11. DNA and Its Role in Heredity

DNA and Its Role in Heredity11 DNA and Its Role in Heredity 11.1 What Is the Evidence that the Gene Is DNA?11.2 What Is the Structure of DNA?11.3 How Is DNA Replicated?11.4 How Are Errors in DNA Repaired?11.5 What Are Some Applications of Our Knowledge of DNA Structure and Replication?11.1 What Is the Evidence that the Gene Is DNA?By the 1920s, it was known that chromosomes consisted of DNA and proteins.A new dye stained DNA and provided circumstantial evidence that DNA was the genetic material: It was in the right place It varied among species It was present in the right amount11.1 What Is the Evidence that the Gene Is DNA?Frederick Griffith, working with two strains of Streptococcus pneumoniae determined that a “transforming principle” from dead cells of one strain produced a heritable change in the other strain.Figure 11.1 Genetic Transformation of Nonvirulent Pneumococci11.1 What Is the Evidence that the Gene Is DNA?Identifying the transforming principle, Oswald Avery:Treated samples to destroy different molecules; if DNA was destroyed, the transforming principle was lost. Figure 11.2 Genetic Transformation by DNA (Part 1)Figure 11.2 Genetic Transformation by DNA (Part 2)11.1 What Is the Evidence that the Gene Is DNA?Hershey-Chase experiment:Determined whether DNA or protein is the genetic material using bacteriophage T2 virus.Bacteriophage proteins were labeled with 35S; the DNA was labeled with 32P.Figure 11.3 Bacteriophage T2: Reproduction CycleFigure 11.4 The Hershey–Chase Experiment (Part 1)Figure 11.4 The Hershey–Chase Experiment (Part 2)11.1 What Is the Evidence that the Gene Is DNA?Next, genetic transformation of eukaryotic cells was demonstrated—called transfection.Use a genetic marker—a gene that confers an observable phenotype.Any cell can be transfected, even an egg cell—results in a transgenic organism.Figure 11.5 Transfection in Eukaryotic Cells11.2 What Is the Structure of DNA?The structure of DNA was determined using many lines of evidence.One crucial piece came from X-ray crystallography.A purified substance can be made to form crystals; position of atoms is inferred by the pattern of diffraction of X-rays passed through it.Figure 11.6 X-Ray Crystallography Helped Reveal the Structure of DNA11.2 What Is the Structure of DNA?Chemical composition also provided clues:DNA is a polymer of nucleotides: deoxyribose, a phosphate group, and a nitrogen-containing base.The bases:Purines: adenine (A), guanine (G)Pyrimidines: cytosine (C), thymine (T)Figure 3.23 Nucleotides Have Three Componentsrepeat fig 3.23 here11.2 What Is the Structure of DNA?1950: Erwin Chargaff found in the DNA from many different species: amount of A = amount of T amount of C = amount of GOr, the abundance of purines = the abundance of pyrimidines—Chargaff’s rule.Figure 11.7 Chargaff’s Rule11.2 What Is the Structure of DNA?Model building started by Linus Pauling—building 3-D models of possible molecular structures.Francis Crick and James Watson used model building and combined all the knowledge of DNA to determine its structure.Figure 11.8 DNA Is a Double Helix (A)11.2 What Is the Structure of DNA?X-ray crystallography convinced them the molecule was helical.Other evidence suggested there were two polynucleotide chains that ran in opposite directions—antiparallel.1953—Watson and Crick established the general structure of DNA.Figure 11.8 DNA Is a Double Helix (B)11.2 What Is the Structure of DNA?Key features of DNA:A double-stranded helix, uniform diameterIt is right-handedIt is antiparallelOuter edges of nitrogenous bases are exposed in the major and minor grooves11.2 What Is the Structure of DNA?Complementary base pairing:Adenine pairs with thymine by two hydrogen bonds.Cytosine pairs with guanine by three hydrogen bonds.Every base pair consists of one purine and one pyrimidine. Figure 11.9 Base Pairing in DNA Is Complementary (Part 1)Figure 11.9 Base Pairing in DNA Is Complementary (Part 2)11.2 What Is the Structure of DNA?Antiparallel strands: direction of