Genetics chapter 7 dna structure and replication


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Genetics chapter 7 dna structure and replication

  1. 1. DNA: The Chemical Nature of the Gene
  2. 2. The remarkable stability of DNA makes the extraction and analysis of DNA from ancient remains possible, including Neanderthal bones that are more than 30,000 years old! More on this later… More on this later…
  3. 3. The Genetic Material MUST.. • Store information • Information can vary but be stable • Replicate • DNA replication • Encode the phenotype • Govern expression: gene function
  5. 5. 5 Features of Hereditary Material 1. Localized to the nucleus, component of chromosomes 2. Present in stable form in cells 3. Sufficiently complex to contain information needed for structure, function, development, and reproduction of an organism
  6. 6. 5 Features of Hereditary Material, continued 4. Able to accurately replicate itself so that daughter cells contain the same information as parent cells 5. Mutable, undergoing a low rate of mutations that introduces genetic variation and serves as a foundation for evolutionary change
  7. 7. DNA: The Early Age • Friedrich Meischer (1869): extracted substance from white blood cells • Slightly acidic and phosphorus rich • DNA and protein Friedrich Meischer • Kossel (1901): DNA contains A, G, C, and T • Phoebus Albert Levene (1910): Tetranucleotide Theory Ludwig Karl Martin Leonhard Albrecht Kossel (16 September 1853 – 5 July 1927) • DNA is made of nucleotides • Not variable • Erwin Chargaff (11 August 1905 – 20 June 2002) Phoebus Aaron Theodore Levene, M.D. (25 February 1869 – 6 September 1940)
  8. 8. Chargaff’s Rules In all organisms, the nucleotide bases are found in specific proportions. • Chargaff’s rules are based on two observations. – The amount of A, C, G, and T varies from species to species. – In each species, the amount of A is equal to T and the amount of G is equal to C.
  9. 9. Pneumococcus & Bacteriophage Experiment Series EVIDENCE THAT DNA IS THE MOLECULE RESPONSIBLE FOR CONVEYING HEREDITARY CHARACTERISTICS Is it DNA, RNA, lipids, carbohydrates, etc.?
  10. 10. The Transformation Factor • Frederick Griffith identified two strains of Pneumococcus: S, which caused fatal pneumonia in mice, and R, which did not • S = smooth = protective capsule • A single nucleotide change can convert the S (smooth) strain into R (rough) the strain • R has a mutant allele on the polysaccharide gene
  11. 11. Griffith’s Experimental Results • Mice infected with strain SIII developed pneumonia and died • Mice infected with strain RII or with heat-killed strain SIII survived • Mice infected with heatkilled strain SIII and live strain RII developed pneumonia and died – livetype SIII bacteria were recovered from the mice
  12. 12. Avery, MacLeod, and McCarty • What is the Hereditary Molecule? • Avery, MacLeod, and McCarty used heat-killed SIII bacteria and live RII bacteria and infected mice • The extract of heat-killed SIII bacteria was divided into aliquots and treated to destroy either DNA, RNA, proteins, or lipids and polysaccharides • All aliquots killed the mice except the one with the DNA destroyed
  13. 13. DNA Is the Hereditary Molecule! • Are we sure? Replication and verification is an important part of the scientific process…. • Hershey and Chase, in 1952, showed that DNA is responsible for bacteriophage infection of bacteria cells
  14. 14. Bacteriophages (phages) are viruses that infect bacteria Awesome Awesome Phage Lab Phage Lab here at here at VCU!  VCU! 
  15. 15. They do NOT have RNA!
  16. 16. Phage Infection of Bacteria • Phages must infect bacterial hosts to reproduce • Infection begins when the phage injects DNA into the bacterial cell and leaves its protein shell on the surface (ghost!) • The phage DNA replicates in the bacterium and produces proteins that are assembled into progeny phages – these are released by lysis of the host cell
  17. 17. Hershey and Chase Experiments • Distinguish DNA and protein by their sulfur and phosphorous content • Proteins contain large amounts of sulfur but almost no phosphorus • DNA contains large amounts of phosphorus but no sulfur • Hershey and Chase separately labeled either phage proteins (with 35S) or DNA (with 32P) and then traced each radioactive label in the course of infection
  18. 18. Conclusion • These results demonstrate that phage DNA, not phage protein, is transferred to host bacterial cells and directs synthesis of phage DNA and proteins, the assembly of progeny phage particles, and ultimately lysis of infected cells. • Furthermore, this experiment demonstrated that the transformation factor previously identified by Griffith was DNA • It also showed that Avery, MacLeod and McCarty were correct in concluding that DNA is the hereditary material.
