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Chapter 13

Chapter 13. The Molecular Basis of Inheritance Central Dogma Song Sing along Hip Hip Hooray for DNA Blame it on the DNA . DNA, the substance of inheritance is the most celebrated molecule of our time Hereditary information

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Chapter 13

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  1. Chapter 13 The Molecular Basis of Inheritance Central Dogma Song Sing along Hip Hip Hooray for DNA Blame it on the DNA

  2. DNA, the substance of inheritance • is the most celebrated molecule of our time • Hereditary information • is encoded in the chemical language of DNA and reproduced in all the cells of your body • It is the DNA program • that directs the development of many different types of traits

  3. Early in the 20th century • the identification of the molecules of inheritance loomed as a major challenge to biologists • The role of DNA in heredity • was first worked out by studying bacteria and the viruses that infect them

  4. Frederick Griffith was studying Streptococcus pneumoniae • A bacterium that causes pneumonia in mammals • He worked with two strains of the bacterium • A pathogenic strain (capsulated- smooth) and a nonpathogenic strain (uncapsulated - rough) Evidence That DNA Can Transform Bacteria

  5. Griffith found that when he mixed heat-killed remains of the pathogenic strain (smooth) with living cells of the nonpathogenic strain (rough), some of these living cells became pathogenic

  6. EXPERIMENT RESULTS CONCLUSION Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below: Heat-killed (control) S cells Living R (control) cells Mixture of heat-killed S cells and living R cells Living S (control) cells Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample. Figure 16.2 Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells.

  7. Griffith called the phenomenon transformation • Now defined as a change in genotype and phenotype due to the assimilation of external DNA by a cell

  8. Phage head Tail Tail fiber DNA 100 nm Bacterial cell Figure 16.3 • Additional evidence for DNA as the genetic material • Came from studies of a virus that infects bacteria • Viruses that infect bacteria,bacteriophages • Are widely used as tools by researchers in molecular genetics Evidence That Viral DNA Can Program Cells

  9. Alfred Hershey and Martha Chase • Performed experiments showing that DNA is the genetic material of a phage known as T2

  10. In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells. EXPERIMENT CONCLUSION Agitated in a blender to separate phages outside the bacteria from the bacterial cells. Centrifuged the mixture so that bacteria formed a pellet at the bottom of the test tube. Mixed radioactively labeled phages with bacteria. The phages infected the bacterial cells. 3 2 4 1 Measured the radioactivity in the pellet and the liquid Radioactivity (phage protein) in liquid Radioactive protein Empty protein shell Phage Bacterial cell RESULTS Batch 1: Phages were grown with radioactive sulfur (35S), which was incorporated into phage protein (pink). DNA Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Batch 2: Phages were grown with radioactive phosphorus (32P), which was incorporated into phage DNA (blue). Centrifuge Radioactivity (phage DNA) in pellet Pellet Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells. When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus. Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material. Figure 16.4 The Hershey and Chase experiment

  11. P 32 S 35 P 32 ends up in DNA of new virons

  12. Sugar-phosphate backbone Nitrogenous bases 5 end CH3 O– 5 O H CH2 O P O O 1 4 N O– N H H H H H O 2 3 H Thymine (T) O H H CH2 O O P N O N H O– H N H H H N N H H Adenine (A) H H O H N CH2 O O P H O O– N H N H H H O H Cytosine (C) O 5 H CH2 O P N O O O 1 4 O– H N H Phosphate H H N 2 H 3 DNA nucleotide N H OH N H Sugar (deoxyribose) 3 end H Figure 16.5 Guanine (G) • Prior to the 1950s, it was already known that DNA • Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a Pentose sugar, and a phosphate group Additional Evidence That DNA Is the Genetic Materia

  13. Erwin Chargaff analyzed the base composition of DNA • from a number of different organisms • In 1947, Chargaff reported • that DNA composition varies from one species to the next • The % of Cytosine = the % of Guanine & the % of Thymine = the % of Adenine – Chargaff’s rule • This evidence of molecular diversity among species • made DNA a more credible candidate for the genetic material

  14. Once most biologists were convinced that DNA was the genetic material • The challenge was to determine how the structure of DNA could account for its role in inheritance Building a Structural Model of DNA: Scientific Inquiry

  15. Franklin’s X-ray diffraction Photograph of DNA (b) (a) Rosalind Franklin Figure 16.6 a, b • Maurice Wilkins and Rosalind Franklin • were using a technique called X-ray crystallography to study molecular structure • Rosalind Franklin produced a picture (Photo 51) of the DNA molecule using this technique

  16. Franklin's Photo 51 shows the mysterious "X" shape that inspired Watson and Crick to visualize the double helix structure of DNA. She was able to get this remarkable image—the clearest image of DNA ever created up until that time—with her advanced techniques of X-ray diffraction. Using Franklin's image as physical evidence, Watson and Crick then went on to publish their Nobel Prize-winning theoretical structure of DNA in Nature in 1953 Secret of Photo 51

