CHAPTER 11 LECTURE SLIDES Prepared by Brenda Leady University of Toledo To run the animations you must be in Slideshow View. Use the buttons on the animation to play, pause, and turn audio/text on or off. Please note: once you have used any of the animation functions (such as Play or Pause), you must first click in the white background before you advance the next slide. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Genetic material must meet criteria: 1. Information 2. Replication 3. Transmission 4. Variation 2
Late 1800s scientists postulated a biochemical basis Researchers became convinced chromosomes carry genetic information 1920s to 1940s expected the protein portion of chromosomes to be the genetic material 3
Griffith s bacterial transformation Late 1920s Frederick Griffith was working with Streptococcus pneumoniae S. pneumoniae Strains that secrete capsules look smooth and can cause fatal infections in mice Strains that do not secrete capsules look rough and infections are not fatal in mice 4
Rough strains (R) without capsule are not fatal No living bacteria found in blood Smooth strains (S) with capsule are fatal Capsule prevents immune system from killing bacteria Living bacteria found in blood If mice are injected with heat-killed type S, they survive Mixing live R with heatkilled S kills the mouse Blood contains living S bacteria Transformation 5
Genetic material from the heat-killed type S bacteria had been transferred to the living type R bacteria This trait gave them the capsule and was passed on to their offspring Griffith did not know the biochemical basis of his transforming principle 6
Avery, MacLeod, and McCarty used purification methods to reveal that DNA is the genetic material 1940s interested in bacterial transformation Only purified DNA from type S could transform type R Purified DNA might still contain traces of contamination that may be the transforming principle Added DNase, RNase and proteases RNase and protease had no effect With DNase no transformation DNA is the genetic material
Hershey and Chase 1952, studying T2 virus infecting Escherichia coli Bacteriophage or phage Phage coat made entirely of protein DNA found inside capsid 9
Shearing force from a blender will separate the phage coat from the bacteria 35 S will label proteins only 32 P will label DNA only Experiment to find what is injected into bacteria- DNA or protein? Results support DNA as the genetic material 10
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Levels of DNA structure 1. Nucleotides are the building blocks of DNA (and RNA). 2. A strand of DNA (or RNA) 3. Two strands form a double helix. 4. In living cells, DNA is associated with an array of different proteins to form chromosomes. 5. A genome is the complete complement of an organism s genetic material. 12
Nucleotides 3 components Phosphate group Pentose sugar Nitrogenous base 13
DNA 3 components Phosphate group Pentose sugar Deoxyribose Nitrogenous base Purines Adenine (A), guanine (G) Pyrimidines Cytosine (C), thymine (T) 14
RNA 3 components Phosphate group Pentose sugar Ribose Nitrogenous base Purines Adenine (A), guanine (G) Pyrimidines Cytosine (C), uracil (U) 15
Conventional numbering system Sugar carbons 1 to 5 Base attached to 1 Phosphate attached to 5 16
Strands Nucleotides covalently bonded Phosphodiester bond phosphate group links 2 sugars Phosphates and sugars from backbone Bases project from backbone Directionality- 5 to 3 5 TACG 3 17
Solving DNA structure 1953, James Watson and Francis Crick, with Maurice Wilkins, proposed the structure of the DNA double helix Watson and Crick used Linus Pauling s method of working out protein structures using simple ball-and-stick models Rosalind Franklin s X-ray diffraction results suggested a helical structure with uniform diameter 18
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Erwin Chargoff analyzed base composition of DNA of many different species Results consistently showed that amount of adenine similar to the amount of thymine and amount of cytosine similar to amount of guanine 20
Watson and Crick Put together these pieces of information Found ball-and-stick model consistent with data Watson and Crick awarded Nobel Prize in 1962 Rosalind Franklin had died and the Nobel is not awarded posthumously 21
DNA is Double stranded Helical Sugar-phosphate backbone Bases on the inside Stabilized by hydrogen bonding Base pairs with specific pairing 22
AT/GC or Chargoff s rule A pairs with T G pairs with C Keeps width consistent 10 base pairs per turn 2 DNA strands are complementary 5 GCGGATTT 3 3 CGCCTAAA 5 2 strands are antiparallel One strand 5 to 3 Other stand 3 to 5 23
Space-filling model shows grooves Major groove Where proteins bind and affect gene expression Minor groove 24
Replication 3 different models for DNA replication proposed in late 1950s Semiconservative Conservative Dispersive Newly made strands are daughter strands Original strands are parental strands 25
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In 1958, Matthew Meselson and Franklin Stahl devised experiment to differentiate among 3 proposed mechanisms Nitrogen comes in a common light form ( 14 N) and a rare heavy form ( 15 N) Grew E.coli in medium with only 15 N Then switched to medium with only 14 N Collected sample after each generation Original parental strands would be 15 N while newly made strands would be 14 N Results consistent with semiconservative mechanism 27
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Semiconservative replication During replication 2 parental strands separate and serve as template strands New nucleotides must obey the AT/GC rule End result 2 new double helices with same base sequence as original 29
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Origin of replication Site of start point for replication Bidirectional replication Replication proceeds outward in opposite directions Bacteria have a single origin Eukaryotes require multiple origins 31
DNA replication Origin of replication provides an opening called a replication bubble that forms two replication forks DNA replication proceeds outward from forks Bacteria have single origin of replication Eukaryotes have multiple origins of replication 32
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DNA helicase Binds to DNA and travels 5 to 3 using ATP to separate strand and move fork forward DNA topoisomerase Relives additional coiling ahead of replication fork Single-strand binding proteins Keep parental strands open to act as templates 34
DNA polymerase Covalently links nucleotides Deoxynuceloside triphosphates 35
Deoxynuceloside triphosphates Free nucleotides with 3 phosphate groups Breaking covalent bond to release pyrophosphate (2 phosphate groups) provides energy to connect adjacent nucleotides 36
DNA polymerase 2 other enzymatic features 1. DNA polymerase unable to begin DNA synthesis on a bare template strand DNA primase must make a short RNA primer RNA primer will be removed and replaced with DNA later 2. DNA polymerase can only work 5 to 3 37
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Leading and lagging strands In the leading strand DNA synthesized in as one long continuous molecule DNA primase makes one RNA primer DNA polymerase attaches nucleotides in a 5 to 3 direction as it slides forward In the lagging strand DNA synthesized 5 to 3 but as Okazaki fragments 39
In the lagging strand Okazaki fragments a short RNA primer made by DNA primase at the 5 end and then DNA laid down by DNA polymerase In both strands RNA primers will be removed by DNA polymerase and filled in with DNA DNA ligase will join adjacent DNA fragments 40
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DNA replication is very accurate 3 reasons 1. Hydrogen bonding between A and T or G and C more stable than mismatches 2. Active site of DNA polymerase unlikely to form bonds if pairs mismatched 3. DNA polymerase removes mismatched pairs Proofreading results in DNA polymerase backing up and digesting linkages Other DNA repair enzymes 42
DNA polymerases are a family of enzymes with specialized functions 3 important issues for DNA polymerase are speed, fidelity, and completeness Nearly all living species have more than 1 type of DNA polymerase Genomes of most species have several DNA polymerase genes due to gene duplication Independent genetic changes produce enzymes with specialized functions
E. coli has 5 DNA polymerases DNA polymerase III with multiple subunits responsible for majority of replication DNA polymerase I has a single subunit whose job is to rapidly remove RNA primers and fill in DNA DNA polymerases II, IV and V are involved in DNA repair and replicating damaged DNA DNA polymerases I and III stall at DNA damage DNA polymerases II, IV and V don t stall but go slower and make sure replication is complete
Humans have 12 or more DNA polymerases Designated with Greek letters DNA polymerase α has its own built in primase subunit DNA polymerase δ and ε extend DNA at a faster rate DNA polymerase γ replicates mitochondrial DNA When DNA polymerases α, δ or ε encounter abnormalities they may be unable to replicate Lesion-replicating polymerases may be able to synthesize complementary strands to the damaged area
Telomeres Series of short nucleotide sequences repeated at the ends of eukaryotic chromosomes Specialized form of DNA replication only in eukaryotes in the telomeres Telomere at 3 does not have a complementary strand and is called a 3 overhang 47
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DNA polymerase cannot copy the tip of the DNA strand with a 3 end No place for upstream primer to be made If this replication problem were not solved, linear chromosomes would become progressively shorter Telomerase enzyme attaches many copies of DNA repeat sequence to the ends of chromosomes 49
Progressive shortening of telomeres correlated with cellular senescence Telomerase function reduced as organism ages 99% of all types of human cancers have high levels of telomerase 50
Eukaryotic chromosome structure Typical eukaryotic chromosome may be hundreds of millions of base pairs long Length would be 1 meter Must fit in cell 10-100µm Chromosome Discrete unit of genetic material Chromosomes composed of chromatin DNA-protein complex 51
3 levels of DNA compaction 1. DNA wrapping DNA wrapped around histones to form nucleosome Shortens length of DNA molecule 7-fold 2. 30-nm fiber Current model suggests asymmetric, 3D zigzag of nucleosomes Shortens length another 7-fold 52
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3. Radial loop domains Interaction between 30-nm fibers and nuclear matrix Each chromosome located in discrete territory Level of compaction of chromosomes not uniform Heterochromatin Euchromatin 54
Cell division When cells prepare to divide, chromosomes become even more compacted Euchromatin not as compact Hetrochromatin much more compact Metaphase chromosomes highly compacted 55
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