885024-920-947211041:36age946nac76ma76:385 946 Chapter 24 Genes and Chromosomes Schmid, C W(1996) Alu: structure, origin, evolution, significance Lebowitz, J. (1990) Through the looking glass: the discovery of nd function of one-tenth of human DNA. Prog. Nucleic Acid Res. supercoiled DNA. Trends Biochem. Sci. 15, 202-207 Mol.Biol.53,283-319 A short and interesting historical note Tyler-Smith, C.& Floridia, G(2000)Many paths to the top of Wang, J C(2002) Cellular roles of DNA topoisomerases:a mountain: diverse evolutionary solutions to centromere struc. molecular perspective. Nat. Rev. Mol. Cell Biol. 3, 430-440 ture. Cell 102, 5-8. Details of the diversity of centromere structures from different Chromatin and nucleosomes Filipski, J, Leblanc, J, Youdale, T Sikorska, M,& Walker, P.R(1990) Periodicity of DNA folding in higher order chromatin akin, V.A.(1996)Structure, function, and replication of structures. EMBO J. 9. 1319-1327 Saccharomyces cerevisiae telomeres Annu. Reu. 141-17 Hirano, T(2002) The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion and repair. Genes Dev Supercoiling and Topoisomerases rger,J M.(1998) Type II DNA topoisomerases. CurT: Opin. Description of the rapid advances in understanding of this C, White, J.H., Cozzarelli, N.R.(1990) Structure of hornberg, R D.(1974)Chromatin structure: a repeating unit of plectonemically supercoiled DNA. J. Mol BioL. 213, 931-95 histones and DNA. Science 184, 868-s71 a study that defines several fundamental features of A classic paper that introduced the subunit model for chromatin. supercoiled DNA. Nasmyth, K(2002)Segregating sister genomes: the molecular Champoux, J.J.(2001) DNA topoisomerases: structure, function, biology of chromosome separation Science 297, 559-565 nd mechanism. Annu. Rev. Biochem. 70. 369-413 Wyman, C& Kanaar, R(2002) Chromosome organization: An excellent surmmary of the topoisomerase classes. reaching out to embrace new models. Curr Biol. 12, R446-R448 Cozzarelli, N.R., Boles, T.C.,& White, J H(1990) Primer on A good, short surmmary of chromosome structure and the roles the topology and geometry of DNA supercoiling In DNA Topology of SMC proteins within it. and Its Biological Effects(Cozzarelli, N.R.& Wang, J.C., eds), Zlatanova, J.& van Holde, K(1996) The linker histones and pp. 139-184, Cold Spring Harbor Laboratory Press, Cold Spring chromatin structure: new twists. Prog. Nucleic Acid Res. Mol Harbor. NY. BioL52,217-259 A more advanced and thorough discussion. Problems 1. Packaging of DNA in a Virus Bacteriophage T2 has ( b)one DNA strand is broken,(c) DNa gyrase and ATP are a dna of molecular weight 120 X 10 contained in a head added to the DNA solution, or(d) the double helix is dena about 210 nm long. Calculate the length of the DNA (assume tured by heat he molecular weight of a nucleotide pair is 650)and com- pare it with the length of the T2 head 6. Superhelical Density Bacteriophage A infects E coli by integrating its DNA into the bacterial chromosome. The 2. The DNA of Phage M13 The base composition of success of this recombination depends on the topology of the phage M13 DNA is A, 23%; T, 36%6: G, 21%6; C, 20%6. What E. coli DNA. When the superhelical density (o)of the E. coli does this tell you about the dNa of phage M13? DNA is greater than -0.045, the probability of integra 3. The Mycoplasma Genome The complete genome of plasmid dNA isolated froman. coli culture is found to have the simplest bacterium known, Mycoplasma genitalium, is a length of 13, 800 bp and an Lk of 1,222. Calculate a for this lecular weight and contour length(when relaxed)of this mol. DNA and predict the likelihood that bacteriophage A will be ecule. What is Lko for the Mycoplasma chromosome? g=-006 what is Lk? 7. Altering Linking Number (a) What is the Lk of a 5,000 bp circular duplex DNA molecule with a nick in one 4. Size of Eukaryotic Genes An enzyme isolated from strand?(b)What is the Lk of the molecule in(a)when the rat liver has 192 amino acid residues and is coded for by a nick is sealed (relaxed)?(c)How would the Lk of the mole- gene with 1,440 bp. Explain the relationship between the cule in(b)be affected by the action of a single molecule of number of amino acid residues in the enzyme and the num- E coli topoisomerase I?(d) What is the Lk of the molecule ber of nucleotide pairs in its gene. in(b) after eight enzymatic turnovers by a single molecule of 5. Linking Number A closed-circular DNA molecule in DNA gyrase in the presence of ATP?(e) What is the Lk of the its relaxed form has an Lk of 500. Approximately how many molecule in( d)after four enzymatic turnovers by a single mol- base pairs are in this DNA? How is the linking number altered ecule of bacterial type I topoisomerase?(f What is the Lk of (increases, decreases, doesnt change, becomes undefined) the molecule in(d)after binding of one nucleosome? when(a) a protein complex is bound to form a nucleosome