  17. Figure 16.1 • In 1953, James Watson and Francis Crick shook the world (thanks to Franklin’s photo) • with sheet metal & clamps double-helical model for the structure of deoxyribonucleic acid, or DNA

  18. G C A T T A 1 nm C G 3.4 nm C G A T G C T A T A A T T A G C 0.34 nm A T Figure 16.7a, c (a) Key features of DNA structure (c) Space-filling model • Watson and Crick deduced that DNA was a double helix • through observations of Franklin’s X-ray crystallographic images of DNA

  19. Franklin had concluded that DNA • was composed of two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior • The nitrogenous bases • are paired in specific combinations: adenine with thymine, and cytosine with guanine

  20. 5 end 3 end O OH Hydrogen bond P –O OH O O A T O CH2 O O P O –O O– O P O H2C O O G C O O CH2 O P O O O –O O– O– O– O P P P O O O H2C O O O O C G O O CH2 O P –O O H2C A T O O CH2 OH 3 end (b) Partial chemical structure 5 end Figure 16.7b Structure of DNA

  21. Watson and Crick reasoned that there must be additional specificity of pairing dictated by the structure of the bases • Each base pair forms a different number of hydrogen bonds • Adenine and thymine form two bonds, cytosine and guanine form three bonds • BioRap

  22. H N O H CH3 N N N N H Sugar N N O Sugar Adenine (A) Thymine (T) H O N H N N N H N Sugar N N N O H Sugar H Figure 16.8 Cytosine (C) Guanine (G) PurinesvsPyrimidines

  23. The relationship between structure and function • is manifest in thedouble helix • Since the two strands of DNA are complementary • each strand acts as a template for building a new strand in replication The Basic Principle: Base Pairing to a Template Strand

  24. DNA replication is semiconservative • Each of the two new daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand

  25. EXPERIMENT Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with only 14N, the lighter, more common isotope of nitrogen.Any new DNA that the bacteria synthesized would be lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different densities by centrifuging DNA extracted from the bacteria. 3 2 4 1 Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N RESULTS Less dense DNA sample centrifuged after 40 min (after second replication) DNA sample centrifuged after 20 min (after first replication) More dense The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask in step 2, one sample taken after 20 minutes and one after 40 minutes. Figure 16.11 • Experiments performed by Meselson & Stahl • Supported the semiconservative model of DNA replication

  26. CONCLUSION Meselson and Stahl concluded that DNA replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure 16.10. The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated the dispersive model and supported the semiconservative model. First replication Second replication Conservative model Semiconservative model Dispersive model

  27. T A A A A T A T T T T A G C G C C C C G G G G C A A T T T T A A A A T T T A A A A T A T T T T A C G G G G C G C C C C G (a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. (c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. (d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. (b) The first step in replication is separation of the two DNA strands. Figure 16.9 a–d • In DNA replication • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

  28. The copying of DNA is remarkable in its speed and accuracy • More than a dozen enzymes and other proteins participate in DNA replication • Topoisomerase - Relieves strain ahead of the replication fork due to untwisting of the double helix. Flattens out helix • Helicase - Untwists the double helix at replication forks to make two parental strands available as template strands. Unzips helix • Single stranded binding protein – stabilizes the open strands following helicase- keeps replication fork open • DNA Polymerase I or III - Elongates a new DNA strand at a replication fork. Adds nucleotides one by one to the new and growing DNA strand. Binds nucleotides to exposed bases • DNA ligase - Joins sugar-phosphates of Okazaki fragments. Okazaki fragments are found on the lagging strand. Molecular glue • Primase - Starts an RNA chain from scratch that will eventually be replaced by DNA nucleotides (remember nucleotides come from DNA polymerase). Produces temporary RNA fragments DNA Replication: A Closer Look

  29. The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated by DNA helicase Getting Started: Origins of Replication

  30. Origin of replication Parental (template) strand 0.25 µm Daughter (new) strand Replication begins at specific sites where the two parental strands separate and form replication bubbles. 1 Bubble Replication fork 2 The bubbles expand laterally, as DNA replication proceeds in both directions. Eventually, the replication bubbles fuse, and synthesis of the daughter strands is complete. 3 Two daughter DNA molecules In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome. (a) (b) Figure 16.12 a, b • A eukaryotic chromosome • may have hundreds or even thousands of replication origins

  31. New strand Template strand 3 end 5 end 3 end 5 end Sugar A T A T Base Phosphate C G C G G G C C A T A T OH P P P P 3 end P Pyrophosphate C C OH 2 P 5 end 5 end Figure 16.13 • Elongation of new DNA at a replication fork • Is catalyzed by enzymes called DNA polymerases, which add nucleotides to the 3 end of a growing strand or leading strand DNA Polymerase Elongating a New DNA Strand HHMI animation Nucleoside triphosphate

  32. DNA polymerases add nucleotides • only to the free 3end (sugar first then phosphate group) of a growing strand • Along one template strand of DNA, the leading strand • DNA polymerase III can synthesize a complementary strand continuously, moving toward the replication fork • DNA strands are replicated from the 3’ end to the 5’ end only so the new strands formed goes in the 5’ to 3’ direction How does the antiparallel structure of the double helix affect replication?

