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Chapter 16~ The Molecular Basis of Inheritance Scientific History • The march to understanding that DNA is the genetic material – T.H. Morgan (1908) – Frederick Griffith (1928) – Avery, McCarty & MacLeod (1944) – Erwin Chargaff (1947) – Hershey & Chase (1952) – Watson & Crick (1953) – Meselson & Stahl (1958) The “Transforming 1928 Principle” • Frederick Griffith – Streptococcus pneumonia bacteria • was working to find cure for pneumonia – harmless live bacteria (“rough”) mixed with heat-killed pathogenic bacteria (“smooth”) causes fatal disease in mice – a substance passed from dead bacteria to live bacteria to change their phenotype • “Transforming Principle” The “Transforming Principle” mix heat-killed live pathogenic strain of bacteria A. mice die live non-pathogenic heat-killed strain of bacteria pathogenic bacteria B. C. mice live mice live pathogenic & non-pathogenic bacteria D. mice die Transformation = change in phenotype something in heat-killed bacteria could still transmit disease-causing properties DNA is the “Transforming 1944 Principle” • Avery, McCarty & MacLeod – purified both DNA & proteins separately from Streptococcus pneumonia bacteria • which will transform non-pathogenic bacteria? – injected protein into bacteria • no effect – injected DNA into bacteria • transformed harmless bacteria into virulent bacteria mice die What’s the conclusion? 1944 | ??!! Avery, McCarty & MacLeod • Conclusion – first experimental evidence that DNA was the genetic material Oswald Avery Maclyn McCarty Colin MacLeod 1952 | 1969 Confirmation of DNA • Hershey & Chase – classic “blender” experiment – worked with bacteriophage • viruses that infect bacteria – grew phage viruses in 2 media, radioactively labeled with either Why use Sulfur vs. Phosphorus? • • 35S in their proteins 32P in their DNA – infected bacteria with labeled phages Hershey Protein coat labeled with 35S Hershey & Chase T2 bacteriophages are labeled with radioactive isotopes S vs. P bacteriophages infect bacterial cells bacterial cells are agitated to remove viral protein coats Which radioactive marker is found inside the cell? Which molecule carries viral genetic info? DNA labeled with 32P 35S radioactivity found in the medium 32P radioactivity found in the bacterial cells Blender experiment • Radioactive phage & bacteria in blender – 35S phage • radioactive proteins stayed in supernatant • therefore viral protein did NOT enter bacteria – 32P phage • radioactive DNA stayed in pellet • therefore viral DNA did enter bacteria – Confirmed DNA is “transforming factor” Taaa-Daaa! Hershey & Chase Martha Chase 1952 | 1969 Alfred Hershey Hershey Chargaff • DNA composition: “Chargaff’s rules” – varies from species to species – all 4 bases not in equal quantity – bases present in characteristic ratio • humans: A = 30.9% T = 29.4% G = 19.9% C = 19.8% That’s interesting! What do you notice? Rules A = T C = G 1947 Structure of 1953 | 1962 DNA • Watson & Crick – developed double helix model of DNA • other leading scientists working on question: – Rosalind Franklin – Maurice Wilkins – Linus Pauling Franklin Wilkins Pauling 1953 article in Nature Watson and Crick Watson Crick Rosalind Franklin (1920-1958) Double helix structure of DNA “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick Directionality of DNA • You need to number the carbons! nucleotide PO4 N base – it matters! 5 CH2 This will be IMPORTANT!! O 4 1 ribose 3 OH 2 5 The DNA backbone • Putting the DNA backbone together – refer to the 3 and 5 ends of the DNA • the last trailing carbon Sounds trivial, but… this will be IMPORTANT!! PO4 base 5 CH2 O 4 1 C 3 O –O P O O 5 CH2 2 base O 4 1 2 3 OH 3 Anti-parallel strands • Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons 5 3 3 5 – DNA molecule has “direction” – complementary strand runs in opposite direction Bonding in DNA hydrogen 5 bonds 3 covalent phosphodiester bonds 3 ….strong or weak bonds? How do the bonds fit the mechanism for copying DNA? 5 Base pairing in DNA • Purines – adenine (A) – guanine (G) • Pyrimidines – thymine (T) – cytosine (C) • Pairing –A:T • 2 bonds –C:G • 3 bonds But how is DNA copied? • Replication of DNA – base pairing suggests that it will allow each side to serve as a template for a new strand “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” — Watson & Crick Copying DNA • Replication of DNA – base pairing allows each strand to serve as a template for a new strand – new strand is 1/2 parent template & 1/2 new DNA • semi-conservative copy process Semiconservative replication, • when a double helix replicates each of the daughter molecules will have one old strand and one newly made strand. • Experiments in the late 1950s by Matthew Meselson and Franklin Stahl supported the semiconservative model, proposed by Watson and Crick, over the other two models. (Conservative & dispersive) Let’s meet the team… DNA Replication • Large team of enzymes coordinates replication Replication: 1st step • Unwind DNA – helicase enzyme • unwinds part of DNA helix • stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork Replication: 2nd step Build daughter DNA strand add new complementary bases DNA polymerase III DNA Polymerase III 5 Replication energy • Adding bases – can only add nucleotides to 3 end of a growing DNA strand • need a “starter” nucleotide to bond to – strand only grows 53 3 DNA Polymerase III energy DNA Polymerase III energy DNA Polymerase III energy DNA Polymerase III 3 5 Okazaki Leading & Lagging strands Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand 5 3 5 3 5 3 5 5 5 Lagging strand ligase growing 3 replication fork Leading strand 3 Lagging strand 3 Okazaki fragments joined by ligase “spot welder” enzyme 5 3 DNA polymerase III Leading strand continuous synthesis 3 Replication fork / Replication bubble 5 3 5 DNA polymerase III leading strand 5 3 3 5 3 5 5 5 3 lagging strand 3 5 3 5 lagging strand 5 5 leading strand growing replication fork 5 3 growing replication fork leading strand 3 lagging strand 5 5 5 5 3 Starting DNA synthesis: RNA primers Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand 5 3 3 5 5 3 5 3 5 growing 3 replication fork DNA polymerase III primase RNA 5 RNA primer built by primase serves as starter sequence for DNA polymerase III 3 Replacing RNA primers with DNA DNA polymerase I removes sections of RNA primer and DNA polymerase I replaces with DNA nucleotides 5 3 3 5 5 ligase growing 3 replication fork RNA 5 3 But DNA polymerase I still can only build onto 3 end of an existing DNA strand Houston, we have a problem! Chromosome erosion All DNA polymerases can only add to 3 end of an existing DNA strand DNA polymerase I 5 3 3 5 5 growing 3 replication fork DNA polymerase III RNA Loss of bases at 5 ends in every replication chromosomes get shorter with each replication limit to number of cell divisions? 5 3 Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions 5 3 3 5 5 growing 3 replication fork telomerase 5 Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells high in stem cells & cancers -- Why? TTAAGGG TTAAGGG 3 Replication fork DNA polymerase III lagging strand DNA polymerase I 5’ 3’ ligase primase Okazaki fragments 5’ 3’ 5’ SSB 3’ helicase DNA polymerase III 5’ 3’ leading strand direction of replication SSB = single-stranded binding proteins DNA polymerases • DNA polymerase III – 1000 bases/second! – main DNA builder Roger Kornberg 2006 • DNA polymerase I – 20 bases/second – editing, repair & primer removal DNA polymerase III enzyme Arthur Kornberg 1959 Editing & proofreading DNA • 1000 bases/second = lots of typos! • DNA polymerase I – proofreads & corrects typos – repairs mismatched bases – removes abnormal bases • repairs damage throughout life – reduces error rate from 1 in 10,000 to 1 in 100 million bases Fast & accurate! • It takes E. coli <1 hour to copy 5 million base pairs in its single chromosome – divide to form 2 identical daughter cells • Human cell copies its 6 billion bases & divide into daughter cells in only few hours – remarkably accurate – only ~1 error per 100 million bases – ~30 errors per cell cycle What does it really look like? 1 2 3 4 Any Questions?? 2007-2008