946 Chapter 24 Genes and Chromosomes Schmid, C.W. (1996) Alu: structure, origin, evolution, significance and function of one-tenth of human DNA. Prog. Nucleic Acid Res. Mol. Biol. 53, 283–319. Tyler-Smith, C. & Floridia, G. (2000) Many paths to the top of the mountain: diverse evolutionary solutions to centromere structure. Cell 102, 5–8. Details of the diversity of centromere structures from different organisms, as currently understood. Zakian, V.A. (1996) Structure, function, and replication of Saccharomyces cerevisiae telomeres. Annu. Rev. Genet. 30, 141–172. Supercoiling and Topoisomerases Berger, J.M. (1998) Type II DNA topoisomerases. Curr. Opin. Struct. Biol. 8, 26–32. Boles, T.C., White, J.H., & Cozzarelli, N.R. (1990) Structure of plectonemically supercoiled DNA. J. Mol. Biol. 213, 931–951. A study that defines several fundamental features of supercoiled DNA. Champoux, J.J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu. Rev. Biochem. 70, 369–413. An excellent summary of the topoisomerase classes. Cozzarelli, N.R., Boles, T.C., & White, J.H. (1990) Primer on the topology and geometry of DNA supercoiling. In DNA Topology and Its Biological Effects (Cozzarelli, N.R. & Wang, J.C., eds), pp. 139–184, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. A more advanced and thorough discussion. Lebowitz, J. (1990) Through the looking glass: the discovery of supercoiled DNA. Trends Biochem. Sci. 15, 202–207. A short and interesting historical note. Wang, J.C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat. Rev. Mol. Cell Biol. 3, 430–440. Chromatin and Nucleosomes Filipski, J., Leblanc, J., Youdale, T., Sikorska, M., & Walker, P.R. (1990) Periodicity of DNA folding in higher order chromatin structures. EMBO J. 9, 1319–1327. Hirano, T. (2002) The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion and repair. Genes Dev. 16, 399–414. Description of the rapid advances in understanding of this interesting class of proteins. Kornberg, R.D. (1974) Chromatin structure: a repeating unit of histones and DNA. Science 184, 868–871. A classic paper that introduced the subunit model for chromatin. Nasmyth, K. (2002) Segregating sister genomes: the molecular biology of chromosome separation. Science 297, 559–565. Wyman, C. & Kanaar, R. (2002) Chromosome organization: reaching out to embrace new models. Curr. Biol. 12, R446–R448. A good, short summary of chromosome structure and the roles of SMC proteins within it. Zlatanova, J. & van Holde, K. (1996) The linker histones and chromatin structure: new twists. Prog. Nucleic Acid Res. Mol. Biol. 52, 217–259. 1. Packaging of DNA in a Virus Bacteriophage T2 has a DNA of molecular weight 120 106 contained in a head about 210 nm long. Calculate the length of the DNA (assume the molecular weight of a nucleotide pair is 650) and compare it with the length of the T2 head. 2. The DNA of Phage M13 The base composition of phage M13 DNA is A, 23%; T, 36%; G, 21%; C, 20%. What does this tell you about the DNA of phage M13? 3. The Mycoplasma Genome The complete genome of the simplest bacterium known, Mycoplasma genitalium, is a circular DNA molecule with 580,070 bp. Calculate the molecular weight and contour length (when relaxed) of this molecule. What is Lk0 for the Mycoplasma chromosome? If 0.06, what is Lk? 4. Size of Eukaryotic Genes An enzyme isolated from rat liver has 192 amino acid residues and is coded for by a gene with 1,440 bp. Explain the relationship between the number of amino acid residues in the enzyme and the number of nucleotide pairs in its gene. 5. Linking Number A closed-circular DNA molecule in its relaxed form has an Lk of 500. Approximately how many base pairs are in this DNA? How is the linking number altered (increases, decreases, doesn’t change, becomes undefined) when (a) a protein complex is bound to form a nucleosome, (b) one DNA