DNA Replication Checkpoints, Cyclins, and CDKs Progression through the cell cycle is regulated through a series of checkpoints that control whether the cell continues into the next phase. These checkpoints are situated to ensure that all the necessary steps in each phase of the cycle have been satisfactorily completed before the next phase is entered. If conditions for advancement to the next phase are not met, the cycle is arrested until they are. Checkpoints depend on cyclins and cyclin- dependent protein kinases (CDKs). Cyclin is the name given to a class of proteins synthesized at one phase of the cell cycle and degraded at another. Thus, cyclins appear and then disappear at specific times during the cell cycle. Cyclins are larger than the small CDK protein kinase subunits to which they bind. The vari- ous CDKs are inactive unless complexed with their specific cyclin partners. In turn, these CDKs control events at each phase of the cycle by targeting specific proteins for phosphorylation. Destruction of the phase-specific cyclin at the end of the phase inactivates the CDK. Initiation of Replication Eukaryotic cells initiate DNA replication at multiple ori- gins, and two replication forks arise from each origin. The two replication forks then move away from each other in opposite directions. Initiation of replication de- pends on the origin recognition complex, or ORC, a protein complex that binds to replication origins. Indeed, eukaryotic replication origins are defined as nucleotide sequences that bind ORC. Stable maintenance of the eukaryotic genome demands that DNA replication occurs only once per cell cycle. This demand is met by divid- ing initiation of DNA replication into two steps: (1) the licensing of replication ori- gins during late M or early G 1 , and (2) the activation of replication at the origins during S phase through the action of two protein kinases, Cdc7-Dbf4 and S-CDK (the S-phase cyclin-dependent protein kinase). Licensing involves the highly regulated assembly of prereplication complexes (pre-RCs) on origins of replication. Early in G 1 (just after M), the ORC (a hetero- hexameric complex of Orc1-6) serves as a “landing pad” for proteins essential to replication control. Binding of these proteins to ORC establishes a pre-RC, but only within this window of opportunity during G 1 . Yeast, a simple eukaryote, provides an informative model: ORC binds to origins and recruits Cdc6 (in its phosphorylated form, Cdc6p), Cdt1, and the MCM proteins (Figure 28.12). Cdc6 and Cdt1 are the replicator activator proteins. Cdc6 is degraded following replication initiation, thereby precluding the possibility for errant replication initiation events until after mitosis, when Cdc6 accumulates again. MCM proteins are also known as replication licensing factors, because they “license,” or permit, DNA replication to occur. The MCM proteins assemble as hexameric helicases that render the chromosomes com- petent for replication. Two MCM complexes are active within each origin, one for each replication fork. The pre-RC therefore consists of Cdc6, Cdt1, the MCM com- plexes, and other proteins. DNA replication is the defining characteristic of the S phase of the cell cycle. The switch from G 1 to S is triggered by phosphorylation events carried out by S-CDK and Cdc7-Dbf4. Phosphorylation of the MCM proteins and binding of Cdc45 activates the helicase activity of MCM (Figure 28.12). Phosphorylation of Sld2 and Sld3, a pair S DNA replication and growth G 2 Growth and preparation for cell division G 1 Rapid growth and metabolic activity M Mitosis FIGURE 28.11 The eukaryotic cell cycle.The stages of mi- tosis and cell division define the M phase (M for mitosis). G 1 (G for gap, not growth) is typically the longest part of the cell cycle; G 1 is characterized by rapid growth and metabolic activity.Cells that are quiescent, that is, not growing and dividing (such as neurons), are said to be in G 0 .The S phase is the time of DNA synthesis. S is fol- lowed by G 2 , a relatively short period of growth when the cell prepares for cell division. Cell cycle times vary from less than 24 hours (rapidly dividing cells such as the epithelial cells lining the mouth and gut) to hun- dreds of days.