  19. 19. DNA is the heritable material… BUT WHAT IS THE STRUCTURE OF DNA?
  20. 20. 1951 - Recognize Rosalind Franklin, Yo!
  21. 21. 7.2 The DNA Double Helix Consists of Two Complementary and Antiparallel Strands • Watson and Crick’s model of the secondary structure of DNA shows that it is fairly simple in structure • It is composed of four kinds of nucleotides, joined by covalent phosphodiester bonds with two polynucleotide chains that come together to form a double helix
  22. 22. Nucleotides have a polarized structure Phosphate end: 5’cabon (five prime end) OH in sugar : 3’carbon (three prime end)
  23. 23. Two Types of DNA Bases • Pyrimidines, thymine or cytosine, have a single ring, and purines, adenine or guanine, have a double ring • Deoxynucleotide monophosphates that are part of a polynucleotide chain have single phosphates and are called dNMPs, where N refers to any of the four bases • Deoxynucleotide triphosphates, dNTPs, are not part of a polynucleotide chain
  24. 24. dNTPs are dNTPs are recruited by DNA recruited by DNA polymerase polymerase Pyrophosphate Pyrophosphate group discarded group discarded
  25. 25. The diameter results from the fact that each complementary base pair (A and T or G and C) is 20Å wide
  26. 26. Base-pair stacking creates gaps between the sugar phosphate backbones that partially expose the nucleotides The major groove, approximately 12Å wide, alternates with the minor groove, approximately 6Å wide These grooves are regions where DNA binding proteins can make direct contact with nucleotides
  28. 28. Three Attributes of DNA Replication Shared by All Organisms 1. Each strand of the parental DNA molecule remains intact during replication 2. Each parental strand serves as a template for formation of an antiparallel, complementary daughter strand 3. Completion of replication results in the formation of two identical daughter duplexes composed of one parental and one daughter strand
  29. 29. 7.3 DNA Replication Is Semiconservative and Bidirectional • The general mechanism of DNA replication is the same in all organisms • As organisms diverged and became more complex, some differences did develop in the replication proteins and enzymes • These subtle differences are why antibiotics stop replication in bacteria, but not us!
  30. 30. Origin and direction of replication • Is origin single or multiple? • Does it start at random or specific location? • Is it unidirectional or bidirectional?
  31. 31. We can see DNA replicating! • John Cairns grew bacteria in a medium containing 3H-thymine • He extracted the bacterial chromosomes during replication and placed them on x-ray film (radioactive decay of 3H is slow, this took several months!) • The autoradiographs showed dark lines that revealed the pattern of replicating DNA molecules – these were called theta structures (θ structures)
  32. 32. Evidence of Bidirectional DNA Replication • In bidirectional DNA replication, new DNA is synthesized in both directions from the single origin, creating an expanding replication bubble • At each end of the replication bubble is a replication fork; replication is complete when the replication forks meet 41 Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.
  33. 33. The location of the origin of replication and replication terminus regions on opposite sides of the E. coli chromosome.
  34. 34. Additional Support for Bidirectional Replication • Biochemical studies of DNA polymerase of E. coli show that the polymerase can incorporate about 1000 nucleotides per second into the new DNA strand • At this rate of synthesis, the entire genome can be replicated in approximately 33 minutes • This corresponds well with the generation time of E. coli; unidirectional synthesis would require twice as much time!