  33. To elongate the other new strand of DNA, the lagging strand of 5` side, • DNA polymerase III must work in the direction away from the replication fork (5’ to 3’) • The lagging strand • is synthesized as a series of segments called Okazaki fragments, which are then joined together by DNA ligase

  34. 4 2 3 1 DNA pol Ill elongates DNA strands only in the 5 3 direction. 3 One new strand, the leading strand, can elongate continuously 5 3 (DNA)as the replication fork progresses. 5 Parental DNA 5 3 Okazaki fragments The other new strand, the lagging strand must grow in an overall 3 5 direction by addition of short segments, Okazaki fragments, that grow 5 3 (numbered here in the order they were made). 2 3 1 5 DNA pol III Template strand DNA ligase joins Okazaki fragments by forming a bond between their free ends. This results in a continuous strand. Leading strand Lagging strand 3 1 2 Template strand DNA ligase Figure 16.14 Overall direction of replication Synthesis of leading & lagging strands during DNA replication

  35. DNA polymerases cannot initiate the synthesis of a polynucleotide • They can only add nucleotides to the 3 end • The initial nucleotide strand • Is an RNA or DNA primer to set down a starting point for the DNA fragment to be added on in the 3’ to 5’ direction Priming DNA Synthesis

  36. Only one primer is needed for synthesis of the leading strand • But for synthesis of the lagging strand, each Okazaki fragment must be primed separately

  37. 7 2 3 6 5 1 4 3 5 3 5 Templatestrand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. Primasejoins RNA nucleotides into aprimer. RNA primer 3 5 3 1 5 After reaching the next RNA primer (not shown), DNA pol III falls off. Okazakifragment 3 3 5 1 5 After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off. 5 3 3 2 5 1 DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2. 5 3 3 5 2 1 DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1. The lagging strand in this region is nowcomplete. 5 3 3 2 5 1 Figure 16.15 Overall direction of replication

  38. Table 16.1 • Helicase, topoisomerase, single-strand binding protein • Are all proteins that assist DNA replication Other Proteins That Assist DNA Replication

  39. Overall direction of replication Lagging strand Leading strand Helicase unwinds the parental double helix. Origin of replication 1 Molecules of single- strand binding protein stabilize the unwound template strands. 2 The leading strand is synthesized continuously in the 5 3 direction by DNA pol III. 3 Leading strand Lagging strand OVERVIEW DNA pol III Leading strand 5 Replication fork DNA ligase DNA pol I 3 Primase 2 Parental DNA Lagging strand DNA pol III 1 Primer 3 Primase begins synthesis of RNA primer for fifth Okazaki fragment. 4 3 5 4 DNA pol III is completing synthesis of the fourth fragment, when it reaches the RNA primer on the third fragment, it will dissociate, move to the replication fork, and add DNA nucleotides to the 3 endof the fifth fragment primer. DNA pol I removes the primer from the 5 end of the second fragment, replacing it with DNA nucleotides that it adds one by one to the 3 end of the third fragment. The replacement of the last RNA nucleotide with DNA leaves the sugar- phosphate backbone with a free 3 end. DNA ligase bonds the 3 end of the second fragment to the 5 end of the first fragment. 5 6 7 Figure 16.16 • A summary of DNA replication DNA Replication Song and a good animation

  40. The various proteins that participate in DNA replication • Form a single large complex, a DNA replication “machine” • The DNA replication machine • Is probably stationary during the replication process The DNA Replication Machine as a Stationary Complex

  41. DNA replication inserts an incorrect base on average only once in every 104 to 106bases. • DNA polymerases proofread newly made DNA replacing any incorrect nucleotides • In mismatch repair of DNA, • repair enzymes correct errors in base pairing to 1/100,000,000 bases (our entire genome is ~ 3.2 billion base pairs) Proofreading and Repairing DNA

  42. 1 2 4 A thymine dimer distorts the DNA molecule. • In nucleotide excision repair • Enzymes cut out and replace damaged stretches of DNA • Animation A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease DNA polymerase Repair synthesis by a DNA polymerase fills in the missing nucleotides. 3 DNA ligase DNA ligase seals the Free end of the new DNA To the old DNA, making the strand complete. Figure 16.17

  43. 5 End of parental DNA strands Leading strand Lagging strand 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 New leading strand 3 New lagging strand 5 3 Further rounds of replication Shorter and shorter daughter molecules Figure 16.18 • The ends of eukaryotic chromosomal DNA • get shorter with each round of replication • Explains the process of aging Replicating the Ends of DNA Molecules

  44. 1 µm Figure 16.19 • Eukaryotic chromosomal DNA molecules • have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules just like the tips on your shoelaces

  45. If the chromosomes of germ cells became shorter in every cell cycle • essential genes would eventually be missing from the gametes they produce • An enzyme called telomerase • catalyzes the lengthening of telomeres in germ cells • Animation

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