The Molecular Basis of Inheritance (Ch. 13)

Transcription

1 The Molecular Basis of Inheritance (Ch. 13)

2 Many people contributed to our understanding of DNA T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Erwin Chargaff (1947) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958)

3 T.H. Morgan working with Drosophila associated phenotype with specific chromosome white-eyed male had specific X chromosome

4 Morgan s conclusions genes are on chromosomes but is it the protein or the DNA of the chromosomes that are the genes? initially proteins were thought to be genetic material Why? What s so impressive about proteins?!

5 Frederick Griffith Streptococcus pneumonia bacteria 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 Transforming Principle

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7 1944 Avery, McCarty & MacLeod Purified DNA & proteins from Streptococcus pneumonia bacteria injected protein into bacteria no effect injected DNA into bacteria transformed harmless bacteria into virulent bacteria mice die What s the conclusion?

8 Conclusion first experimental evidence that DNA was the genetic material Oswald Avery Maclyn McCarty Colin MacLeod

9 Hershey & Chase classic blender experiment worked with bacteriophage viruses that infect bacteria grew phages in 2 media, radioactively labeled with either 35S in their proteins 32P in their DNA infected bacteria phages Why use Sulfur vs. Phosphorus?

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11 Radioactive phage & bacteria in blender 35 S phage radioactive proteins stayed in supernatant therefore viral protein did NOT enter bacteria 32 P phage radioactive DNA stayed in pellet therefore viral DNA did enter bacteria Confirmed DNA is transforming factor Taaa-Daaa!

12 Martha Chase Alfred Hershey

13 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% Rules A = T C = G That s interesting! What do you notice?

14 Watson & Crick developed double helix model of DNA other leading scientists working on question: Rosalind Franklin Maurice Wilkins Linus Pauling Franklin Wilkins Pauling

15 Watson and Crick 1953 article in Nature Watson Crick

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17 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

18 Alternative models become experimental predictions Can you design a nifty experiment to verify? conservative semiconservative dispersive P 1 2

19 Meselson & Stahl label parent nucleotides in DNA strands with heavy nitrogen = 15 N label new nucleotides with lighter isotope = 14 N Franklin Stahl Matthew Meselson

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21 March to understanding that DNA is the genetic material T.H. Morgan (1908): genes are on chromosomes Frederick Griffith (1928): a transforming factor can change phenotype Avery, McCarty & MacLeod (1944): transforming factor is DNA Erwin Chargaff (1947): Chargaff rules: A = T, C = G Hershey & Chase (1952): confirmation that DNA is genetic material Watson & Crick (1953): determined double helix structure of DNA Meselson & Stahl (1958): semi-conservative replication

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23 Flow of genetic information in a cell transcription translation DNA RNA protein replication

24 You need to number the carbons! it matters! PO 4 nucleotide N base This will be IMPORTANT!! 5 4 CH 2 O sugar 1 3 OH 2

25 5 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!! 5 PO 4 CH 2 4 O 3 5 C O O P O O CH 2 O 4 2 base 1 base 1 3 OH 3 2

26 Nucleotides in DNA backbone are bonded from phosphate to sugar between 3 & 5 carbons DNA molecule has direction complementary strand runs in opposite direction

27 5 hydrogen bonds 3 covalent phosphodiester bonds 3 5.strong or weak bonds? How do the bonds fit the mechanism for copying DNA?

28 Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T 2 bonds C : G 3 bonds

29 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

30 Let s meet the team Large team of enzymes coordinates replication

31 Unwind DNA helicase enzyme unwinds part of DNA helix I d love to be helicase & unzip your genes stabilized by single-stranded binding proteins helicase single-stranded binding proteins replication fork

32 Build daughter DNA strand add new complementary bases DNA polymerase III DNA Polymerase III But Where s the We re missing ENERGY something! for the bonding! What?

33 Where does energy for bonding usually come from? You remember ATP! Are there other energy ways nucleotides? to get energy You out of bet! it? We come with our own energy! energy energy GTP TTP CTP ATP modified nucleotide And we leave behind a nucleotide! ADP AMP GMP TMP CMP

34 The nucleotides arrive as nucleosides DNA bases with P P P P-P-P = energy for bonding DNA bases arrive with their own energy source for bonding bonded by enzyme: DNA polymerase III ATP GTP TTP CTP

35 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 B.Y.O. ENERGY! The energy rules the process 5 energy DNA Polymerase III energy DNA Polymerase III energy DNA Polymerase III DNA Polymerase III energy 3 3 5

36 Okazaki Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand ligase Lagging strand 5 growing replication fork Lagging strand Okazaki fragments joined by ligase 3 spot welder enzyme DNA polymerase III Leading strand Leading strand continuous synthesis

37 DNA polymerase III leading strand lagging strand growing replication fork lagging strand leading strand 5 leading strand lagging strand growing replication fork 5 3

38 Limits of DNA polymerase III can only build onto 3 end of an existing DNA strand growing replication fork 3 DNA polymerase III primase RNA 5 RNA primer built by primase serves as starter sequence for DNA polymerase III 3

39 DNA polymerase I removes sections of RNA primer and replaces with DNA nucleotides DNA polymerase I ligase 5 growing replication fork 3 RNA 5 But DNA polymerase I still can only build onto 3 end of an existing DNA strand 3

40 Houston, we have a problem! All DNA polymerases can only add to 3 end of an existing DNA strand DNA polymerase I growing replication fork 3 DNA polymerase III RNA 5 Loss of bases at 5 ends in every replication chromosomes get shorter with each replication limit to number of cell divisions? 3

41 Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions growing replication fork 3 telomerase 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 5 3

42 DNA polymerase I DNA polymerase III Okazaki fragments lagging strand 5 ligase 3 5 primase 3 SSB 3 helicase leading strand direction of replication DNA polymerase III SSB = single-stranded binding proteins

43 DNA polymerase III 1000 bases/second! main DNA builder DNA polymerase I 20 bases/second editing, repair & primer removal Thomas Kornberg?? DNA polymerase III enzyme Arthur Kornberg 1959

44 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

45 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 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

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