  35. 35. Multiple Replication Origins in Eukaryotes • Autoradiograph analysis shows multiple origins of replication on eukaryotic chromosomes • Large eukaryotic genomes contain thousands of origins of replication separated by 40,000 to 50,000 base pairs • The human genome contains more than 10,000 origins! • DNA replication rate varies among different types of cells Multiple origins of replication on a single chromosome from Drosophila melanogaster
  36. 36. Bacterial Replication Origins • Replication origins have sequences that attract replication enzymes • The origin of replication sequence of E. coli is called oriC, and it contains about 245 bp of A-T rich DNA • The origin is divided into three 13-bp sequences followed by four 9-bp sequences
  37. 37. Replication Initiation in Bacteria • Replication in E. coli requires that replication-initiating enzymes locate and bind to oriC consensus sequences • Enzymes DnaA, DnaB, DnaC bind at oriC and initiate DNA replication • DnaA • Binds the 9-mer, bends the DNA, • breaks hydrogen bonds in the A-T rich 13mer sequences • DnaB is a helicase • uses ATP energy • separate the strands and unwind the helix • DnaB is carried to the DNA helix by DnaC • Single-stranded binding protein (SSBP) • Prevents reannealing of unwound strands
  38. 38. Topoisomerases Topoisomerases Unwinding of circular chromosomes will create torsional stress, potentially leading to supercoiled DNA Enzymes called topoisomerases catalyze controlled cleavage and rejoining of DNA that prevents over-winding
  39. 39. Eukaryote Replication Origins • Saccharomyces cerevisiae (yeast) has the most fully characterized originof-replication sequences • The multiple origins of replication are called autonomously replicating sequences (ARS) • These sequences help orient the replication machinery before replication begins!
  40. 40. RNA Primers Are Needed for DNA Replication • DNA polymerase elongates DNA strands by adding nucleotides to the 3′ end of a preexisting strand • They cannot initiate DNA strand synthesis on their own • RNA primers are needed; these are synthesized by a specialized RNA polymerase called primase
  41. 41. DNA Polymerase III • In E. coli, daughter DNA strands are synthesized by the DNA polymerase III (pol III) holoenzyme • Holoenzyme refers to a multiprotein complex in which a core enzyme is associated with the additional components needed for full function • The replisome is found at each replication fork and contains two copies of pol III
  42. 42. ANIMATION: DNA Replication-4 Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.
  43. 43. Leading and Lagging Strand Synthesis • Leading strand: pol III synthesizes one daughter strand continuously in the same direction as fork progression • Lagging strand: pol III elongates the daughter strand discontinuously, in the opposing direction to fork progression, via short segments (Okazaki fragments)
  44. 44. RNA Primer Removal and Okazaki Fragment Ligation
  45. 45. Simultaneous Synthesis of Leading and Lagging Strands • Each replisome complex carries out replication of the leading and lagging strand simultaneously • The DNA pol III holoenzyme contains 11 protein subunits, with the two pol III core polymerases each tethered to a different copy of the tau (τ) protein • The tau proteins are joined to a protein complex called the clamp loader; two additional proteins form the sliding clamp
  46. 46. The Sliding Clamp • The sliding clamp can close around the double-stranded DNA during replication • It has a “doughnut hole” of about 35Å, into which the DNA fits • The sliding clamp anchors the DNA pol III core enzyme to the template • It is required for the high level of pol III activity
  47. 47. ANIMATION: Molecular Model of DNA Replication Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.
  48. 48. FOUNDATION FOUNDATION FIGURE 7.24 FIGURE 7.24 The Trombone Model of DNA Replication http://www.yo atch? v=5VefaI0Lrg E&feature=rel ated
  49. 49. DNA Proofreading • DNA replication is very accurate, mainly because DNA polymerases undertake DNA proofreading, to correct occasional errors • Errors in replication occur about one every billion nucleotides in E. coli • Proofreading ability of DNA polymerase enzymes is due to a 3′ to 5′ exonuclease activity
  50. 50. You should know the You should know the functions of these functions of these molecules; spend less molecules; spend less time on naming time on naming details! details!
