DNA Structure and Replication Ch. 14

1. DNA Structure and ReplicationCh. 14

2. DNA and Heredity • Mendel set the stage for inheritance patterns, but it was not yet known that this is through DNA • Proteins were considered the better source of variation, why? • More possible variation; 20 AA vs. 4 Base pairs • A series of researches over decades showed it really DNA: • Griffith • Avery • Hershey and Chase

3. Frederick Griffith Kills Mice • Worked with Streptococcus pneumoniae • S-Type (smooth) virulent; deadly • R-Type (rough) non-virulent; pretty okay • Ran for tests on mice: • S-type injected into mice • Mice die • R-type injected into mice • Mice live • Heat-killed S-type injected into mice • Mice live • Heat-killed S-type and R-type injected into mice • Mice die Conclusion: • Some material from dead S-type changed R-type into S-type (transformed)

4. Avery Doesn’t Kill Mice • Works with Streptococcus pneumoniaetoo, but only in test tubes • Used enzymes to destroy either the proteins, DNA, or RNA of the cells and then tried to transform them • R-type + S-type with destroyed proteins transformation • R-type + S-type with destroyed RNA transformation • R-type + S-type with destroyed DNA no transformation Conclusion: • DNA must be needed to transform bacteria cells, so it must be the key to heredity

5. Hershey and Chase End the Debate • Worked with E. coliand a bacteriophage called T2 • Virus that infects only bacteria; made of just DNA and a protein coat • Labeled DNA and protein coat of virus with radioactive P and S isotopes and traced them through the virus life cycle • E.coli + Virus with labeled protein coat no radioactivity in offspring • E.coli + Virus with labeled DNA radioactivity in offspring Conclusion: • DNA, not proteins, are passed on to offspring

6. Structure of DNA • Discovered by Watson and Crick with help from Franklin and Wilkins • X-Ray diffraction image of DNA • X-rays shot through crystal containing molecule • Photograph film catches areas exposed when x-rays deflect • What is DNA’s shape? • Double helix • What is it made of? • Nucleotides : Adenine (A), Guanine (G), Thymine (T), and Cytosine (C) • How are the nucleotides connected? • Phosphodiester bonds in a sugar-phosphate backbone; creates 5’ end and a 3’ end • Strands held together by H-bonds

7. Structure of DNA • How do nucleotides match up? • Purines (two rings) with Pyrimidines (one ring) • A-T; G-C • What is the vocab word for this type of pairing? • Complementary base pairing • In order for pairing to happen, the two strands in DNA must run opposite directions. What is this called? • Antiparallel • Other dementions: • DNA is 2n wide (only Purine-Pyrimidine combination makes this length) • Full twist is 3.4 nm long • Distance between each nucleotide is 0.34nm • SO…there are 10 bases/turn

8. Semiconservative Replication? • In replication, one strand is used as a template (guide) to build a new strand • Unzipping DNA allows both strands to be copied at the same time, thus producing copies that each have one full strand from the starting DNA • Other options existed… • Conservative model DNA is template but original DNA reforms and new DNA has no original strands • Dispersive Replication model old and new DNA strands mix as they form

9. Meselson and Stahl • Worked with DNA made of “heavy” 15N isotope • Mixed “heavy” DNA with 14N, did one replication, and then separated DNA types by centrifuge (heavy ones sink more) • Allowed DNA to replicate again and centrifuged again • Semiconservative 1st; lighter DNA (half 15N and half 14N), 2nd; lightest appears (all 14N) • Conservative 1st; heavy DNA (all 15N) and lightest (all 14N), 2nd; same • Dispersive all DNA gets lighter as more 14N is used

10. Vocabulary Explosion! • Deoxyribosenucleoside triphosphates building blocks of DNA (dATP, dGTP, dCTP, dTTP) • DNA Helicase breaks H-bonds and unwinds DNA • Topoisomerse untwists downstream DNA; DNA twists as it is unzipped • SSBs (Single-stranded binding proteins) hold unzipped DNA strands so they don’t adhere • DNA polymerase III main enzyme used to copy DNA • Sliding DNA Clamp helps DNA polymerase stay attached to DNA

