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13 DNA and Its Role in Heredity 13 DNA and Its Role in Heredity 13.1 What Is the Evidence that the Gene Is DNA? 13.2 What Is the Structure of DNA? 13.3 How Is DNA Replicated? 13.4 How Are Errors in DNA Repaired? 13.5 How Does the Polymerase Chain Reaction Amplify DNA? 13 DNA and Its Role in Heredity Lance Armstrong’s cancer was stopped by a drug called cisplatin that forms linkages between DNA strands and prevents replication. Without DNA replication cells can’t divide, and undergo programmed cell death. Opening Question: How does cisplatin work? 13.1 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 that stained DNA provided evidence that DNA is the genetic material. • It was in the right place • It varied among species • It was present in the right amounts 13.1 What Is the Evidence that the Gene Is DNA? Experimental evidence came from work on two strains of Streptococcus pneumoniae. A substance from cells of one strain (even when dead) could produce a heritable change in the other strain. Figure 13.1 Genetic Transformation 13.1 What Is the Evidence that the Gene Is DNA? To identify this substance, Oswald Avery treated samples to destroy different molecules. If DNA was destroyed, the transforming activity was lost. There was no loss of activity with destruction of proteins or RNA. Figure 13.2 Genetic Transformation by DNA (Part 1) Figure 13.2 Genetic Transformation by DNA (Part 2) 13.1 What Is the Evidence that the Gene Is DNA? Hershey-Chase experiment: used bacteriophage T2 virus to determine whether DNA, or protein, is the genetic material. Part of the virus enters E. coli cells and converts the cell into a virus replication machine. Figure 13.3 Bacteriophage T2: Reproduction Cycle 13.1 What Is the Evidence that the Gene Is DNA? Bacteriophage were grown with either 35S to label the proteins, or with 32P to label the DNA. After infection, bacterial cells and viral remains were separated—the bacteria cells were labeled with 32P, indicating that DNA had entered the cells. Figure 13.4 The Hershey–Chase Experiment (Part 1) Figure 13.4 The Hershey–Chase Experiment (Part 2) 13.1 What Is the Evidence that the Gene Is DNA? Eukaryotic cells can also be transformed (transfection). A genetic marker (a gene that confers an observable phenotype, such as antibiotic resistance) is used to demonstrate transfection. Any cell can be transfected, even an egg cell, resulting in a transgenic organism. Figure 13.5 Transfection in Eukaryotic Cells 13.2 What Is the Structure of DNA? The structure of DNA was determined using many lines of evidence. One crucial piece came from X-ray diffraction. A purified substance can be made to form crystals. When X-rays are passed through it, position of atoms is inferred from the pattern of diffraction. Figure 13.6 X-Ray Crystallography Helped Reveal the Structure of DNA 13.2 What Is the Structure of DNA? Rosalind Franklin prepared crystallographs from DNA samples. Her images suggested a doublestranded helix with 10 nucleotides in each full turn. The diameter of 2 nm suggested that the sugar-phosphate backbone of each strand must be on the outside. 13.2 What Is the Structure of DNA? Chemical composition: Biochemists knew that DNA is a polymer of nucleotides. Each nucleotide consists of deoxyribose, a phosphate group, and a nitrogen-containing base. 13.2 What Is the Structure of DNA? The four different nucleotides differed only in the bases: • Purines: adenine (A), guanine (G) • Pyrimidines: cytosine (C), thymine (T) 13.2 What Is the Structure of DNA? Erwin Chargaff noticed that in all DNA, the amount of purines = the amount of pyrimidines. Chargaff’s rule 13.2 What Is the Structure of DNA? Francis Crick and James Watson used model building, plus the physical and chemical evidence to solve the structure of DNA. They published their results in 1953. Figure 13.7 DNA Is a Double Helix (Part 1) 13.2 What Is the Structure of DNA? The X-ray diffraction data indicated that the bases are on the inside and the sugar-phosphate groups on the outside of each strand, and that the chains run in opposite directions—antiparallel. 