strand is determined by the sugar–phosphate bonds.Phosphate groups connect to the 3′ C of one sugar, and the 5′ C of the next sugar.At one end of the chain—a free 5′ phosphate group; at the other end a free 3′ hydroxyl.11.2 What Is the Structure of DNA?The flat base pairs are exposed in the major and minor grooves—accessible for hydrogen bonding.The C═O group in thymine, the N group in adenine, and others offer hydrogen bonding sites. Key to DNA–protein interactions in replication and gene expression.11.2 What Is the Structure of DNA?Functions of DNA:Store genetic material—millions of nucleotides; base sequence stores and encodes huge amounts of informationSusceptible to mutation—change in information11.2 What Is the Structure of DNA?Genetic material is precisely replicated in cell division—by complementary base pairing.Genetic material is expressed as the phenotype—nucleotide sequence determines sequence of amino acids in proteins.11.3 How Is DNA Replicated?Kornberg showed that DNA contains information for its own replication.In a test tube: DNA, the four deoxyribonucleoside triphosphates, and DNA polymerase enzyme. The DNA is a template for synthesis of new DNA.11.3 How Is DNA Replicated?Three possible replication patterns:Semiconservative replicationConservative replicationDispersive replicationFigure 11.10 Three Models for DNA Replication11.3 How Is DNA Replicated?Meselson and Stahl showed that semiconservative replication was the correct model.They used density labeling to distinguish parent DNA strands from new DNA strands.DNA was labeled with 15N, making it more dense.Figure 11.11 The Meselson–Stahl Experiment (Part 1)Figure 11.11 The Meselson–Stahl Experiment (Part 2)11.3 How Is DNA Replicated?Results of their experiment can only be explained by the semiconservative model.If it was conservative, the first generation of individuals would have all been high or low density, but not intermediate.If dispersive, density in the first generation would be half, but this density would not appear in subsequent generations.11.3 How Is DNA Replicated?Two steps in DNA replication:The double helix is unwound, making two template strands.New nucleotides are added to the new strand at the 3′ end; joined by phosphodiester linkages. Sequence is determined by complementary base pairing.Figure 11.12 Each New DNA Strand Grows from Its 5′ End to Its 3′ End (Part 1)Figure 11.12 Each New DNA Strand Grows from Its 5′ End to Its 3′ End (Part 2)11.3 How Is DNA Replicated?A large protein complex—the replication complex—catalyzes the reactions of replication.All chromosomes have a base sequence called origin of replication (ori).Replication complex binds to ori at start.DNA replicates in both directions, forming two replication forks.Figure 11.13 Two Views of DNA Replication11.3 How Is DNA Replicated?DNA helicase uses energy from ATP hydrolysis to unwind the DNA.Single-strand binding proteins keep the strands from getting back together.11.3 How Is DNA Replicated?Small, circular chromosomes have a single origin of replication.As DNA moves through the replication complex, two interlocking circular chromosomes are formed.DNA topoisomerase separates the two chromosomes.Figure 11.14 Replication in Small Circular and Large Linear Chromosomes (A)11.3 How Is DNA Replicated?Large linear chromosomes have many origins of replication.DNA is replicated simultaneously at the origins.Figure 11.14 Replication in Small Circular and Large Linear Chromosomes (B)11.3 How Is DNA Replicated?DNA polymerases are much larger than their substrates.Shape is like a hand; the “finger” regions have precise shapes that recognize the shapes of the nucleotide bases.Figure 11.15 DNA Polymerase Binds to the Template Strand (Part 1)Figure 11.15 DNA Polymerase Binds to the Template Strand (Part 2)11.3 How Is DNA Replicated?A primer is required to start DNA replication—a short single strand of RNA.Primer is synthesized by primase.Then DNA polymerase begins adding nucleotides to the 3′ end of the primer.Figure 11.16 No DNA Forms without a Primer11.3 How Is DNA Replicated?Cells have several DNA polymerases.One is for DNA replication; others are involved in primer removal and DNA repair.Other proteins are involved in the replication process.Figure 11.17 Many Proteins Collaborate in the Replication Complex11.3 How Is DNA Replicated?At the replication fork:The leading strand is pointing in the “right” direction for replication.The lagging strand is in the “wrong” direction.Synthesis of the lagging strand occurs in small, discontinuous stretches—Okazaki fragments.Figure 11.18 The Two New Strands Form in Different Ways11.3 How Is DNA Replicated?Each Okazaki fragment requires a primer.The final phosphodiester linkage between fragments is catalyzed by DNA ligase.Figure 11.19 The Lagging Strand Story (Part 1)Figure 11.19 The Lagging Strand Story (Part 2)11.3 How Is DNA Replicated?DNA polymerases work very fast:They are processive: catalyze many polymerizations each time they bind to DNANewly replicated strand is stabilized by a sliding DNA clamp (a protein)Figure 11.20 A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization11.3 How Is DNA Replicated?The new chromosome has a bit of single stranded DNA at each end (on the lagging strand)—this region is cut off.Eukaryote chromosomes have repetitive sequences at the ends called telomeres. Figure 11.21 Telomeres and Telomerase11.3 How Is DNA Replicated?Human chromosome telomeres (TTAGGG) are repeated about 2500 times.Chromosomes can lose 50–200 base pairs with each replication. After 20–30 divisions, the cell dies.11.3 How Is DNA Replicated?Some cells—bone marrow stem cells, gamete-producing cells—have telomerase that catalyzes the addition of telomeres.90% of human cancer cells have telomerase; normal cells do not. Some anticancer drugs target telomerase.11.4 How Are Errors in DNA Repaired?DNA polymerases make mistakes in replication, and DNA can be damaged in living cells.Repair mechanisms:ProofreadingMismatch repairExcision repair11.4 How Are Errors in DNA Repaired?As DNA polymerase adds a nucleotide to a growing strand, it has a proofreading function—if bases are paired incorrectly, the nucleotide is removed.Figure 11.22 DNA Repair Mechanisms (A)11.4 How Are Errors in DNA Repaired?The newly replicated DNA is scanned for mistakes by other proteins.Mismatch repair mechanism detects mismatched bases—the new strand has not yet been modified (e.g., methylated in prokaryotes) so it can be recognized.If mismatch repair fails, the DNA is altered.Figure 11.22 DNA Repair Mechanisms (B)11.4 How Are Errors in DNA Repaired?DNA can be damaged by radiation, toxic chemicals, and random spontaneous chemical reactions.Excision repair: enzymes constantly scan DNA for mispaired bases, chemically modified bases, and extra bases—unpaired loops.Figure 11.22 DNA Repair Mechanisms (C)11.5 What Are Some Applications of Our Knowledge of DNA Structure and Replication?Copies of DNA sequences can be made by the polymerase chain reaction (PCR) technique.PCR is a cyclical process:DNA fragments are denatured by heating.A primer, plus nucleosides and DNA polymerase are added.New DNA strands are synthesized.Figure 11.23 The Polymerase Chain Reaction11.5 What Are Some Applications of Our Knowledge of DNA Structure and Replication?PCR results in many copies of the DNA fragment—referred to as amplifying the sequence.Primers are 15–20 bases, made in the laboratory. The base sequence at the 3′ end of the DNA fragment must be known.11.5 What Are Some Applications of Our Knowledge of DNA Structure and Replication?DNA polymerase that does not denature at high temperatures (90°C) was taken from a hot springs bacterium, Thermus aquaticus.11.5 What Are Some Applications of Our Knowledge of DNA Structure and Replication?DNA sequencing determines the base sequence of DNA molecules.Relies on altered nucleosides with fluorescent tags that emit different colors of light. DNA fragments are then denatured and separated by electrophoresis.Figure 11.24 Sequencing DNA (Part 1)Figure 11.24 Sequencing DNA (Part 2)