strand is broken, (c) DNA gyrase and ATP are added to the DNA solution, or (d) the double helix is denatured by heat? 6. Superhelical Density Bacteriophage infects E. coli by integrating its DNA into the bacterial chromosome. The success of this recombination depends on the topology of the E. coli DNA. When the superhelical density () of the E. coli DNA is greater than 0.045, the probability of integration is 20%; when is less than 0.06, the probability is 70%. Plasmid DNA isolated from an E. coli culture is found to have a length of 13,800 bp and an Lk of 1,222. Calculate for this DNA and predict the likelihood that bacteriophage will be able to infect this culture. 7. Altering Linking Number (a) What is the Lk of a 5,000 bp circular duplex DNA molecule with a nick in one strand? (b) What is the Lk of the molecule in (a) when the nick is sealed (relaxed)? (c) How would the Lk of the molecule in (b) be affected by the action of a single molecule of E. coli topoisomerase I? (d) What is the Lk of the molecule in (b) after eight enzymatic turnovers by a single molecule of DNA gyrase in the presence of ATP? (e) What is the Lk of the molecule in (d) after four enzymatic turnovers by a single molecule of bacterial type I topoisomerase? (f) What is the Lk of the molecule in (d) after binding of one nucleosome? Problems 8885d_c24_920-947 2/11/04 1:36 PM Page 946 mac76 mac76:385_reb:
8885dc24920-9472/11/041:36 PM Page947mac76mac76:385 Chapter 24 Problems 947 8. Chromatin Early evidence that helped researchers 9. DNA Structure Explain how the underwinding of a B- efine nucleosome structure is illustrated by the agarose gel DNA helix might facilitate or stabilize the formation of Z-DNA. below, in which the thick bands represent DNA. It was gen- ated by briefly treating chromatin with an enzyme that 10. Maintaining DNA Structure (a) Descnbe two struc- tural features required for a dNa molecule to maintain a neg purified DNA to electrophoresis. Numbers at the side of the atively supercold state. (b)List three structural changes gel denote the position to which a linear DNA of the indicated that become more favorable when a dNa molecule is nega size would migrate. What does this gel tell you about chro- tively supercoiled. (c)What enzyme, with the aid of ATP,can matin structure? Why are the dna bands thick and spread out rather than sharply defined? physical mechanism by which this enzyme acts 11. Yeast Artificial Chromosomes (YACs) YACs are used to clone large pieces of DNA in yeast cells. What three ypes of DNa sequences are required to ensure proper repli- cation and propagation of a YAc in a yeast cell
Chapter 24 Problems 947 8. Chromatin Early evidence that helped researchers define nucleosome structure is illustrated by the agarose gel below, in which the thick bands represent DNA. It was generated by briefly treating chromatin with an enzyme that degrades DNA, then removing all protein and subjecting the purified DNA to electrophoresis. Numbers at the side of the gel denote the position to which a linear DNA of the indicated size would migrate. What does this gel tell you about chromatin structure? Why are the DNA bands thick and spread out rather than sharply defined? 9. DNA Structure Explain how the underwinding of a BDNA helix might facilitate or stabilize the formation of Z-DNA. 10. Maintaining DNA Structure (a) Describe two structural features required for a DNA molecule to maintain a negatively supercoiled state. (b) List three structural changes that become more favorable when a DNA molecule is negatively supercoiled. (c) What enzyme, with the aid of ATP, can generate negative superhelicity in DNA? (d) Describe the physical mechanism by which this enzyme acts. 11. Yeast Artificial Chromosomes (YACs) YACs are used to clone large pieces of DNA in yeast cells. What three types of DNA sequences are required to ensure proper replication and propagation of a YAC in a yeast cell? 200 bp 400 bp 600 bp 800 bp 1,000 bp 8885d_c24_920-947 2/11/04 1:36 PM Page 947 mac76 mac76:385_reb:
8885dc25948-9942/11/041:57 PM Page957mac76mac76:385 25.1 DNA Replication 957 and by the requirements for accuracy. The main classes of replication enzymes are considered here in terms the pre they overcome ccess to the dna strands that are to act as tem- plates requires separation of the two parent strands This is generally accomplished by helicases, enzymes hat move along the dNa and separate the strands, us ing chemical energy from ATP Strand separation cre- ates topological stress in the helical DNA structure(see Fig. 24-12), which is relieved by the action of topo- isomerases. The separated strands are stabilized by DNA-binding proteins. As noted earlier, before DNA polymerases can begin synthesizing DNA, primers must be present on the template--generally short segments FIGURE 25-8 Large( Klenow) fragment of DNA polymerase I.This polymerase is widely distributed in bacteria. The Klenow fragment, Nick produced by proteolytic treatment of the polymerase, retains the poly- nerization and proofreading activities of the enzyme. The Klenow RNA or DNA fragment shown here is from the thermophilic bacterium Bacillus stearothermophilus(PDB ID 3BDP). The active site for addition of nu- cleotides is deep in the crevice at the far end of the bound dNA. The dark blue strand is the template DNA strand DNA another set of subunits, a clamp-loading complex, or y complex, consisting of five subunits of four different types, T2y68. The core polymerases are linked through the T(tau) subunits. Two additional subunits, x(chi)and dNTPs a(psi), are bound to the clamp-loading complex. The entire assembly of 13 protein subunits (nine different types) is called DNa polymerase Ill(Fig. 25-10a) DNA polymerase I can polymerize DNA, but with He a much lower processivity than one would expect for the organized replication of an entire chromosome. The necessary increase in processivity is provided by the ad- dition of the B subunits, four of which complete the dNA polymerase II holoenzyme. The B subunits associate in pairs to form donut-shaped structures that encircle the DNA and act like clamps(Fig. 25-10b) Each dimer as- Nick sociate with a core subassembly of polymerase ll(one dimeric clamp per core subassembly) and slides along the DNA as replication proceeds. The B sliding clamp prevents the dissociation of dna polymerase Ill from DNA, dramatically increasing processivity--to greater FIGURE 25-9 Nick translation. In this process, an RNA or DNA strand than500,000(able25-1) paired to a DNA template is simultaneously degraded by the 5-3 exonuclease activity of DNA polymerase I and replaced by the poly- DNA Replication Requires Many Enzymes merase activity of the same enzyme. These activities have a role in and Protein factors both DNA repair and the removal of RNA primers during replication (both described later). The strand of nucleic acid to be removed (ei. Replication in E. coli requires not just a single DNA ther DNA or RNA) is shown in green, the replacement strand in red polymerase but 20 or more different enzymes and pro- DNA synthesis begins at a nick (a broken phosphodiester bond, leav- teins, each performing a specific task. The entire com- ing a free 3 hydroxyl and a free 5 phosphate) Polymerase I extends plex has been termed the DNa replicase system or the nontemplate DNA strand and moves the nick along the dNA-a replisome. The enzymatic complexity of replication re- process called nick translation. A nick remains where DNA polymeras flects the constraints imposed by the structure of DNa I dissociates, and is later sealed by another enzyme
another set of subunits, a clamp-loading complex, or complex, consisting of five subunits of four different types, 2. The core polymerases are linked through the (tau) subunits. Two additional subunits, (chi) and (psi), are bound to the clamp-loading complex. The entire assembly of 13 protein subunits (nine different types) is called DNA polymerase III* (Fig. 25–10a). DNA polymerase III* can polymerize DNA, but with a much lower processivity than one would expect for the organized replication of an entire chromosome. The necessary increase in processivity is provided by the addition of the subunits, four of which complete the DNA polymerase III holoenzyme. The subunits associate in pairs to form donut-shaped structures that encircle the DNA and act like clamps (Fig. 25–10b). Each dimer associates with a core subassembly of polymerase III* (one dimeric clamp per core subassembly) and slides along the DNA as replication proceeds. The sliding clamp prevents the dissociation of DNA polymerase III from DNA, dramatically increasing processivity—to greater than 500,000 (Table 25–1). DNA Replication Requires Many Enzymes and Protein Factors Replication in