DNA The Genetic Material

Transcription

1 DNA The Genetic Material

2 Chromosomes related to phenotype T.H. Morgan working with Drosophila fruit flies associated phenotype with specific chromosome white-eyed male had specific X chromosome

3 Genes are on chromosomes 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?

4 The Transforming Principle 1928 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

5 The Transforming Principle mix heat-killed pathogenic & live pathogenic strain of bacteria live non-pathogenic strain of bacteria heat-killed pathogenic bacteria non-pathogenic bacteria A. B. C. D. mice die mice live mice live mice die Transformation = change in phenotype something in heat-killed bacteria could still transmit disease-causing properties

6 DNA is the Transforming 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 1944 mice die

7 Avery, McCarty & MacLeod Conclusion 1944??!! first experimental evidence that DNA was the genetic material Oswald Avery Maclyn McCarty Colin MacLeod

8 Confirmation of DNA Hershey Hershey & Chase classic blender experiment worked with bacteriophage viruses that infect bacteria grew phage viruses in 2 media, radioactively labeled with either 35 S in their proteins 32 P in their DNA infected bacteria with labeled phages

9 Hershey & Chase Protein coat labeled with 35 S T2 bacteriophages are labeled with radioactive isotopes S vs. P DNA labeled with 32 P bacteriophages infect bacterial cells Which radioactive marker is found inside the cell? bacterial cells are agitated to remove viral protein coats Which molecule carries viral genetic info? 35 S radioactivity found in the medium 32 P radioactivity found in the bacterial cells

10 Blender experiment 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

11 Hershey & Chase Hershey Martha Chase Alfred Hershey

12 Chargaff 1947 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

13 Rosalind Franklin ( )

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

15 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

16 Watson and Crick 1953 article in Nature Watson Crick

17 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

18 Models of DNA Replication Alternative models become experimental predictions conservative semiconservative dispersive P 1 2

19 Semiconservative replication Meselson & Stahl label parent nucleotides in DNA strands with heavy nitrogen = 15 N label new nucleotides with lighter isotope = 14 N 1958 The Most Beautiful Experiment in Biology parent replication 15 N/ 15 N 15 N parent strands

20 Predictions 1st round of replication 14 N/ 14 N 15 N/ 15 N 15 N/ 14 N 15 N/ 14 N P 1 2nd round of replication 15 N/ 15 N conservative 14 N/ 14 N 15 N/ 15 N semiconservative dispersive 14 N/ 14 N 15 N/ 14 N 15 N/ 14 N 2 15 N parent strands conservative semiconservative dispersive

21 Meselson & Stahl Matthew Meselson Franklin Stahl Franklin Stahl Matthew Meselson

22 Scientific History 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

23 DNA Replication

24 The DNA backbone Putting the DNA backbone together refer to the and ends of the DNA the last trailing carbon PO 4 CH 2 O base 4 O C O P O O CH 2 O base 1 OH 2

25 Anti-parallel strands Nucleotides in DNA backbone are bonded from phosphate to sugar between & carbons DNA molecule has direction complementary strand runs in opposite direction

26 Bonding in DNA hydrogen bonds covalent bonds.strong or weak bonds? How do the bonds fit the mechanism for copying DNA?

27 Base pairing in DNA Purines adenine (A) guanine (G) Pyrimidines thymine (T) cytosine (C) Pairing A : T 2 bonds C : G 3 bonds

28 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

29 DNA Replication Large team of enzymes coordinates replication

30 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

31 Replication: 2nd step 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?

32 Energy of Replication 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

33 Replication Adding bases can only add nucleotides to end of a growing DNA strand need a starter nucleotide to bond to strand only grows energy DNA Polymerase III energy DNA Polymerase III energy DNA Polymerase III DNA Polymerase III energy

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

35 Replication fork / Replication bubble DNA polymerase III leading strand lagging strand growing replication fork lagging strand leading strand leading strand lagging strand growing replication fork

36 Starting DNA synthesis: RNA primers Limits of DNA polymerase III can only build onto end of an existing DNA strand growing replication fork DNA polymerase III primase RNA RNA primer built by primase serves as starter sequence for DNA polymerase III

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

38 Replication fork 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

39 DNA polymerases DNA polymerase III 1000 bases/second! main DNA builder DNA polymerase I 20 bases/second editing, repair & primer removal DNA polymerase III enzyme Roger Kornberg 2006 Arthur Kornberg 1959

40 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

41 Chromosome erosion All DNA polymerases can only add to end of an existing DNA strand DNA polymerase I growing replication fork DNA polymerase III RNA Loss of bases at ends in every replication chromosomes get shorter with each replication limit to number of cell divisions?

42 Telomeres Repeating, non-coding sequences at the end of chromosomes = protective cap limit to ~50 cell divisions growing replication fork telomerase Telomerase enzyme extends telomeres can add DNA bases at end different level of activity in different cells high in stem cells & cancers -- Why? TTAAGGG TTAAGGG