13.2 What Is the Structure of DNA? Antiparallel chains: 13.2 What Is the Structure of DNA? To satisfy Chargaff’s rule, the model paired a purine on one strand with a pyrimidine on the opposite strand, resulting in uniform width. Figure 13.7 DNA Is a Double Helix (Part 2) 13.2 What Is the Structure of DNA? Four key features of DNA structure: • It is a double-stranded helix • It is right-handed • It is antiparallel • The outer edges of the bases are exposed in major and minor grooves 13.2 What Is the Structure of DNA? The two chains are held together by: 1. Hydrogen bonding between bases – complementary base pairing: One purine (A or G) with one pyrimidine (T or C) In-Text Art, Ch. 13, p. 266 (1) 13.2 What Is the Structure of DNA? 2. Van der Waals forces between adjacent bases on the same strand. When the base rings come near one another, they tend to stack like poker chips. 13.2 What Is the Structure of DNA? Antiparallel strands: direction of strand is determined by the sugar–phosphate bonds. 13.2 What Is the Structure of DNA? Phosphate groups connect to the 3′ C of one sugar, and the 5′ C of the next sugar. Results in one chain with a free 5′ phosphate group—the 5′ end; The other chain has is a free 3′ hydroxyl group—the 3′ end. Figure 4.5 DNA Replication and Transcription 13.2 What Is the Structure of DNA? The backbones of the two DNA strands are closer together on one side of the double helix (forming the minor groove) than on the other (forming the major groove). There are four possible configurations of the base pairs in the grooves. Figure 13.8 Base pairs in DNA Can Interact with Other Molecules 13.2 What Is the Structure of DNA? The outer edges of the base pairs are exposed and accessible for additional hydrogen bonding. The surfaces of the A-T and C-G base pairs are chemically distinct. Binding of proteins to specific base pair sequences is the key to protein-DNA interactions, which are necessary for the replication and expression of DNA. 13.2 What Is the Structure of DNA? The double-helix structure is essential to DNA function: • Stores genetic information: with millions of nucleotides, the base sequences store a huge amount of information • Susceptible to mutations: alterations in base sequences 13.2 What Is the Structure of DNA? • Precisely replicated in cell division by complementary base pairing • Genetic information is expressed as the phenotype—nucleotide sequence determines sequence of amino acids in proteins 13.3 How Is DNA Replicated? The mechanism of DNA replication was confirmed by replicating DNA in a test tube. Ingredients needed: • Deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP (dNTPs, the monomers of DNA) 13.3 How Is DNA Replicated? • DNA molecules to serve as templates for the sequence of nucleotides • DNA polymerase enzyme • Salts and a pH buffer These experiments confirmed that DNA contains the information needed for its own replication. 13.3 How Is DNA Replicated? Three possible replication patterns: • Semiconservative: Each parent strand is a template; new molecules have one old and one new strand • Conservative: Original molecule serves as a template only • Dispersive: Fragments of DNA are templates, old and new parts are assembled into new molecules Figure 13.9 Three Models for DNA Replication 13.3 How Is DNA Replicated? Meselson and Stahl showed that semiconservative replication was the correct model: E. coli cultures were grown with 15N (a heavy, stable isotope that makes DNA more dense), then transferred to a medium with 14N. DNA densities could only be explained by the semiconservative model. Figure 13.10 The Meselson–Stahl Experiment (Part 1) Figure 13.10 The Meselson–Stahl Experiment (Part 2) Working with Data 13.1: The Meselson–Stahl Experiment In the Meselson–Stahl experiment, DNA with 14N was separated from DNA with 15N using an ultracentrifuge to create a density gradient of cesium chloride. Working with Data 13.1, Figure A Working with Data 13.1: The Meselson–Stahl Experiment DNA bands from successive generations of E. coli after centrifugation. Plots show quantitative analysis of the bands, where height indicates amount of DNA. Working with Data 13.1, Figure B Working with Data 13.1: The Meselson–Stahl Experiment Question 1: Use the heights of the peaks to estimate the percent of total DNA that was