E. coli requires not just a single DNA polymerase but 20 or more different enzymes and proteins, each performing a specific task. The entire complex has been termed the DNA replicase system or replisome. The enzymatic complexity of replication reflects the constraints imposed by the structure of DNA and by the requirements for accuracy. The main classes of replication enzymes are considered here in terms of the problems they overcome. Access to the DNA strands that are to act as templates requires separation of the two parent strands. This is generally accomplished by helicases, enzymes that move along the DNA and separate the strands, using chemical energy from ATP. Strand separation creates topological stress in the helical DNA structure (see Fig. 24–12), which is relieved by the action of topoisomerases. The separated strands are stabilized by DNA-binding proteins. As noted earlier, before DNA polymerases can begin synthesizing DNA, primers must be present on the template—generally short segments 25.1 DNA Replication 957 5 3 3 5 OH P RNA or DNA Template DNA strand (PPi) n 5 3 3 5 OH P dNTPs dNMPs or rNMPs 5 3 3 5 OH P 5 3 3 5 OH P Nick Nick DNA polymerase I FIGURE 25–8 Large (Klenow) fragment of DNA polymerase I. This polymerase is widely distributed in bacteria. The Klenow fragment, produced by proteolytic treatment of the polymerase, retains the polymerization and proofreading activities of the enzyme. The Klenow fragment shown here is from the thermophilic bacterium Bacillus stearothermophilus (PDB ID 3BDP). The active site for addition of nucleotides is deep in the crevice at the far end of the bound DNA. The dark blue strand is the template. FIGURE 25–9 Nick translation. In this process, an RNA or DNA strand paired to a DNA template is simultaneously degraded by the 5n3 exonuclease activity of DNA polymerase I and replaced by the polymerase activity of the same enzyme. These activities have a role in both DNA repair and the removal of RNA primers during replication (both described later). The strand of nucleic acid to be removed (either DNA or RNA) is shown in green, the replacement strand in red. DNA synthesis begins at a nick (a broken phosphodiester bond, leaving a free 3 hydroxyl and a free 5 phosphate). Polymerase I extends the nontemplate DNA strand and moves the nick along the DNA—a process called nick translation. A nick remains where DNA polymerase I dissociates, and is later sealed by another enzyme. 8885d_c25_948-994 2/11/04 1:57 PM Page 957 mac76 mac76:385_reb:
8885dc25948-9942/11/041:57 PM Page949 hapter 25 DNA Metabolism 949 Mismatch rotein mutL Single-stranded dna-bi rotein ssb DNA repair wUrA Helicase dnaB holc DNA polymerase III subunit RNA polymerase rpoB holD DNA polymerase Ill subunit dnac Primosome component DNA polymerase polB DNA polymerase II DNA helicase/mismatch repair uurD mutT dnap polc (dnaE) DNA polymerase III subuni Helicase3'→>5′rep dnaQ DNA polymerase Ill subunit dinB DNA polymerase Iv recR Recombinational repair (Replication origin oric 100/0 holA DNA polymerase III subunit Replication initiation phr DNA photolyase dnaA Recombinational repair recF uurB DNA repair holB DNA polymerase III subunit DNA gyrase subuni Methylation dam umud DNA polymerase v RNA polymerase∫rpaA subunit ogt O-G alkyltransferase Ter(Replication termination Mismatch repair proteins mutH holE DNA polymerase III subunit Recombination and ruA Recombination and recombinational repair DNA ligase lig Recombination and combinational repair recA /reco Recombinational urc DNA repair sbcb Exonuclease I DNA gyrase subunit gyra nfo AP endonuclease FIGURE 25-1 Map of the E coli chromosome. The map shows the DNA molecule of E coli. The three-letter names of genes and other relative positions of genes encoding many of the proteins important elements generally reflect some aspect of their function. These include DNA metabolism. The number of genes known to be involved pro- mut, mutagenesis; dna, DNA replication; pol, DNA polymerase; rpo, vides a hint of the complexity of these processes. The numbers 0 to RNA polymerase; uvr, U/V resistance; rec, recombination; dam, DNA 100 inside the circular chromosome denote a genetic measurement adenine methylation; lig, DNA ligase; Ter, termination of replication called minutes. Each minute corresponds to-40, 000 bp along the and ori, origin of replication. A Word about Terminology Before