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Topic 1: DNA Structure & Replication (Chp 7) Image:http://thewildcarrot.ca/putting-on-my-summer-genes-do-you-have-seasonal-dna/  1.1 DNA is the Hereditary Molecule of Life 5 essential characteristics of hereditary mat...

Topic 1: DNA Structure & Replication (Chp 7) Image:http://thewildcarrot.ca/putting-on-my-summer-genes-do-you-have-seasonal-dna/  1.1 DNA is the Hereditary Molecule of Life 5 essential characteristics of hereditary material: 1. Found in the nucleus 2. Stable 3. Sufficiently complex 4. Can accurately replicate itself 5. Mutable  DNA as Hereditary Material 3 foundational experiments: 1. Griffith’s experiment: identified the ‘transformation factor’ 2. Avery’s experiment: identified DNA as the ‘transformation factor’ 3. Hersey & Chase experiment: identified DNA as the hereditary material  1. Griffith’s Experiment Frederick Griffith identified two strains of Pneumococcus: S strain R strain Each strain has four antigenic types (I, II, III, and IV) Figure 7.1.  The transformation factor Figure 7.2 Conclusion? Does Griffith’s experiment tell us anything about the nature of the ‘transformation factor’?  2. Avery’s Experiment - Same S and R strains as Griffith - Systematically destroy either lipids, proteins, RNA or DNA - Does transformation still occur? Figure 7.3 Application Transformation is frequently used to alter the genetics of bacterial cells in molecular research. 1. Do a 'Google search' to find what methods researchers use to transform bacterial cells. 2. Find or brainstorm a practical application of bacterial transformation.  3. Hershey and Chase experiment Bacteriophage (phages) are viruses that infect bacteria Used T2 phage  Hershey and Chase Experiment Proteins: contain sulfur, but little phosphorus DNA: contains phosphorus but no sulfur Hershey and Chase separately labeled either: phage proteins (with 35S) or phage DNA (with 32P) Then, traced each radioactive label in the course of infection Figure 7.4  Topic 1.1 review questions What results from Griffith’s experiment provide the strongest evidence that a transformation factor is responsible for heredity? If Hershey and Chase used a phage that contained both DNA and RNA, would they have been able to draw the same conclusion? Why/why not?  1.2 The DNA Double Helix: 2 Complementary, Antiparallel Strands Fairly simple in structure Very complex informational molecule Figs 1.3 & 1.4 Fig 1.6  BIOL 1217 Review Questions 1. What 3 chemical groups make up a nucleotide? 2. How many different nitrogenous bases are there? 3. What enzyme adds nucleotides to a growing polynucleotide chain?  DNA nucleotides Fig 7.5  Covalent bonds Fig 7.6  DNA double helix structure 1. Complementary base-pairs between 2 DNA strands (A pairs with T and G pairs with C) 2. The two strands are antiparallel with respect to their 5' and 3' ends The DNA Double Helix - Axis of helical symmetry - Conserved diameter - Conserved spacing between base pairs - Base stacking - Major & minor groove Figure 7.7b Topics 1.1 & 1.2 Review questions (Chp 7): Topic Practice questions 2nd Ed Practice questions 3rd Ed 1.1 1, 2, 3, 4 1, 2, 3, 4 1.2 5, 7, 8, 9, 11, 13, 17 5, 7, 8, 9, 13, 17, 35  Topic 1.2 review questions Consider the sequence: 3’ ACGCTACGTC 5’ a) What is the double-stranded DNA sequence? b) What is the number of phosphodiester bonds joining the nucleotides in each strand? c) What is the total number of hydrogen bonds joining the nucleotides of complementary strands?  1.3 DNA Replication is Semiconservative and Bidirectional The general mechanism of DNA replication is the same in all organisms.  3 attributes of DNA replication shared by all organisms: 1. Parental DNA strands remains intact 2. Each parental strand is a template for synthesis of an antiparallel, complementary daughter strand 3. Two identical daughter molecules composed of one parental and one daughter strand produced  Three proposed mechanisms of DNA replication Figure 7.8  Meselson-Stahl Experiment Cesium chloride (CsCl) gradient and ultracentrifugation technique Can separate molecules with only slightly different molecular weights Used ‘heavy’ nitrogen (15N) and ‘light’ nitrogen (14N) Figure 7.9  Three proposed mechanisms of DNA replication Figure 7.8  Origin and Directionality of Replication in Bacterial DNA DNA replication is usually bidirectional in bacteria: Proceeds in 2 directions from a single origin of replication Figure 7.10  Visual Evidence for Bidirectional Replication Figure 7.11 Experimental Evidence for Bidirectional Replication Pulse-chase labeling provided the first evidence of bidirectional replication Cells were exposed to high levels (pulse) and then low levels (chase) of a radioactive tracer Figure 7.12  Biochemical Evidence of Bidirectional Replication in Bacteria Speed of replication E. coli DNA polymerase - can incorporate about 1000 nucleotides per second Entire genome can be replicated in ~33 min (about the generation time of E. coli)  Multiple Replication Origins in Eukaryotes Large eukaryotic genomes can contain thousands of origins of replication Human genome? 10,000 origins of replication. DNA replication rate can vary between different types of cells. Figure 7.13 a Figure 7.13 b  Topic 1.3 Review questions: Topic Practice questions 2nd Practice questions 3rd Ed Ed 1.3 20, 21, 22, 23, 32, 35 20, 21, 22, 23, 32, 36 Describe 1 of the 3 pieces of evidence that support bi- directional DNA replication If DNA replication was conservative, what results would have been observed in the Meselson & Stahl experiment ?  1.4 Mechanism of DNA Replication Focus on the bacterial system Many similarities between all 3 domains of life General mechanism is the same Different proteins & enzymes have evolved; the replication process is not identical  Replication initiation Replication origins have sequences that attract replication enzymes Consensus sequence: nucleotides found most often at each position of DNA in the conserved region. Figure 7.15 How to make a consensus sequence 5’ 3’ E. coli TTATCCACA B. subtilis TTATCCACT H. pylori TCATTCACA M. tuberculosis TTGTCCACA  Replication initiation enzymes Replication-initiating enzymes locate and bind to oriC consensus sequences: DnaA – binds consensus sequences, bends the DNA and breaks the H- bonds DnaB - a helicase that unwinds the DNA strands by breaking H-bonds DnaC – carries DnaB to the DNA helix Figure 7.17  Summary of DNA Replication The “replisome” Campbell Text: Figure 16.17  RNA Primers Are Needed for DNA Replication DNA polymerase: adds nucleotides to the 3' end of a pre- existing strand Cannot initiate DNA strand synthesis on its own Requires an available 3’ OH to add to Primase: RNA polymerase that synthesizes RNA primers Can initiate RNA synthesis on its own  Leading and Lagging Strand Synthesis Leading strand: One copy of pol III continuously synthesizes one daughter strand in the same direction as fork progression Lagging strand: the other copy of pol III discontinuously synthesizes the daughter strand, in the opposite direction as fork progression. Uses short DNA segments (Okazaki fragments)  © 2015 Pearson Education, Inc. Figure 7.18  RNA Primer Removal and Okazaki Fragment Ligation DNA polymerase I (pol I) uses two activities to complete replication: 1. 5'- 3' exonuclease activity: removes the RNA primers 2. 5'- 3' polymerase activity: adds DNA nucleotides to the gaps Figure 7.19  DNA ligase seals the gap between the resulting DNA segments  Topoisomerase enzyme – catalyze controlled cleavage and rejoining of DNA to prevent supercoiling. Figure 7.22  Processivity of DNA polymerase Sliding clamp: a protein that increases the processivity of DNA pol III enzyme Figure 7.20  DNA Proofreading DNA replication is very accurate DNA polymerases undertake DNA proofreading Errors in replication occur once about every billion nucleotides in E. coli Proofreading ability of DNA polymerase enzymes is due to 3'-to-5' exonuclease activity  Wrong nucleotide added during replication = mismatch base pair 3'-OH of strand moves into exonuclease “site” of the enzyme Several nucleotides (including the incorrect one) are removed and new nucleotides incorporated Figure 7.21 Finishing replication of linear chromosomes Linear ends of chromosomes can’t be replicated Telomeres: repetitive sequences at the ends of chromosomes Elizabeth Blackburn, Carol Greider, & Jack Szostak awarded Nobel Prize in Physiology or Medicine in 2009 for work on telomeres and telomerase Campbell Text Fig Your text: Figure 7.23  Telomeres are synthesized by a Protein-RNA complex called telomerase T loop: ‘knotted’ DNA fold Figure 7.24  Telomerase Activity Telomerase is active in germ-line cells and some stem cells in eukaryotes Differentiated somatic cells have virtually no telomerase activity; such cells have limited life spans Hayflick limit: limit to the length of a cell’s lifespan 50-70 cycles before dying Application: Telomeres, aging and cancer Premature aging condition – dyskeratosis congenita Scientists found the gene responsible for normal telomerase function is defective Abnormal reactivation of telomerase? If telomerase is active when it shouldn’t be, cells will continue to proliferate and escape apoptosis Many cancer cells reactivate expression of telomerase → results in cells that continue to divide. Most frequent genetic mutation in all cancer types Telomerase activity upregulated in 80-90% of all cancers! Image: https://news.berkeley.edu/2018/04/25/long-sought-structure-of-telomerase-paves-way-for-drugs-for-aging-cancer/  Topic 1.4 review questions: Topic Practice questions 2nd Ed Practice questions 3rd Ed 1.4 10, 12, 14, 15, 18, 26 10, 11, 14, 15, 18, 26, 37