heavy, intermediate, and light at each generational stage. Create a table summarizing these calculations and discuss whether they support the authors’ conclusions. Working with Data 13.1: The Meselson–Stahl Experiment Question 2: What would the data look like if the bacteria had been allowed to divide for three more generations? Working with Data 13.1: The Meselson–Stahl Experiment Question 3: If Meselson and Stahl had done their experiment starting with light DNA and then added 15N for succeeding generations, what would the bands look like? Draw them alongside the actual data, above. Working with Data 13.1: The Meselson–Stahl Experiment Question 4: What would the data look like if conservative replication were the correct model? What would the data look like if dispersive replication were correct? Draw these alongside the actual data above. 13.3 How Is DNA Replicated? Two steps in DNA replication: • Double helix is unwound, making two template strands • New nucleotides form complementary base pairs with template DNA and are linked by phosphodiester bonds 13.3 How Is DNA Replicated? Nucleotides are added to the new strand at the 3′ end. Formation of the phosphodiester linkage is a condensation reaction. Bonds linking the phosphate groups of the triphosphate nucleosides are broken, releasing energy that drives the reaction. Figure 13.11 Each New DNA Strand Grows from Its 5 End to Its 3 End (Part 1) Figure 13.11 Each New DNA Strand Grows from Its 5 End to Its 3 End (Part 2) 13.3 How Is DNA Replicated? DNA replication starts when a large protein complex (pre-replication complex) binds to a region called origin of replication (ori). In E. coli, DNA is unwound and replication proceeds in both directions, forming two replication forks. 13.3 How Is DNA Replicated? Eukaryote chromosomes are much longer, and have multiple origins of replication. Otherwise, it would take weeks to replicate chromosomes, which have up to a billion base pairs. Figure 13.12 The Origin of DNA Replication 13.3 How Is DNA Replicated? DNA polymerase requires a primer, a short starter strand—usually RNA. The primer is complementary to the DNA template and is synthesized by an enzyme called a primase. DNA polymerase then adds nucleotides to the 3′ end until that section is complete, and the primer is degraded. Figure 13.13 DNA Forms with a Primer 13.3 How Is DNA Replicated? DNA polymerases are larger than their substrates, the dNTPs, and template DNA. The enzyme is shaped like an open right hand—the “palm” brings the active site and the substrates into contact. The “fingers” recognize the nucleotide bases. Figure 13.14 DNA Polymerase Binds to the Template Strand 13.3 How Is DNA Replicated? Other proteins have roles in replication: • DNA helicase uses energy from ATP hydrolysis to unwind the DNA • Single-strand binding proteins keep the strands from getting back together Figure 13.15 Many Proteins Collaborate in the Replication Complex 13.3 How Is DNA Replicated? At the replication fork DNA opens up like a zipper in one direction. The leading strand grows at its 3′ end as the fork opens. In the lagging strand the exposed 3′ end gets farther from the fork, and an unreplicated gap forms. Figure 13.16 The Two New Strands Form in Different Ways 13.3 How Is DNA Replicated? Synthesis of the lagging strand occurs in small, discontinuous stretches called Okazaki fragments. Each fragment requires its own primer, synthesized by the primase. DNA polymerase III adds nucleotides to the 3′ end, until reaching the primer of the previous fragment. 13.3 How Is DNA Replicated? DNA polymerase I then replaces the primer with DNA. The final phosphodiester linkage between fragments is catalyzed by DNA ligase. Figure 13.17 The Lagging Strand Story (Part 1) Figure 13.17 The Lagging Strand Story (Part 2) 13.3 How Is DNA Replicated? The enzymes of replication work very fast and accurately. In E. coli, new DNA is made at a rate in excess of 1,000 base pairs per second, with errors in fewer than one base in a million. 13.3 How Is DNA Replicated? DNA polymerases work fast because: • They are processive: they catalyze many linkages each time they bind to DNA, rather than just one. • The polymerase-DNA complex is stabilized by a sliding DNA clamp, a protein that keeps the enzyme and DNA in close contact. Figure 13.18 A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization 13.3 How Is DNA Replicated? In eukaryotes, the replication complex remains stationary while the DNA moves. It goes into the complex as one doublestranded molecule, and emerges as two double-stranded molecules. 13.3 How Is DNA Replicated? Eukaryote chromosomes have repetitive sequences at the ends called telomeres. In humans the sequence is TTAGGG, repeated about 2,500 times. It prevents the DNA repair system from seeing the chromosome end as a break. 13.3 How Is DNA Replicated? On lagging strands, when the terminal Okazaki primer is removed, no DNA can be synthesized to replace it (no 3′ end). The short piece of DNA is removed, and the chromosome becomes shorter with each replication. After many divisions, genes may be lost and the cell dies. Figure 13.19 Telomeres and Telomerase 13.3 How Is DNA Replicated? Continuously dividing cells, such as bone marrow stem cells, have telomerase, which catalyzes addition of lost telomeres. Telomerase is expressed in most cancer cells, and is important in their ability to keep dividing. It is a target for anti-cancer drugs. 13.4 How Are Errors in DNA Repaired? DNA polymerases initially make many mistakes, and DNA can be damaged by chemicals, UV radiation, and other threats. Cells have three repair mechanisms: • Proofreading • Mismatch repair • Excision repair 13.4 How Are Errors in DNA Repaired? DNA proofreading: As DNA polymerase adds a nucleotide to a growing strand, it can recognize mismatched pairs. If bases are paired incorrectly, the nucleotide is removed. Figure 13.20 DNA Repair Mechanisms (A) 13.4 How Are Errors in DNA Repaired? Mismatch repair: The newly replicated DNA is scanned for mistakes by other proteins, and mismatches can be corrected. If mismatch repair fails, the DNA is altered. Figure 13.20 DNA Repair Mechanisms (B) 13.4 How Are Errors in DNA Repaired? Excision repair: Enzymes constantly scan DNA for damaged bases—they are excised and DNA polymerase I adds the correct ones. Lack of excision repair mechanisms can lead to skin cancers. Figure 13.20 DNA Repair Mechanisms (C) 13.5 How Does the Polymerase Chain Reaction Amplify DNA? Principles of DNA replication were used to develop the polymerase chain reaction (PCR) technique. An automated process makes multiple copies of short DNA sequences for genetic manipulation and research (DNA amplification). 13.5 How Does the Polymerase Chain Reaction Amplify DNA? A PCR mixture contains: • A sample of double-stranded DNA (the template) • Two artificially synthesized primers • The four dNTPs • DNA polymerase that can tolerate high temperatures • Salts and buffer to maintain neutral pH 13.5 How Does the Polymerase Chain Reaction Amplify DNA? PCR amplification is a cyclical process: • DNA strands are separated (denatured) by heating • Reaction is cooled to allow primers to bind (anneal) to template strands • Reaction is warmed to temperature for DNA polymerase to catalyze new strands • The sequence is repeated many times Figure 13.21 The Polymerase Chain Reaction 13.5 How Does the Polymerase Chain Reaction Amplify DNA? Base sequences at the 3ʹ ends of the DNA strands must be known, so that primers can be made. The specificity of the primers is a key to the power of PCR. 13.5 How Does the Polymerase Chain Reaction Amplify DNA? An initial problem with PCR: the temperature needed to denature the DNA destroyed most DNA polymerases. A DNA polymerase that does not denature at high temperatures (90°C) was taken from a hot springs bacterium, Thermus aquaticus. 13.5 How Does the Polymerase Chain Reaction Amplify DNA? PCR has had a huge impact on genetic research. Applications range from identification of individual persons, to detection of diseases. 13 Answer to Opening Question Cisplatin has a platinum atom bonded to two chlorines and two amino groups. The chlorines can be displaced easily by nitrogens from guanine bases to form strong covalent bonds. This results in cross-linking the DNA strands, so that replication can’t occur. The cross-linking can’t be repaired by the cell’s normal repair mechanisms. Figure 13.22 Cisplatin: A Small but Lethal Molecule