beginning to look reflecting their order of discovery rather than their or- closely at replication, we must make a short digression der in a reaction sequence. into the use of abbreviations in naming genes and pro- During genetic investigations, the protein product teins. By convention, bacterial genes generally are of each gene is usually isolated and characterized. Many named using three lowercase, italicized letters that of bacterial genes have been identified and named before ten reflect their apparent function. For example, the the roles of their protein products are understood in dna, uur and rec genes affect DNA replication, resist- detail. Sometimes the gene product is found to be a pre- ance to the damaging effects of Uv radiation, and re- viously isolated protein, and some renaming occurs combination respec tively. Where several genes affect Often the product turns out to be an as yet unknown the same process, the letters A, B, C, and so forth, are protein, with an activity not easily described by a sim- added-as in dnaA, dnaB, dnaQ, for example--usually ple enzyme name. In a practice that can be confusing
A Word about Terminology Before beginning to look closely at replication, we must make a short digression into the use of abbreviations in naming genes and proteins. By convention, bacterial genes generally are named using three lowercase, italicized letters that often reflect their apparent function. For example, the dna, uvr, and rec genes affect DNA replication, resistance to the damaging effects of UV radiation, and recombination, respectively. Where several genes affect the same process, the letters A, B, C, and so forth, are added—as in dnaA, dnaB, dnaQ, for example—usually reflecting their order of discovery rather than their order in a reaction sequence. During genetic investigations, the protein product of each gene is usually isolated and characterized. Many bacterial genes have been identified and named before the roles of their protein products are understood in detail. Sometimes the gene product is found to be a previously isolated protein, and some renaming occurs. Often the product turns out to be an as yet unknown protein, with an activity not easily described by a simple enzyme name. In a practice that can be confusing, Chapter 25 DNA Metabolism 949 Mismatch repair protein mutL Single-stranded DNA–binding protein ssb Helicase dnaB RNA polymerase subunits rpoB rpoC DNA polymerase I polA mutU dnaP rep (Replication origin) oriC Replication initiation dnaA dnaN Recombinational repair recF Methylation dam RNA polymerase subunits rpoA rpoD Primase dnaG Mismatch repair proteins mutH mutS recC Recombination and recombinational repair recB recD recA Recombination and recombinational repair DNA repair uvrA DNA helicase/mismatch repair uvrD DNA gyrase subunit Primosome assembly gyrB priA Ter (Replication termination) DNA ligase lig Uracyl glycosylase ung recO Recombinational repair nfo AP endonuclease DNA gyrase subunit gyrA sbcB Exonuclease I uvrC DNA repair ruvC ruvA Recombination and recombinational repair holE DNA polymerase III subunit xthA AP endonuclease ogt O6 -G alkyltransferase ruvB umuC umuD uvrB DNA repair phr DNA photolyase holB DNA polymerase III subunit holA DNA polymerase III subunit recR Recombinational repair dinB DNA polymerase IV dnaQ DNA polymerase III subunit polC (dnaE) DNA polymerase III subunit mutT polB DNA polymerase II holC DNA polymerase III subunit dnaJ, dnaK dnaC Primosome component holD DNA polymerase III subunit 100/0 50 75 25 Helicase 3 5 DNA polymerase V FIGURE 25–1 Map of the E. coli chromosome. The map shows the relative positions of genes encoding many of the proteins important in DNA metabolism. The number of genes known to be involved provides a hint of the complexity of these processes. The numbers 0 to 100 inside the circular chromosome denote a genetic measurement called minutes. Each minute corresponds to ~40,000 bp along the DNA molecule of E. coli. The three-letter names of genes and other elements generally reflect some aspect of their function. These include mut, mutagenesis; dna, DNA replication; pol, DNA polymerase; rpo, RNA polymerase; uvr, UV resistance; rec, recombination; dam, DNA adenine methylation; lig, DNA ligase; Ter, termination of replication; and ori, origin of replication. 8885d_c25_948-994 2/11/04 1:57 PM Page 949 mac76 mac76:385_reb:
8885dc25948-9942/11/041:57 PM Page950mac76mac76:385 Chapter 25 DNA Metabolism these bacterial proteins often retain the name of their 1957. Meselson and Stahl grew E. coli cells for many genes. When referring to the protein, roman type is used generations in a medium in which the sole nitrogen and the first letter is capitalized: for example, the dnaa source (NH Ci contained N, the " heavy"isotope of and reca gene products are called the Dnaa and reca nitrogen, instead of the normal, more abundant " light proteins, respectively. You will encounter many such ex- isotope, N. The DNA isolated from these cells had a amples in this chapter. density about 1% greater than that of normal [ndna ofc: similar conventions exist for the naming of eukary-(Fig. 25-2a). Although this is only a small difference, a genes, although the exact form of the abbreviations mixture of heavy NDNa and light [NDNA can be may vary with the species and no single convention ap separated by centrifugation to equilibrium in a cesium plies to all eukaryotic systems chloride density gradient The E. coli cells grown in the N medium were transferred to a fresh medium containing only the N 25.1 DNA Replication isotope, where they were allowed to grow until the cell population had just doubled. The DNA isolated from Long before the structure of DNA was known, scientists these first-generation cells formed a single band in the wondered at the ability of organisms to create faithful CsCl gradient at a position indicating that the double- copies of themselves and, later, at the ability of cells to helical DNA molecules of the daughter cells were hy- produce many identical copies of large and complex brids containing one new 4N strand and one parent 5N macromolecules. Speculation about these problems cen strand (Fig. 25-2b) ered around the concept of a template, a structure that would allow molecules to be lined up in a specific This result argued against conservative replication, an alternative hypothesis in which one progeny dNA order and joined, to create a macromolecule with a unique sequence and function. The 1940s brought the revelation that dNa was the genetic molecule, but not DNA extracted and centri until James Watson and francis crick deduced its struc density gradient ture did the way in which DNA could act as a template for the replication and transmission of genetic informa- tion become clear: one strand is the complement of the other: The strict base-pairing rules mean that each strand provides the template for a sister strand with a Heavy predictable and complementary sequence(see Fig DNA(ON Original parent 8-16, 8-17). 0 Nucleotides: Building Blocks of Nucleic Acids The fundamental properties of the dNa replication process and the mechanisms used by the enzymes that catalyze it have proved to be essentially identical in all species. This mechanistic unity is a major theme as w Hybrid DNA (b) proceed from general properties of the replication process, to E. coli replication enzymes, and, finally, to First-generation replication in eukaryotes daughter molecules DNA Replication Follows a Set of Fundamental Rules Early research on bacterial DNA replication and its en- zymes helped to establish several basic properties that DNA (N have proven applicable to dna synthesis in every DNA Replication Is Semiconservative Each DNA strand daughter molecules serves as a template for the synthesis of a new strand, FIGURE 25-2 The Meselson-Stahl experiment (a) Cells were grown producing two new DNA molecules, each with one new for many generations in a medium containing only heavy nitrogen, strand and one old strand. This is semiconservative I5N, so that all the nitrogen in their dNA was ISN, as shown by a sin replication. gle band (blue) when centrifuged in a CsCl density gradient. (b)Once Watson and Crick proposed the hypothesis of semi- the cells had been transferred to a medium containing only light ni- 1953 paper on the structure of DNA, and the hypothe- at a higher position in the density gradient (purple band s quilibrated conservative replication soon after publication of their trogen,N, cellular DNA isolated after one generation sis was proved by ingeniously designed experiments car- uation of replication for a second generation yielded two hybrid DNAs ried out by Matthew Meselson and Franklin Stahl in and two light DNAs (red), confirming semiconservative replication