11. Vocabulary Explosion 2! The Sequel • DNA polymerase I removes RNA primers at 5’ end • Primase makes primers; RNA nucleotides that mark the start of replication • DNA ligase fixes breaks in sugar-phosphate backbone • Leading strand DNA strand continuously replicated • Lagging strand DNA strand replicated in fragments (Okazaki fragments)

12. DNA Replication: Getting Started • DNA strands have a 5’ and 3’ end, but nucleotides can only be added to the 3’ (free –OH ready for dehydration reaction) • New DNA is built 5’3’, so the template is “read” 3’5’ • Replication starts at ori region of the DNA (origin of replication) • Eukaryotic DNA is too long to replicate from end to end • Hundreds ori sights exist and replication goes in both directions (replication bubbles) • DNA helicase unwinds the strands creating a replication fork (Y-structure) • SSBs hold stands apart

13. DNA Replication: Primers • Pulling apart strands causes the DNA to twist and bundle up • Topoisomerase cuts DNA ahead of the replication fork, untwists it, and rebinds it • DNA Poly III needs a 3’ end to start replication • Primers (RNA) are base paired at ori and provide a 3’ for Poly III • Primers are built by Primase • Primers are removed by DNA Polymerase I (exonuclease) and replaced with DNA

14. DNA Replication: Two Types of Synthesis • DNA is antiparallel, so one strand is read 3’5’ (leading strand) while the other runs 5’3’ (lagging strand) • DNA cannot be added in the 5’3’ direction • Leading stand as continuous replication • Lagging strand is replicated in sections (Okazaki Fragments) • Leaves gaps which are filled in by DNA Poly I and Ligase

15. DNA Replication: Finishing Up • When bubbles meet, the enzymes detach and DNA re-adheres • If DNA is liner, what happens to the starting stands on either end? • They are not be replicated; short section is lost after each replication DNA gets shorts with time • Telomere noncoding area at the end of DNA (5’-TTAGGG-3’) that protect against this • Shortening is believed to be the main cause of aging and death • Telomerase enzyme that adds more telomeres; only active as an embryo • What type of cell is telomerase also active in? • Cancer; If we can turn these off cancer will divide itself to death

16. DNA Replication: Opps…Mistake… • Polymerase is not perfect; makes a base-pair mismatch 1: 1,000 nucleotides • Proofreading mechanism Poly III can backup and use exonuclease to replace mistakes • Lowers mutation rates to 1:1 million nucleotides • What if Poly III miss the mistake? • DNA repair mechanisms run along the DNA double checking it • Any area wider or narrower than 2nm must have a mistake and replaces the nucleotide

17. DNA Compaction • DNA is around 2 meter long and must fit in a 10mm nucleus • Most is compacted and only opened for making proteins • Chromatin DNA and Chromosomal proteins • Histone small, positively charged proteins; bind negative backbone of DNA • Histones join together and wrap DNA around them to make nucleosomes • Strings of nucleosomes connected by linkers string of beads • Decreases DNA size by a factor of 7! • Nonhistone proteins effect histone binding so regions of DNA become accessible

18. DNA Compaction • Nucleosome strings (10-nm chromatin fibers) can wrap around a H1 histone to make 30-nm chromatin fiber • Solenoid model helix of nucleosomes • This level of condensing protects against damage • Euchromatin loosely packed regions (light color band on chromosome); often expressed • Heterchromatin densely packed regions (dark color band on chromosome); often deactivated genes

19. Bacterial DNA • Prokaryotes have no need for histones • DNA is one circular ring (bacterial chromosome) that is short enough • Kept compacted in a mass called the nucleoid • Addition al DNA can be absorbed by bacteria • Plasmids short DNA rings • Can be copied and exchanged with other bacteria of the same species or genus • Can help form drug resistant bacteria

20. Homework • Suggested Homework: • Test Your Knowledge Ch. 14 • Actual Homework: • Interpret the Data Ch. 14 • Discuss the Concepts #1 and #4 • Lab Reports due 12/11 • Papers due. 12/13