  51. 51. Telomeres • The leading strand of linear chromosomes can be replicated to the end • The lagging strand requirement for a primer means that lagging strands cannot be completely replicated • This problem is resolved by repetitive sequences at the ends of chromosomes, called telomeres • These repeats ensure that incomplete chromosome replication does not affect vital genes
  52. 52. Telomerase • Telomeres are synthesized by the ribonucleoprotein telomerase • Blackburn, Greider, and Szostak received the 2009 Nobel Prize for the discovery of telomeres and telomerase • The RNA in telomerase is complementary to the telomere repeat sequence and acts as a template for addition of DNA
  53. 53. Telomerase Function • The template RNA of telomerase allows new DNA replication, to lengthen the telomere sequences • Once telomeres are sufficiently elongated, the α polymerase synthesizes additional RNA primers • New DNA replication then fills out the chromosome ends • Telomere sequences in most organisms are quite similar
  54. 54. Importance of Telomerase Activity • Mice that are homozygous for loss-of-function mutations of the TERT (telomerase reverse transcriptase) gene give rise to developmental defects • The defects are first observed in the fourth and fifth generations, due to loss of telomere length with each generation • By the sixth generation, shortening of chromosomes is critical and apoptosis is induced Left, 48-week-old TERT-ER mouse with activated telomerase. Right, 35week-old TERT-ER mouse, not treated. (Dana-Farber Cancer Institute) Chromosome spreads showing telomere elongation after telomerase activation 68 (right). DNA in blue, telomeres in red. (Dana-Farber Cancer Institute)
  55. 55. Telomeres, Aging, and Cancer • Telomere length is important for chromosome stability, cell longevity, and reproductive success • Telomerase is active in germ-line cells and some stem cells in eukaryotes • Differentiated somatic cells and cells in culture have virtually no telomerase activity; such cells have limited life spans (30 to 50 cell divisions) • Hayflick Limit: limitation on the growth of most cells grown in culture
  56. 56. Werner Syndrome • Telomerase inactivity is associated with normal aging of cells • A condition known as Werner syndrome causes early onset of some features of aging • A mutation in RECQL2, a gene encoding a helicase required for telomerase activity, is the cause of Werner syndrome
  57. 57. Dyskeratosis Congenita • Dyskeratosis congenita is a disorder associated with a loss of function of a gene, DKC1, that encodes a protein needed for normal telomerase function • Patients with this disorder have skin and nail abnormalities, loss of vision and hearing, and abnormalities of blood cell formation
  58. 58. Abnormal Reactivation of Telomerase Activity • Telomerase is normally turned off in somatic cells • Reactivation of telomerase can lead to aging cells that continue to proliferate, a feature of many types of cancer • TERT reactivation is one of the most common mutations in cancers of all types
  59. 59. 7.5 Molecular Genetic Analytical Methods Make Use of DNA Replication Processes • Molecular biologists have used their understanding of DNA replication to develop new methods of molecular analysis • Two widely used methods include polymerase chain reaction (PCR) and dideoxynucleotide DNA sequencing
  60. 60. The Polymerase Chain Reaction • The polymerase chain reaction (PCR) is an automated version of DNA replication that produces millions of copies of a short target DNA segment • There are numerous applications of PCR • -Genetic testing, oncogenes, detection of disease (HIV, etc), forensics (genetic fingerprinting), research (sequencing, cloning, gene expressions, phylogenetic analysis, rapid DNA production) • PCR reactions are carried out in small volumes (less than 100 µ l)
  61. 61. The Process of PCR • PCR uses two DNA sequences called PCR primers that provide a starting point for Taq polymerase to add nucleotides • The PCR primers define the 5′ and 3′ boundaries of the replication products • PCR is composed of three steps that result in exponential amplification of large numbers of the target DNA
  62. 62. Components of PCR • PCR requires • A double-stranded DNA template containing the target sequence to be amplified • A supply of the four DNA nucleotides • A heat-stable DNA polymerase • Two different single-stranded DNA primers • A buffer solution • The most commonly used DNA polymerase, Taq, is isolated from Thermus aquaticus, which occurs naturally in hot springs*
  63. 63. Steps of PCR 1. Denaturation: the reaction is heated to ∼ 95⁰ C to denature the DNA into single strands 2. Primer annealing: the reaction temperature is reduced to ∼ 45-68⁰ C to allow primers to hybridize to their complementary sequences in the target DNA 3. Primer extension: the reactions temperature is raised to 72⁰ C to allow Taq polymerase to synthesize DNA
  64. 64. Limitations of PCR 1. Some knowledge of the target DNA sequences is required in order to determine primer sequences 2. Amplification products longer than 10 to 15 kb are difficult to produce • Despite the limitations, PCR is a practical way to obtain large quantities of DNA from a particular gene for molecular analysis
  65. 65. Separation of PCR Products • Amplified DNA fragments are separated from the rest of the reaction mixture by gel electrophoresis and visualized by ethidium bromide staining • PCR product sizes are measured in base pairs (bp) • Differences in the size of DNA amplified by a pair of primers are related to the amount of DNA between the primers
  66. 66. What do we do with PCR Products? • Genetic testing • Human consoling: amniocentesis • Disease detection: viruses • Tissue typing: organ transplantation • Cancer: oncogenes • Forensic applications r-testing.php • DNA Fingerprinting: VNTRs (next!) • RESEARCH! • Hybridization probes, DNA sequencing, clonining, phylogenetic analysis of ancient DNA (coming soon!) & MORE!!
  67. 67. ANIMATION: Polymerase Chain Reaction (PCR)-1 Genetics Analysis: An Integrated Approach Copyright © 2012 Pearson Education Inc.
  68. 68. Variable Number Tandem Repeats • A Variable Number Tandem Repeat (or VNTR) is a location in a genome where a short nucleotide sequence is organized as a tandem repeat. These can be found on many chromosomes, and often show variations in length between individuals. Each variant acts as an inherited allele, allowing them to be used for personal or parental identification. Their analysis is useful in genetics and biology research, forensics, and DNA fingerprinting. • Codominant fingerprint! Different father Adopted Within the VNTRs there are sites where an enzyme can cut the DNA, and the location of these sites also varies from person to person. Cutting with the enzyme will lead to DNA fragments of different lengths, which are called Restriction Fragment Length Polymorphisms (RFLPs).
  69. 69. Variable Number Tandem Repeats • VNTRs have variable numbers of repeats of DNA of up to 20 bp in length • VNTRs are inherited, as are other types of alleles, and can be detected through PCR
  70. 70. Hereditary transmission of VNTR alleles follows a codominant pattern
  71. 71. Dideoxynucleotide DNA Sequencing • The ultimate description of a DNA molecule is its precise sequence of bases • The first DNA-sequencing protocols were developed by Maxam and Gilbert, and another by Sanger in 1977 • The Sanger (dideoxynucleotide) Method was most amenable to automation and is the method of choice today Frederick Sanger Born 1918, won two Nobel Prizes
  72. 72. Sanger Sequencing • Dideoxynucleotide DNA sequencing (dideoxy sequencing) uses DNA polymerase to replicate new DNA from a single-stranded template • The four standard deoxynucleotide bases (dNTPs) are present in large amounts • Each reaction contains a small amount of one dideoxynucleotide (ddNTP), which lacks a 3′-OH group • Didexoy = TWO deoxygenated sites!
  73. 73. The Principle of Sanger Sequencing • When replication starts, most fragments will incorporate a dNTP and continue replication • BUT, whenever a ddNTP is incorporated into the product DNA molecule, replication ceases
  74. 74. Sanger Sequencing • A separate reaction is carried out for A, T, G, and C, using the corresponding small amount of ddNTP • Each reaction tube produces a series of partial DNA molecules, each of which ends with that nucleotide • All four reactions must be run side by side on a gel in order to determine the complete sequence
  75. 75. Visualization of DNA Sequence • After the reactions are complete, the reactions are run side by side on a gel • Autoradiograph bands are visualized by labeling the 5’ primers with radioactive isotopes or fluorescent tags (end-labeling) • The shortest bands are the DNA products closest to the primer and these travel fastest on the gel; the gel is read from the bottom up, all four lanes together
  76. 76. Automated DNA Sequencing • Automated DNA sequencers use a single reaction for each DNA sequence, in which all four ddNTPs are included • Each ddNTP is labeled with a unique fluorescent marker • The DNA is synthesized, and a mixture of fragments is produced and run on a DNA gel • The fluorescent label on each ddNTP has a different wavelength, and a laser light excites the fluorescent tag on each fragment as it passes • The fluorescence pattern produced shows the sequence of the DNA
  77. 77. Questions?