these bacterial proteins often retain the name of their genes. When referring to the protein, roman type is used and the first letter is capitalized: for example, the dnaA and recA gene products are called the DnaA and RecA proteins, respectively. You will encounter many such examples in this chapter. Similar conventions exist for the naming of eukaryotic genes, although the exact form of the abbreviations may vary with the species and no single convention applies to all eukaryotic systems. 25.1 DNA Replication Long before the structure of DNA was known, scientists wondered at the ability of organisms to create faithful copies of themselves and, later, at the ability of cells to produce many identical copies of large and complex macromolecules. Speculation about these problems centered around the concept of a template, a structure that would allow molecules to be lined up in a specific order and joined, to create a macromolecule with a unique sequence and function. The 1940s brought the revelation that DNA was the genetic molecule, but not until James Watson and Francis Crick deduced its structure did the way in which DNA could act as a template for the replication and transmission of genetic information become clear: one strand is the complement of the other. The strict base-pairing rules mean that each strand provides the template for a sister strand with a predictable and complementary sequence (see Figs 8–16, 8–17). Nucleotides: Building Blocks of Nucleic Acids The fundamental properties of the DNA replication process and the mechanisms used by the enzymes that catalyze it have proved to be essentially identical in all species. This mechanistic unity is a major theme as we proceed from general properties of the replication process, to E. coli replication enzymes, and, finally, to replication in eukaryotes. DNA Replication Follows a Set of Fundamental Rules Early research on bacterial DNA replication and its enzymes helped to establish several basic properties that have proven applicable to DNA synthesis in every organism. DNA Replication Is Semiconservative Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand. This is semiconservative replication. Watson and Crick proposed the hypothesis of semiconservative replication soon after publication of their 1953 paper on the structure of DNA, and the hypothesis was proved by ingeniously designed experiments carried out by Matthew Meselson and Franklin Stahl in 1957. Meselson and Stahl grew E. coli cells for many generations in a medium in which the sole nitrogen source (NH4Cl) contained 15N, the “heavy” isotope of nitrogen, instead of the normal, more abundant “light” isotope, 14N. The DNA isolated from these cells had a density about 1% greater than that of normal [14N]DNA (Fig. 25–2a). Although this is only a small difference, a mixture of heavy [15N]DNA and light [14N]DNA can be separated by centrifugation to equilibrium in a cesium chloride density gradient. The E. coli cells grown in the 15N medium were transferred to a fresh medium containing only the 14N isotope, where they were allowed to grow until the cell population had just doubled. The DNA isolated from these first-generation cells formed a single band in the CsCl gradient at a position indicating that the doublehelical DNA molecules of the daughter cells were hybrids containing one new 14N strand and one parent 15N strand (Fig. 25–2b). This result argued against conservative replication, an alternative hypothesis in which one progeny DNA 950 Chapter 25 DNA Metabolism DNA extracted and centrifuged to equilibrium in CsCl density gradient Original parent molecule First-generation daughter molecules Second-generation daughter molecules Heavy DNA (15N) Hybrid DNA (15N–14N) Hybrid DNA Light DNA (14N) (a) (b) (c) FIGURE 25–2 The Meselson-Stahl experiment. (a) Cells were grown for many generations in a medium containing only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N, as shown by a single band (blue) when centrifuged in a CsCl density gradient. (b) Once the cells had been transferred to a medium containing only light nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient (purple band). (c) Continuation of replication for a second generation yielded two hybrid DNAs and two light DNAs (red), confirming semiconservative replication. 8885d_c25_948-994 2/11/04 1:57 PM Page 950 mac76 mac76:385_reb: